Dr Daniel Picot

Prof. Winfried Weissenhorn

Prof. Ilme Schlichting

Dr Diana Kirilovsky

Dr Michel Sliwa

Ninon

Giorgio

Anne-Sophie Kyprianos

Elke Kevin

Rory Tadeo

Keywords: Adenosine triphosphate Bt: Bacillus thuringiensis Bti: Bacillus thuringiensis subsp. israelensis Fluorescence Recovery Protein HCP: Helical Carotenoid Protein Reactive Oxygen Species LHC: Light Harvesting Complex Serial Femtosecond Crystallography SSX: Serial Synchrotron Crystallography SAXS: Small-angle X-ray scattering WAXS: Wide-angle X-ray scattering WT: wild-type Orange Carotenoid Protein (OCP), photoactivation mechanism, X-ray scattering, Xray crystallography, oligomerization, in vivo nanocrystallography, Bacillus thuringiensis

I would like to express my most sincere acknowledgment to my supervisor, Dr. Jacques-Philippe Colletier, for allowing me to conduct this research from the very beginning, and for giving me the freedom, the constant encouragement and helpful advices, needed for the accomplishment of this project. My thesis would not have reached its present form without his support, his illuminating instructions and especially his constructive criticism. I sincerely thank him for having managed to repeatedly convincing me, as well as my ideas during all these (almost) five years. Jacques taught me to work hard and consistently, to appreciate the importance of being present and consent as a scientist, as well as to cherish every moment, every step towards the road to success. Je te remercie profondément de m'avoir tant soutenue, et surtout de m'avoir permis l'obtention de ce premier "collier de perles". Je te remercie, Jacques! J'ai vécu presque cinq années de doctorat mémorables grâce à tous les membres, passés et présents, du groupe DYNAMOP avec qui j'ai eu le plaisir de tant interagir, tant discuter et tant partager. Merci à Guillaume,

I would like to express my profound gratitude to Prof. Ilme Schlichting, who allowed me to continue working on this project over the past two years. For me it was a great honor to learn directly from her, as well as from the members of her team at the Max Planck Institute in Heidelberg. Since we first met, Prof. Schlichting has always been very fair regarding my work and my competences. She didn't just share with me her knowledge about protein crystallization, but she offered me the opportunity to develop my curiosity and skills as a scientist. Thanks to her trust, I was able to go until the end of my project. I thank Prof. Schlichting for her comprehension and her accuracy in difficult moments. I wish to express my gratitude to her. I would like to express my sincere gratitude to Martin Weik, who welcomed me in his group DYNAMOP, at IBS. Martin always offered me valuable suggestions for my project. During these years, he spent much time to improve my work, and always provided me relevant advices. Martin has always known how to support and respect students, and especially how to raise their courage. Often a bowl of Cup Noodles was more than enough. I would also like to acknowledge Dr. Diana Kirilovsky, Dr. Michel Sliwa and Dr. Adjélé Wilson for the fruitful collaboration, for all scientific discussions we have had together and for generously sharing their expertise with me. The curiosity and passion that I had for OCP (especially for its excited states) was also possible thanks to you.

Pendant ce projet, j'ai côtoyé de nombreuses personnes qui m'ont beaucoup apporté scientifiquement. Des remerciements particuliers iront à Nicolas Coquelle, Johanna Timmins et Paul Schanda qui ont beaucoup contribué à l'avancement de mon travail.

de rire, trois femmes avec qui, comme dirait Leo Ferré, j'ai souvent ''pris la rosée pour du rosé d´Anjou''.

Je tiens à remercier du plus profond de mon coeur, Stéphane, sans qui rien n'aurait pu être possible ces derniers 8 ans. Stéphane, mon français préféré, mon ami, mon confident avec qui on a tant vécu, tant voyagé et tant ri. Je le remercie tout particulièrement pour sa confiance imbattable, son positivisme et sa bienveillance. Il m'a surtout appris à avoir de l'empathie envers l'autre et à savoir qu'il vaut mieux avoir des remords que des regrets. Merci, Steph ! Je tiens à remercier tout particulièrement mes parents, qui m'ont toujours apporté le soutien et l'amour inconditionnel sans faille ni faiblesse tout au long du chemin que j'ai parcouru jusqu'à présent. Elitza, ma mère, une vraie forteresse qui m'as enseigné l'amour, la curiosité, la force et l'élégance. Alexandre, mon père, qui m'a appris à ne jamais abandonner, à travailler et à suivre ma voie. Tous les deux, mes parents, qui m'ont si soigneusement donné des ailes, ces ailes qui m'ont permis de m'envoler, et de voler très haut depuis. Mes parents, que j'espère tellement ne pas avoir déçu. Благодаря ви за всичко! Je dédie cette thèse à mon frère, Andrey, qui a seulement 9 ans et que vous ne connaissez certainement pas, mais lui vous connait tous. Je voudrais qu'il sache que tout est possible, et qu'il faut toujours prendre les opportunités que ce monde lui présente. Je sais qu'il va le faire, car grâce à son esprit et son coeur grand ouvert il sera capable de ''faire de la vie un rêve et du rêve, une réalité''. The most important biological process that allows the maintenance of life on our Earth is photosynthesis. All living organisms require a constant flux of energy in order to persist, develop and reproduce. In photosynthetic organisms, this energy gradient is provided by photosynthesis which consists in bringing together a set of biophysical and biochemical reactions allowing to make use of the sunlight energy and carbon dioxide (CO2) to generate oxygen (O2) through the decomposition of water (H2O) [START_REF] Hohmann-Marriott | Evolution of photosynthesis[END_REF] . During this oxidation-reduction reaction, the released electrons are subsequently used to reduce inorganic carbon and form organic compounds, essential for the cell metabolism and survival.

Before water became the main electron source, the first living organisms used hydrogen (H), ferrous iron (Fe) and hydrogen sulfide (H2S) as electron donors, all of which are present in comparatively small amounts compared to water. Even today, a vast number of non-oxygenevolving (anoxygenic) photosynthetic organisms, including various group of bacteria (e.g. purple bacteria, green sulfur bacteria, green filamentous phototrophs, heliobacteria and acidobacteria)

as well as some archaea (e.g. halobacterium), still use these atoms and molecules as electron donors.

In plants and green algae, oxygenic photosynthesis takes place in and around the thylakoid membranes (TM) of chloroplasts. The chloroplast is a lens-shaped organelle with a size ranging from 5 to 10 µm, which is composed by a pair of concentric membranes. The outer and inner membranes (Figure 1A) contain multiple carriers which ensure the flows of ions and metabolites between the cytosol and the chloroplast. The most abundant carrier present in the inner membrane is a phosphate translocator, the role of which is to supply the cell with products from photosynthesis (e.g. sugars, adenosine triphosphate (ATP)). The internal milieu (called stroma) of the chloroplast is majorly occupied by a membrane system, named thylakoid membranes (Figure 1B). The thylakoid membrane is composed of flattened structures in the form of saccules (thylakoids), which communicate between each other in a closed and continuous way. A feature common to the thylakoids is the formation of multiples piles of saccules called grana (granum in singular), which result from the stacking of thylakoids one upon the other. The grana are interconnected by unstacked thylakoids, that constitute the stromal thylakoids. Within the granum (singular), the thylakoid membrane encloses an internal space, fully isolated from the stromal environment (lumen) (Figure 1B). The thylakoid membranes support the multimeric complexes which are involved in the photosynthetic light reactions and related electron transport. Precisely, these complexes include the Photosystem II (PSII), the Cytochrome b6f complex (Cytb6f), the Photosystem I (PSI) and the ATP synthase. The first three are the main actors in a series of reactions, commonly named 'light reactions', whereas the latter is a transmembrane pump which use the proton gradient generated aside the electron flow to synthesize ATP (Figure 2).

Both PSII and PSI are associated with their respective light-harvesting complexes (LHC), i.e.

LHCII and LHCI, whose principal role consists in driving the absorbed light energy to the photosystem reaction centers (RC) where charge separation takes place. In this way, a transmembrane electrochemical proton gradient is generated which will be use to fuel production of ATP by the transmembrane ATP synthase, thereby accumulating promptly-accessible energy in the cell. The following section provides a brief overview of each protein complex which constitutes the photosynthetic machinery. The photosynthetic electron transport chain. In the thylakoid membranes of chloroplasts, photosystem II (PSII) is excited by absorption of a photon light energy. In the reaction center of PSII (P680) a chlorophyll molecule transiently loses an electron, which is transmitted to the plastoquinone pool (PQ). The chlorophyll is a very strong oxidizing agent, thus upon oxidation it is able to accept electrons from water. Water splitting occurs in the reaction centers, situated on the lumen side of the thylakoid membrane and results in generation of oxygen and protons. The reduced plastoquinone (PQ) move through the membrane, from PSII to cytochrome b6f (Cytb6f), where PQ is oxidized and its proton is released in the lumen. This enables transport from the stroma to the lumen. Electrons are further transferred to photosystem I (PSI), where the electron transfer allows reduction of the excited chlorophyll localized in the PSI reaction center. Upon excitation, this chlorophyll transfers its electrons to the stromal ferredoxin (Fd) which can reduce NADP + through the ferredoxin NADP reductase (FNR). The photolysis of water photolysis and the proton transport (via PQ) contribute to the establishment of a proton gradient. This gradient is used as an energy source for the generation of ATP via ATP synthase.

The Photosystem II (PSII) is a dimeric water-plastoquinone (PQ) photo-oxidoreductase, wherein each monomer is composed of around 20 integral membrane protein subunits, 35 chlorophyll a (Chla) and ~12 β-carotenes molecules, 1 oxygen-evolving center (OEC), ~3 PQ and redox cofactors, such as Cl -and Ca 2+ , all of them involved in the electron transfer. The first structure of a PSII reaction center, that from the cyanobacteria Thermosynechococcus elongatus, was published in 2001 3 revealing the presence of a heterodimer of ~700 kDa, composed of two monomers, i.e. D1 (i.e. PsbA) and D2 (i.e. PsbD) which form the reaction core of PSII. While D2 is homologous (around 30% of identity) to D1, it is less involved in binding active cofactors.

However, under extreme conditions (e.g. high-light irradiation), D1 is replaced by a repair process.

In addition, via its two histidine residues, i.e. His214 and His268, PsbD is able to provide ligands to non-heme iron. Few years later, two more PSII structures from the cyanobacteria Thermosynechococcus vulcanus were released, allowing to obtain further information about PSII OEC. Briefly, the first step throughout the electron transfer across PSII consists in charge separation between an excited Chla (P680*) and an electron-acceptor molecule associated to D1, the phoehytin. From there, the electron is transferred to quinone A (QA, associated to D1)) and then to quinone B (QB, also associated to D1)). QB needs to receive two electrons reaching full protonation to leave PSII, under the form of plastoquinol (PQH2). Chlorophyll P680 + regains an electron by reducing tyrosine Z (TyrZ, associated to D1) and the OEC. The OEC is a Mn4CaO5 cluster which will in return reduce TyrZ + generating the formation of O2. At each cycle, TyrZ is reduced four times and two water molecules are oxidized, releasing four H+ protons into the lumen of the thylakoid.

The Cytochrome b6f complex is a dimeric, helical, thylakoid membrane-embedded protein, in which each monomer is composed of 8 subunits, whereby each one is composed of four main sub-units, the cytochrome f, cytochrome b6, the Rieske iron-sulfur [2Fe2S] protein and subunit IV, and of four smaller sub-units coined PetG, PetM, PetL and PetN 4 . The complex owes its name to the fact that among the protein subunits, the presence of cytochromes is also observed. The cytochromes are small proteins that are able to covalently bind heme groups. The Cyt b6f is involved in the electronic connection between the photosystem I and photosystem II reaction centers and its role consists in reducing the pool of PQ, thereby contributing to the electrochemical gradient, and in transferring electrons to PSI. Briefly, the Cytb6f oxidizes PQH2 by donation of electrons to the Rieske Fe carrier, releasing protons into the thylakoid lumen. One electron passes through the Rieske iron-sulfur [2Fe2S] protein before transferring to the external Plastocyanin (PC), while the second electron passes through three heme groups following the reduction of PQ bound to the Qi site of Cyt b6f 5 . For one oxidized PQH2, two H + protons are released in the lumen of the thylakoid.

The third complex involved in oxygenic photosynthesis is the photosystem I (PSI), which is a transmembrane pigment-protein complex involved in the electron transfer from the lumenlocalized PC to the stromal-localized ferredoxin (Fd). Depending on both growth conditions and type of photosynthetic organism, PSI can be present as a monomer, dimer or a trimer where one functional unit features 96 Chla molecules or in other terms, ~2.5 times more absorption units than PSII. The first published crystallographic structure of PSI, from the cyanobacteria Thermosynechococcus elongatus, revealed that the functional and structural core of PSI is composed of two large protein subunits, namely PsaA and PsaB, with 10 transmembrane PsaA and PsaB helices forming a barrier around the electron transfer chain 6 . The PsaA/PsaB core is surrounded by seven small membrane-embedded subunits, each one containing one to three transmembrane helices, stabilizing the antenna system and mediating trimerization. The electron transport chain of PSI is highly conserved among plants and cyanobacteria and it is composed of six chlorophylls, two molecules of phylloquinone and three iron-sulfur clusters [4Fe4S] 7 .

In contrast to PSII, the absorption maximum of the PSI Chla molecule is 700 nm, explaining reference to this reaction center as P700. In PSI, charge separation takes place after excitation of P700 and concomitant reduction of chlorophyll A0, yielding the excited P700* state. The electrons are transferred to phylloquinone, then successively to the three [4Fe4S] clusters, namely FX, FA/FB.

At the end of the electron transfer chain is Fd, which upon reduction will itself feed electrons to the FNR, allowing production of NADPH which is used in a variety of metabolic processes, e.g. in the assimilation of CO2 in the Calvin-Benson cycle, nitrate metabolism and amino acid synthesis.

The fourth and last complex involved in photosynthesis is the ATP synthase, powering the synthesis of adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and inorganic phosphate (Pi) by use of the proton gradient resulting from either the linear electron flow (LEF) or the cyclic electron flow (CEF). Generally, the sunlight reinforces the linear electron flow (LEF) able to generate NADPH and ATP from H2O and NADP + with the participation of both PSII and PSI.

All photosynthetic oxygenic organisms have evolved to use secondary electron transfer mechanisms, such as CEF, in order to regulate photosynthesis and maintain the equilibrium between light availability and metabolism. During CEF, generated electrons from PSI are reinjected into the PQ pool, generating a proton gradient that allows the synthesis of ATP while downregulating LEF at the level of PSII.

The produced NADPH and ATP are the substrates of Calvin-Benson Cycle, the latter playing a central role in carbon fixation in oxygenic photosynthesis. Thus, fixation of CO2 allows the conversion of inorganic carbon atom to reduced organic carbon, leading to the production of carbohydrates. The Calvin-Benson cycle involves eleven enzymes enabling catalyzation of 13 reactions comprised in three main phases, i.e. (i) the fixation of CO2, (ii) the reduction and formation of 3-phosphoglycerate glyceraldehyde, respectively, and (iii) the reassembly of RuBP (ribulose -1,5-biphosphate).

As it will be explicated below, a major difference between various oxygen producing photosynthetic organisms is the means by which they harvest the sunlight energy, rather than utilize it. In plants the photosystem II reaction centers are closely associated with the lightharvesting complexes (LHC). The photosystem II of plants and green algae feature an integralmembrane antenna, as their LHC, which is called the light-harvesting-chlorophyll antenna (LHCII) and is composed of hydrophobic, membrane-embedded polypeptides, namely CP47 and CP43, both binding Chla and Chlb molecules. In addition to LHCII, three other minor antennas have been described that are localized within the thylakoid membranes, at the junction between LHCII and PSII reaction centers but are present in smaller quantities. These are CP29, CP26 and CP24 which all present a lower Chlb concentration than LHCII [START_REF] Bassi | Chlorophyllproteins of the photosystem II antenna system[END_REF] . The photosystem I light-harvesting complex (LHCI) is composed of two chlorophyll-containing proteins that also partake in the formation of PSI reaction center, namely PsaA and PsaB. Again, these are integral membrane proteins pointing to light harvesting having/energy evolved to take place inside of membranes, shelved from the bulk [START_REF] Roach | Photosynthetic Regulatory Mechanisms for Efficiency and Prevention of Photo-Oxidative Stress[END_REF][START_REF] Kirilovsky | Photoprotection in cyanobacteria: the orange carotenoid protein (OCP)-related non-photochemical-quenching mechanism[END_REF] . The LHC proteins are accordingly encoded by nucleus-encoding genes that share an important level of homology.

Cyanobacteria

Cyanobacteria are prokaryotic oxygen-producing bacteria, which appeared around 3 billion years ago, and are able to use sunlight energy to convert CO2 into organic matter. It is firmly believed that their photosynthetic activity prompted the oxygenation of the atmosphere -a process known as the Great Oxidation Event [START_REF] Kopp | The Paleoproterozoic snowball Earth: a climate disaster triggered by the evolution of oxygenic photosynthesis[END_REF][12][13] .

Precisely, the beginning of oxygen-evolving photosynthesis is attributed to the ancestor of cyanobacteria, present approximately 3.2 to 3.5 billion years ago 14, . Initially, cyanobacteria were classified as "blue-green algae" based mainly on two essential criteria: (i) they present a morphological resemblance to eukaryotic algae; (ii) they possess the ubiquitous green pigment chlorophyll a and other accessory pigments, such as phycocyanin (blue), phycoerythrin (red) and yellow-orange carotenoids. The unique combination of phycobilin and chlorophyll a provides the cyan, blue-green color. In 1970, the prokaryotic nature of cyanobacteria began to be fully recognized, based on morphological and cytological information, and the latter were classified as Gram-negative bacteria (Bergey's Manual).

Notwithstanding the overall gram-negative structure, the cyanobacterial cell wall is yet unusual compared to that of most gram-negative bacteria. Usually, the peptido-glycan (PG) layer of a gram-negative bacteria does not exceed 2-6 nm in thickness, however in cyanobacteria, the PG layer thickness can range from tens (10 nm in Synechoccus) to few hundreds of nm (700 nm in Oscillatoria princeps) [START_REF] Hoiczyk | Cyanobacterial cell walls: news from an unusual prokaryotic envelope[END_REF][START_REF] Golecki | Studies on ultrastructure and composition of cell walls of the cyanobacterium Anacystis nidulans[END_REF][START_REF] Hoiczyk | Envelope structure of four gliding filamentous cyanobacteria[END_REF] . As in plants, the cyanobacterial photosynthetic machinery lodges on the thylakoid membranes (parallel arrangement to the cell membrane), where each thylakoid consists of a double-layer membrane surrounding an intrathylakoid space. In the center of the cyanobacterial cell resides the nucleoplasm, hosting the DNA.

Molecular analyses based on a comparison of 16S ribosomal RNA gene sequence, permitted the reconstruction of the cyanobacterial phylogeny and their classification, despite their overall morphological resemblance. First, cyanobacteria may be unicellular (e.g. Synechococcus elongatus, Thermosynechococcus elongatus), spherical (e.g. Synechocystis PCC 6803) or filamentous (Arthrospira PCC 7345, Planktothrix agardhii, Nostoc punctiforme PCC 7120).

Cyanobacteria normally reproduce via binary fission [START_REF] Springstein | Structural Determinants and Their Role in Cyanobacterial Morphogenesis[END_REF] , however, filamentous cyanobacteria of the order Nostocales have evolved other reproductive strategies whereby cell differentiation can happen through the formation of either vegetative cells (involved in photosynthetic processes), or differentiated cells, such as heterocyst (involved in anaerobic fixation of atmospheric nitrogen; e.g.

Anabaena PCC 7120) and akinetes (spores formed under unfavorable environmental conditions) 19, 20 .

The photosynthetic apparatus of cyanobacteria is the same as that of other oxygen evolving photosynthetic organisms and it is also composed of PSII, PSI, Cyt b6f and ATP synthase. In addition, cyanobacteria feature: (i) an intracellular non-membrane-bound granules (glycogen), (ii) cyanophycin (cyanobacterial nitrogen reserve polymer) and (iii) carboxysomes. The carboxysomes are intracellular inclusions in which occurs the accumulation of ribulose 1,5bisphosphate carboxylase/oxygenase (RubisCO), the enzyme involved in the initial carboxylation step in the Calvin cycle [START_REF] Garcia-Pichel | [END_REF] .

Light harvesting in cyanobacteria: The antennae complex

Cyanobacteria inhabit a variety of aquatic environments where water depth and clarity influence light transmission. To adapt, most cyanobacteria (and red algae) exploit phycobilisomes (PBS) (Figure 3) which are highly ordered membrane-attached soluble supramolecular structures [START_REF] Gantt | Phycobiliprotein localization in algae[END_REF] , capable of harvesting light in the domain of visible light (450 -670 nm) thanks to their multiple phycobilin pigments 2,[START_REF] Adir | Elucidation of the molecular structures of components of the phycobilisome: reconstructing a giant[END_REF] . Thereby, PBS enable to cope with the spectral gap that exists between the two major chlorophyll absorption bands (450 and 600 nm) of membrane-embedded lightharvesting antennas of photosystems. Specifically, and as introduced earlier, photosystems I and II light-harvesting complexes (LHCs) are integrated in the thylakoid membrane, while the PBS is an extrinsic water-soluble antenna, localized on the cytoplasmic side of the thylakoid membrane although strongly associated to it.

The PBS are composed of water-soluble phycobiliproteins (PBPs), representing 85% of the total PBS composition. The phycobiliproteins consist of hetero-monomers which are composed of two subunits, i.e. 𝛼 and β that bind covalently one or more phycobilin pigments. The PBP subunits can assemble and form disc-like trimers (𝛼β)3 or hexamers (𝛼β)6. While these polypeptide subunits are present in an equal stoichiometry ratio (1:1), their amino acid composition and chromophore content strongly differ. The chromophore content and the related absorption maximum allows to divide PBPs in four distinct groups, comprising (i) the phycoerythrin ((PE) Amax = 560 nm), (ii) the phycocyanin ((PC), Amax = 620 nm), (iii) the phycoerythrocyanin ((PEC), Amax = 575 nm) and (iv)

the allophycocyanin ((Apc) Amax = 650 nm) (Figure 3B) [START_REF] Adir | Elucidation of the molecular structures of components of the phycobilisome: reconstructing a giant[END_REF][START_REF] Glazer | Phycobilisome a macromolecular complex optimized for light energy transfer[END_REF] .

To date, only electron microscopy techniques has allowed to elucidate PBS structural organization as well as their morphology. PBS are composed of two sub-structures, i.e. a core which is composed of trimeric (𝛼β)3 APC, and multiple rods, composed of stacked (𝛼β)6 discs, emanating from the core. Even if the number of core and rods components vary among different species, the general composition and architecture of the PBS is conserved [START_REF] Adir | Elucidation of the molecular structures of components of the phycobilisome: reconstructing a giant[END_REF][START_REF] Anderson | A model for early events in the assembly pathway of cyanobacterial phycobilisomes[END_REF] . To date, various structures of PBS have been described and classified into four family groups, based on their morphology.

These families include the hemidiscoidal, the hemiellipsoidal, the block-type and the bundle-type PBS.

The family of hemidiscoidal PBS is the most common amongst cyanobacteria, and to date best described. The cyanobacteria Synechocystis PCC 6803, used as primary model system for molecular and spectroscopic studies, present a hemidiscoidal PBS and its structure was mostly studied by electron microscopy. An agreement between previous and recently published results

allowed to obtain more detailed information into PBS core and rods structure and their arrangements.

The PBS core is composed of three allophycocyanin cylinders (Figure 3A), wherein each cylinder consists of four trimer Apc discs. The top (upper) cylinder is composed of two discs of Apc trimers (αβ)3, and two APC trimers (αβ)3 associated with the linker protein ApcC (LC). The upper cylinder stacks on top of the two basal cylinders, which are characterized by different Apc composition compared to the top cylinder and are in direct contact with the thylakoid membrane. The basal cylinder is composed of two discs of pure Apc trimer (αβ)3, one disc of trimer in which the α-subunits is replaced by ApcE and one of the β-subunits by ApcF, and one trimer disc in which one α-subunit is replaced by ApcD. ApcE present a linker domain, called core-membrane linker (LCM) which ensures the stability of the core by connecting the basal with the upper cylinders. ApcD interacts with ApcE and also play a role in stabilizing the three cylinders. ApcD, ApcE and ApcF feature red-shifted spectrum compared to Apc. It has been previously suggested and recently demonstrated [START_REF] Dominguez-Martin | Structure of the Quenched Cyanobacterial OCP-Phycobilisome Complex[END_REF] , that PBS interacts with the stromal side of the thylakoid membrane through ApcE, the chromophore of which serves as terminal emitter allowing the transfer of absorbed energy straight to the PS reaction centers.

From the core, six rods radiate, wherein each rode is composed of three stacked PC hexamers connected by rod linker proteins (LR) (Figure 3A). Recent studies have reported on three different rod conformations, two of them have not been described previously. In the previously well-studied "up-up" (or static) conformation (Figure 3A), the rods are positioned upwards. In the newly reported conformers the rods can present either an "up-down" conformation, characterized with a movement of a mobile rod and adoption of position parallel to the other rods, or a "down-down" conformation in which all rods connected to an upper core cylinder are in down position. Different cyanobacteria and red algae species may present different phycobiliprotein compositions of their rods. For instance, in the cyanobacteria Anabaena PCC 7120 the distal to core discs of the rod feature PE and PEC as phycobiliproteins, respectively, while PBS from most marine cyanobacteria (Synechocystis PCC 6803, Synechococcus elongatus PCC 7942) present PE and PC as phycobiliproteins [START_REF] Glazer | Phycobilisome a macromolecular complex optimized for light energy transfer[END_REF] . To ensure an optimal light-harvesting, photosynthetic reaction centers and related light-harvesting antennae exploit a variety of chromophores, able to absorb sunlight energy and subsequently transfer it to reaction centers. This energy is then used for the initiation of the primary events of photosynthesis. Being found in all photosynthetic organisms, chlorophylls (Chls) are the most ubiquitous pigments. They are formed from an aromatic tetrapyrrole macrocycle which encases a magnesium (Mg) as a central metal. Chls are mainly presented in the reaction centers of plants and eubacteria, and as all chromophores involved in photosynthesis, they allow the efficient absorption of light and the transfer of the resulting excitation energy to the reaction centers.

Additionally, Chls undertake a third major role, i.e. primary charge separation across the thylakoid membrane [START_REF] Scheer | An Overview of Chlorophylls and Bacteriochlorophylls: Biochemistry, Biophysics, Functions and Applications[END_REF] . The classification of chlorophylls in different classes is mainly based on their spectroscopic characteristics which are themselves related to the degree of unsaturation of their tetrapyrrole macrocycles.

The most abundant chlorophyll is Chla, which is the major component of PSII and PSI reaction centers, as well as of core and peripheral antenna complexes. Chla presents two narrows absorption maxima, at 430 and 680 nm, i.e. it absorbs at the blue and red edges of the UV-Vis spectrum. In order to expand the absorption range, Chla are accompanied by other pigments, generally other Chls, such as Chlb, which is present in cyanobacteria and plants. With absorbance bands shifted at 460 and 650 nm, Chlb reduces and partly compensates the "green" absorption gap in photosynthetic organisms. In addition to Chlb, c-type Chls (Chlc), which mainly absorb at 450 nm, are also involved in the reduction of the "green gap". As opposed to Chla and Chlb, ctype Chls are phytoporphyrins and they are mainly found in brown algae where water clarity is limited [START_REF] Dougherty | The structure, properties, and distribution of chlorophyll c[END_REF]2 . In red algae and oxygenic prokaryotes, another chlorophyll is present, viz. Chld, whose absorption is red shifted, compared to Chla and may serve as a substitute for the latter as a primary donor in PSI.

Phycobilins

Cyanobacterial and red algae phycobiliproteins feature covalently attached linear tetrapyrroles chromophores, commonly named phycobilins [START_REF] Scheer | An Overview of Chlorophylls and Bacteriochlorophylls: Biochemistry, Biophysics, Functions and Applications[END_REF][START_REF] Sidler | Phycobilisome and Phycobiliprotein Structures[END_REF]29 . The latter bind the 𝛼β polypeptide chains at conserved positions and are maintained in extended conformation within the protein structure, conferring to the bilin chromophore an increased fluorescence, i.e. excited state lifetime and structure stability 29 . It has been known for a long time that the precursor of phycobilins is biliverdin, the degradation product of heme, suggesting that structural homology may exist between the heme-protein myoglobin and phycobiliproteins. So far, nine groups of bilins have been identified, based on their absorption properties and structural characteristics, including the number of conjugated double bonds and the side chains harbored by the tetrapyrrole groups. In cyanobacteria and red algae, however, only four of the nine groups are present: phycocyanobilin (PCB), phycourobilin (PUB), phycobiliviolin (PXB) and phycoerythrobilin (PEB) [START_REF] Sidler | Phycobilisome and Phycobiliprotein Structures[END_REF] . By combining the structural diversity and chemical properties of bilin chromophores, their binding affinity to the various phycobiliproteins and the variety of possible chromophore-chromophore interactions, phycobilins present an unusual spectral coverage that enables them to ensure optimal lightharvesting and energy transfer in/from the green domain of visible light.

Carotenoids

Synthesized in plants, algae, bacterial and fungal cells, the carotenoids are the third group of natural pigments, participating in the light-harvesting and in the photoprotection of photosynthetic organisms [START_REF] Polívka | Molecular factors controlling photosynthetic light harvesting by carotenoids[END_REF] . Carotenoids additionally play important roles in antioxidant and immune activities 31 .

Most studied has however been their role in photosynthesis, as will be described below.

Among the hundreds of carotenoids that have been identified so far, only ~50 were found to play a role in light-harvesting and in the governance of electron flow across the photosynthetic apparatus. Structurally, the carotenoids feature a linear central 40-carbon chain of alternating conjugated double bonds (common to all carotenoids). Divergence arises from the number of conjugated double bonds, and the type and number of functional groups attached to the carbon chain. To perform their light-harvesting function, photosynthetic organisms use a combination of unoxygenated, e.g. β-carotene, and oxygenated (xanthophylls) carotenoids, e.g. lutein and zeaxanthin 31,[START_REF] Polívka | Ultrafast dynamics of carotenoid excited States-from solution to natural and artificial systems[END_REF] . Additionally, cyanobacteria and some microalgae synthesize and use a special group of carotenoids, commonly named ketocarotenoids which are characterized by the presence of one or several carbonyl groups in their β-ionone rings.

Because of the resonance conferred by alternating double bonds, carotenoids have the ability to strongly absorb visible light in the blue and green region of the UV-Vis spectrum, explaining their yellow, orange and red colors. Unlike most other pigments, which upon absorption of the actinic photon transition from the ground state (S0 energy level) to the S1 singlet excited state energy level, carotenoids transition to the S2 excited state due to symmetry considerations that render transition to the S1 state optically forbidden. Briefly, due to their central backbone of conjugated single and double carbon bonds that form a conjugated π-electron system, carotenoids present a C2h symmetry which results in two low-lying singlet excited states, i.e. 2 1 Ag -(covalent) and 1 [START_REF] Hohmann-Marriott | Evolution of photosynthesis[END_REF] Bu + (ionic). The ground state of the carotenoids is 1 Ag -(equivalent to S0) and direct transition to 1 1 Bu + (equivalent to S2) is allowed, whereas that to 2 1 Ag -(equivalent to S1) is optically forbidden [START_REF] Polívka | Ultrafast dynamics of carotenoid excited States-from solution to natural and artificial systems[END_REF][START_REF] Chábera | Effect of carotenoid structure on excitedstate dynamics of carbonyl carotenoids[END_REF] .

The S2 excited state (Figure 4) will nonetheless decay to the S1 excited state within hundreds of femtoseconds (50-200 fs), which itself will decay within 1 -200 ps to the S0 ground state. The S2 and S1 lifetimes depend mainly on the number of conjugated double bonds on the central backbone, but also on the environment. The insertion of carbonyl groups in the central conjugated backbone results in changes in the excited-states properties of the carotenoids, which may lead to the formation of an intramolecular charge-transfer (ICT) state, shown to be strongly-coupled to the S1 state but shorter lived (≃0.5 ps). Besides the ICT state, an additional intermediate state has been described, termed S*, whose exact nature remains under debate. Spectroscopic studies have suggested that S* could either be a hot ground state, or a distorted excited state featuring a twisted conformation [START_REF] Polívka | Molecular factors controlling photosynthetic light harvesting by carotenoids[END_REF][START_REF] Hashimoto | Understanding/unravelling carotenoid excited singlet states[END_REF] . S* is predominant in carotenoids whose conjugated 𝜋-system extends to at least one of the terminal cycles. Interestingly, transient absorption spectroscopy studies performed on two carbonyl carotenoids presenting different conjugation lengths, e.g. β-carotene (n=10) and canthaxanthin (n=11) have reported similar relaxation rate for S* (~9 ps) but not for S1, respectively, -in the two molecules [START_REF] Chábera | Effect of carotenoid structure on excitedstate dynamics of carbonyl carotenoids[END_REF] . These results suggest that unlike S1, the lifetime of the S* state is independent of the conjugation length. Hashimoto H. et al., 2018 34 ).

In photosynthetic organisms (plants, algae and cyanobacteria), the carotenoids present a dual function: they can either function as light-harvesting pigments or can serve in the photoprotection of photosynthetic cells in case of energy overflow. Their light-harvesting function consists in transferring energy from their singlet excited states, acting as energy donors, to neighboring chlorophylls, playing the acceptor role (Figure 4). Despite the extremely short lifetime of the S2 and S1 excited states, the efficient energy transfer is ensured by an optimal positioning and related distances between the carotenoid conjugation system and the Chls. The measured distance is between 3 to 10 Angstroms (Å). The energy transfer occurs on the tens to hundreds of fs timescale and is transferred to the QY and QX states of Chls [START_REF] Polívka | Molecular factors controlling photosynthetic light harvesting by carotenoids[END_REF] .

Besides their function as light-harvesting pigments, carotenoids play a crucial role in the maintenance of normal cell functioning, thereby reducing the probability of formation of free radicals. During photosynthesis, the electron transport chain may trigger formation and accumulation of radical oxygen species (ROS), usually originating from H2O splitting. This photoprotection property is mainly due to the very short lifetime of their excited states which enables rapid conversion of the excess of light energy into heat.

Photoprotection: from oxidative stress to non-photochemical quenching (NPQ)

Oxidative stress and generation of radical oxygen species (ROS) in photosynthetic organisms

Light is the main source of energy for all photosynthetic organisms, but it may become a threat for the photosynthetic apparatus in case of light excess. A major consequence of electron overflow is the production of radical oxygen species (ROS) which can damage cellular components, proteinaceous and nucleic alike. The ROS derive from O2 and are divided in two groups: (i) free radical species, such as hydroxyl radical ( • OH) and superoxide anion ( • O2) and (ii) nonradical species like singlet oxygen ( 1 O2) and hydrogen peroxide (H2O2) 35,36, 37 38 . When present at low concentrations, ROS possess useful properties [START_REF] Krieger-Liszkay | Singlet oxygen production in photosynthesis[END_REF] . For instance, 1 O2 may act as an intracellular messenger thus promoting the expression of stress-related genes. However, high concentrations of ROS species are detrimental to the cells. In the present section the formation of 1 O2 in the photosynthetic reaction centers and antennae will be briefly discussed, as well as potential molecular targets.

In photosynthetic organisms the reaction centers of PSII and PSI are the main site of ROS generation. In the ground state, the atomic oxygen presents two unpaired electrons (biradical) with parallel spins, one of which can reverse after an absorption of sufficient energy, leading to the formation of 1 O2. During photosynthesis, singlet oxygen is produced in the PSII antenna system and reaction centers, upon quenching by O2 of the triplet-excited state of Chl. As previously discussed, Chls are the main light-absorbing pigments in LHCs and they present long-lived excited state (ns time-scale) as required to allow the conversion of excitation energy into reducing power.

In case of excess absorbed energy, the electron spins in the Chls excited state ( 1 Chl) can reverse leading to the formation of a low energy excited state, namely the Chl triplet state ( 3 Chl), characterized by a lifetime of orders of magnitude larger (µs time-scale). The 3 Chl can form either from a singlet excited chlorophyll (via intersystem crossing, e.g. reversion of spins), or by charge recombination reaction in the reaction center of PSII due to a reduced plastoquinone pool and an oxidation of quinol in the Cytb6f complex [START_REF] Krieger-Liszkay | Singlet oxygen production in photosynthesis[END_REF] .

Studies aiming to characterize the formation and activity of 1 O2, conducted on Arabidopsis thaliana leaves [START_REF] Hideg | Singlet oxygen imaging in Arabidopsis thaliana leaves under photoinhibition by excess photosynthetically active radiation[END_REF] and Chlamydomonas reinhardtii cells [START_REF] Hideg | Singlet oxygen imaging in Arabidopsis thaliana leaves under photoinhibition by excess photosynthetically active radiation[END_REF][START_REF] Trebst | A specific role for tocopherol and of chemical singlet oxygen quenchers in the maintenance of photosystem II structure and function in Chlamydomonas reinhardtii[END_REF] , respectively, demonstrated that accumulation of singlet oxygen results in photoinhibition of PSII. To prevent the damage of PSII, a physiological defense mechanism takes place that involves interaction between the 1 O2 and the PSII reaction center protein D1 and leads to the degradation of D1. This pathway enables complete restoration of the PSII activity, provided that new D1 subunits are synthesized or readily available [START_REF] Aro | Photoinhibition of Photosystem II. Inactivation, protein damage and turnover[END_REF] . However, the chlorophyll triplet state and the singlet oxygen species can be neutralized before reaching that point, by antioxidant molecules, e.g. carotenoids (β-carotene and zeaxanthin) and ɑ-tocopherol (Vitamin E) [START_REF] Trebst | A specific role for tocopherol and of chemical singlet oxygen quenchers in the maintenance of photosystem II structure and function in Chlamydomonas reinhardtii[END_REF] .

Photoprotective mechanisms in photosynthetic organisms

To avoid damages caused by excess of absorbed light energy, photosynthetic organisms have established several strategies and photoprotection mechanisms which enable them to adapt to stressful conditions. One of these mechanisms is the tuning of the effective size of their lightharvesting antennas, which are essential for an optimal light capture. This adjustment can depend either on gene-expression or proteolysis 2,[START_REF] Scholes | Lessons from nature about solar light harvesting[END_REF][START_REF] Müller | Non-Photochemical Quenching. A Response to Excess Light Energy[END_REF] . Another adaptive response is the so-called chromatic acclimation observed in cyanobacteria [START_REF] Gutu | Emerging perspectives on the mechanisms, regulation, and distribution of light color acclimation in cyanobacteria[END_REF] , that consists of the synthesis of diverse pigments to maximize or reduce photon capture.

In addition to the strategies aiming the optimization of light absorption, photosynthetic organisms have evolved additional mechanisms to get rid of the excess of absorbed light energy. One of these is non-photochemical quenching (NPQ), which allows the dissipation into heat of the excess energy absorbed by the light-harvesting antennae, before it reaches photosystem reaction centers 43,45 . According to the relaxation rates in darkness, preceded by a certain illumination, and the response to inhibitors, NPQ is formed by three components: (i) the pH-or energy-dependent component (qE); (ii) the state-transition component (qT) and (iii) the photoinhibitory component (qI). In plants, the energy-dependent component is the fastest one (µs -min). qE is triggered by the generation of a transmembrane pH gradient across thylakoid membranes that subsequently results in decrease of the lumenal pH. Lowering the pH leads to two important events: first, the activation of enzymes involved in the biosynthesis of the carotenoid zeaxanthin and second, the protonation of the PSII light-harvesting protein complex, the PsbS [START_REF] Niyogi | Safety valves for photosynthesis[END_REF][START_REF] Roach | Photosynthetic Regulatory Mechanisms for Efficiency and Prevention of Photo-Oxidative Stress[END_REF][START_REF] Roach | The role of the PsbS protein in the protection of photosystems I and II against high light in Arabidopsis thaliana[END_REF] . Both synthesis of zeaxanthin and protein protonation result in de-excitation of [START_REF] Hohmann-Marriott | Evolution of photosynthesis[END_REF] Chl by thermal dissipation of its excitation energy. The state-transition NPQ component (qT) involves the displacement of LHCII between PSII and PSI with aim to ensure that the absorbed energy is equally distributed between the two photosystems. The detachment from PSII and migration toward PSI of LHCII is triggered by phosphorylation, and it is accompanied by a reorganization of the thylakoid membrane [START_REF] Roach | Photosynthetic Regulatory Mechanisms for Efficiency and Prevention of Photo-Oxidative Stress[END_REF] .

Studies demonstrated that qE and qT NPQ mechanisms overlap and occur together in case of high-light stress conditions. The photoinhibitory quenching qI is the slowest (several hours) NPQ component. It is generated following inactivation and/or degradation of the D1 protein in PSII [START_REF] Krause | Photoinhibition of photosynthesis. An evaluation of damaging and protective mechanisms[END_REF] .

Non-Photochemical Quenching (NPQ) mechanism in cyanobacteria

As in plants and algae, photosynthetic processes in cyanobacteria occur at the level of PSII and PSI, with the important difference that energy absorption is accomplished by the membraneattached but soluble antennae, the PBS, and directly transferred to the reaction centers. Because of the lack of xanthophyll cycle in cyanobacteria, it has been considered for a long time that cyanobacteria are disadvantaged of photoprotection mechanism.

In 2000, El Bissati and coworkers [START_REF] El Bissati | Photosystem II fluorescence quenching in the cyanobacterium Synechocystis PCC 6803: involvement of two different mechanisms[END_REF] reported on a possible photoprotection mechanism in cyanobacteria, involving thermal dissipation of the energy absorbed by the PBS. At this time, two pathways were known enabling dissipation of light-harvested excitation energy, i.e. the photochemical reactions, and the non-photochemical quenching (NPQ) [START_REF] El Bissati | Photosystem II fluorescence quenching in the cyanobacterium Synechocystis PCC 6803: involvement of two different mechanisms[END_REF] . The fluorescence quenching is modulated either by the oxidoreduction state of quinones or by non-photochemical processes, i.e. energy-quenching (qE), state transition (qT) and photoinhibition (qI). As qE cannot occur in cyanobacteria, qT and qI were considered as possible mechanisms in this study. After exposing Synechocystis cells to high intensities of blue-green light, El Bissati et al. observed a decrease of PSII fluorescence in conditions where the PQ pool was oxidized. Additionally, the occurring fluorescence recovery was still observed even if the study was held in presence of protein synthesis inhibitors. Their results were thus suggestive of blue-green light being capable of inducing a quenching mechanism unrelated to photoinhibition, nor state transitions. In their study the authors proposed that high light intensities of white or blue-green light induce a decrease in the PBS fluorescence, which is furthermore accompanied by a decrease in the energy transferred from the PBS to the photosystems (Figure 5). Few years later, a study was performed on Synechocystis mutants that lacked PSII reaction centers and/or CP43 and CP47 antennas.

The reported results showed that irradiation with blue-green light enables reversible increase in the PBS fluorescence emission. In addition, the recorded actinuous spectrum of PBS exhibited formation of three distinct peaks, i.e. at 496, 470 and 430 nm, characteristic for carotenoid absorption. Thus, the result was suggestive of a carotenoid molecule being possibly involved in the photoprotective cyanobacterial NPQ mechanism.

In 2006, Wilson and al. 50 reported that the cyanobacterial NPQ mechanism involves the a small soluble carotenoid-encasing protein, called the Orange Carotenoid Protein (OCP), recently classified in 3 clades based on phylogenetic studies, i.e. OCP1, OCP2 and OCPX [START_REF] Bao | Additional families of orange carotenoid proteins in the photoprotective system of cyanobacteria[END_REF] . Besides its quenching properties, it was shown that OCP acts as a light sensor owing to presence of a ketocarotenoid pigment absorbing light in the blue-green region, fueling for conformational changes that elicit the quenching state [START_REF] Wilson | A photoactive carotenoid protein acting as light intensity sensor[END_REF][START_REF] Gwizdala | In vitro reconstitution of the cyanobacterial photoprotective mechanism mediated by the Orange Carotenoid Protein in Synechocystis PCC 6803[END_REF] . This mechanism was coined OCP-related NPQ mechanism. Briefly, upon absorption of a blue-green photon, OCP is photoactivated and converts from a dark-adapted inactive orange state (OCP O ) to a light-adapted metastable red state (OCP R ) that is capable of binding to the PBS and of quenching its excess excitation (Figure 5) [START_REF] Wilson | A photoactive carotenoid protein acting as light intensity sensor[END_REF][START_REF] Gwizdala | In vitro reconstitution of the cyanobacterial photoprotective mechanism mediated by the Orange Carotenoid Protein in Synechocystis PCC 6803[END_REF] . In vitro reconstitution of the OCP-related NPQ mechanism in Synechocystis PCC 6803 [START_REF] Gwizdala | In vitro reconstitution of the cyanobacterial photoprotective mechanism mediated by the Orange Carotenoid Protein in Synechocystis PCC 6803[END_REF] , followed by fluorescent measurements [START_REF] Gwizdala | In vitro reconstitution of the cyanobacterial photoprotective mechanism mediated by the Orange Carotenoid Protein in Synechocystis PCC 6803[END_REF][START_REF] Tian | Site, rate, and mechanism of photoprotective quenching in cyanobacteria[END_REF] and transient absorption spectroscopy [START_REF] Berera | The photophysics of the orange carotenoid protein, a light-powered molecular switch[END_REF] studies, suggested that in the cell, one or more OCP can bind per two or three PBS. Furthermore, the study suggested that energy-quenching involve charge and energy transfer from an excited bilin state in the APC660 PBS core to the carotenoid in OCP R state. Recently, the first structure of the OCP-PBS complex, obtained by cryo-electron microscopy, suggested that one OCP bind one PBS [START_REF] Dominguez-Martin | Structure of the Quenched Cyanobacterial OCP-Phycobilisome Complex[END_REF] . In vivo, unbinding of OCP R from the PBS and recovery of the dark-adapted OCP O state is chaperoned by the so-called Fluorescence Recovery Protein (FRP) [START_REF] Boulay | Identification of a protein required for recovery of full antenna capacity in OCP-related photoprotective mechanism in cyanobacteria[END_REF] . The capacity of OCP to interact with FRP was shown to be a key feature of the OCP1 clade, as members of both OCP2 and OCPX (vide infra) clades are incapable of binding to FRP.

The Orange Carotenoid Protein (OCP)

Insights into OCP photoactivation mechanism and structure

The Orange Carotenoid Protein (OCP) was described for the first time by Holt and Krogman in 1981. At the time, it was shown that this carotenoid-binding protein is present in three cyanobacterial species, i.e. Spirulina maxima, Aphanizomenon flos-aquae and Microcystis aeruginosa [START_REF] Holt | A carotenoid-protein from cyanobacteria[END_REF] . Specifically, the bound carotenoid was determined to be the ketocarotenoid 3'hydroxyechinenone (3'OH-ECH) conferring to the protein absorption properties in the blue-green domain of the UV-Vis spectral region. Few years later, Wu and Krogman identified the OCP encoding gene slr 1963 and showed that it is constitutively expressed in Synechocystis PCC 6803 cells [START_REF] Wu | The orange carotenoid protein of Synechocystis PCC 68031Publication No. 15377.1[END_REF] . Mass spectrometry studies revealed the molecular weight of the protein (35 kDa) and furthermore confirmed that OCP is functionalized by non-covalently bound 3'OH-ECH ketocarotenoid.

Sequence homology studies performed on the then-available cyanobacterial genomes showed the existence of multiple homologs of the Synechocystis slr1963 gene in other cyanobacteria, the majority of them featuring a PBS as the main light-harvesting system [START_REF] Boulay | Occurrence and function of the orange carotenoid protein in photoprotective mechanisms in various cyanobacteria[END_REF][START_REF] Kirilovsky | The orange carotenoid protein in photoprotection of photosystem II in cyanobacteria[END_REF] . It was shown that OCP plays an important role in cell survival when cyanobacteria are exposed under conditions of high light. The identity scores between Synechocystis OCP and OCP homologs was shown to range between 50 and 80% and to correlate with the cyanobacterial habitat. For instance, the OCP from the marine cyanobacteria Synechococcus presents low similarity with the OCP of the freshwater Synechocystis, whereas marine cyanobacteria feature OCP sequences that are very similar to one another. In contrast, OCPs from the cyanobacteria Nodularia and the wetrock Gloeobacter violaceus, the two of them inhabiting saline soils, present very low homologies with both freshwater and marine ones [START_REF] Boulay | Occurrence and function of the orange carotenoid protein in photoprotective mechanisms in various cyanobacteria[END_REF] . This observation suggests that the OCP sequence and structure are tuned to level the quenching properties as a function of the natural habitat of the exposing cyanobacteria.

While around ~ 40% of PBS-containing cyanobacteria present OCP homologs, few strains such as Thermosynechococcus elongatus and Synechococcus elongatus PCC 7942 and PCC 6301 lack the entire OCP gene. As a consequence, the blue-green light induced NPQ mechanism is absent in these strains that may therefore endure severe damages at the PSII level. Contrastingly, in a variety of other strains, such as Synechocystis PCC 6803, the slr1963 gene is constitutively expressed and its level of transcription dramatically increases upon stress conditions, such as high light irradiance [START_REF] Hihara | DNA microarray analysis of cyanobacterial gene expression during acclimation to high light[END_REF] or iron starvation [START_REF] Wilson | Light-induced energy dissipation in iron-starved cyanobacteria: roles of OCP and IsiA proteins[END_REF] , meaning that this mechanism can be mobilized at any time and further amplified.

Structure of the OCP dark-adapted state (OCP O )

In 2003 Kerfeld et al. reported the isolation, crystallization and structure determination of the OCP from Arthrospira maxima, in the dark-adapted OCP O state (PDB id: 5ui2) [START_REF] Kerfeld | Crystals of the carotenoid protein from Arthrospira maxima containing uniformly oriented pigment molecules[END_REF][START_REF] Kerfeld | The crystal structure of a cyanobacterial water-soluble carotenoid binding protein[END_REF] . At 2.1 Å resolution, the structure revealed formation of OCP homodimer with modular organization of the monomers.

The OCP O monomer is composed of two functional domains: an α-helical N-terminal domain (NTD; residues 19-160) unique to cyanobacteria and an α-helical/β-sheet C-terminal domain (CTD; residues 190-317) that structurally resembles the fold of the NTF2 (nuclear transport factor 2) superfamily. Both domains are covalently attached by a flexible linker (residues 161-185). At their interface the two domains encapsulate a single non-covalently bound ketocarotenoid, the 3'hydroxyechinenone (3'-hECN). In the monomer, domain interaction is stabilized through two main interfaces. The central domain interface features two hydrogen bonds (including a salt bridge) between Asn104 and Trp279, and Arg155 and Glu246, whereas the second interface comprises an interaction between a short α-helical N-terminal extension (NTE; residues 1-19) and the Cterminal β-sheet. Additionally, the hydrophobic tunnel encasing the ketocarotenoid pigment also contributes to the stabilization of the dark structure. Inside the tunnel, the carotenoid is almost entirely buried, with only ~ 4% of its surface being exposed to the solvent [START_REF] Kerfeld | The crystal structure of a cyanobacterial water-soluble carotenoid binding protein[END_REF] . In the CTD, via its carbonyl end, the hECN hydrogen-bonds to the two absolutely conserved Trp290 and Tyr203 (Arthrospira OCP residue numbering). In the NTD, however, the carotenoid does not hydrogenbond to the protein, but is instead stabilized by π-π stacking interaction between the aromatic rings of the conserved Tyr44 and Trp110 [START_REF] Kerfeld | Structural and EPR characterization of the soluble form of cytochrome c-550 and of the psbV2 gene product from the cyanobacterium Thermosynechococcus elongatus[END_REF][START_REF] Wilson | Structural Determinants Underlying Photoprotection in the Photoactive Orange Carotenoid Protein of Cyanobacteria[END_REF] .

Inside crystals, OCP O assemble as a homodimer burying ~1400 Å 2 of surface area per monomer.

The dimeric interactions are of both polar and hydrophobic nature and are formed between three absolutely conserved residues, i.e. Asp19, Arg27 and Asn 134. These three are among the 22 residues involved in dimeric interaction, furthermore suggesting that OCP exists as physiological dimer [START_REF] Kerfeld | Structural and EPR characterization of the soluble form of cytochrome c-550 and of the psbV2 gene product from the cyanobacterium Thermosynechococcus elongatus[END_REF] . In addition to OCP from Arthrospira maxima, the crystal structures of other OCPs have been solved, all of them featuring a crystallographic dimer. These OCPs are naturally occurring in various cyanobacterial strains, including Synechocystis, Anabaena and Tolypothrix. It has been previously reported that in Synechocystis and Arthrospira cells, OCP can bind 3'-hECN as functional pigment. Interestingly, the protein may also be functionalized by other ketocarotenoids, featuring similar length compared to 3'-hECN, the variety of which depend on the cyanobacterial species. When recombinantly expressed in E. coli cells, OCP from Synechocystis can bind three distinct pigments, e.g. ECN, canthaxanthin (CAN) or zeaxanthin (ZEA), depending on the set of carotenoid-producing genes that are co-transformed with the gene coding for the OCP. This characteristic applies for all OCP variants that are produced recombinantly in carotenoidproducing E. coli cells. To date, OCP structures from Synechocystis PCC 6803 (PDB id: 4XB5), Anabaena PCC 7120 (PDB id: 5hgr) and Tolypothrix PCC 7601 (PDB id: 6pq1;) where determined in the CAN functionalized states and those of Synechocystis PCC 6803 (PDB id: 3mg1) and Arthrospira maxima (PDB id: 5ui2) in the ECN and hECN functionalized states, respectively. (coloured in black). The hECN is shown in sticks and is coloured in wheat, binds in a ~ 35 Å long hydrophobic tunnel spanning the two domains and H-bonds to In the CTD, hECN establish two H-bond interactions with Y203 and W290 in the CTD. The interaction between the NTD and the CTD within the OCP O monomer is supported by two strictly-conserved interactions, i.e. a salt-bridge between R155 and E246, and H-bonds between N104 and W279 (shown as sticks in inset and green dashes). The two H-bonds of the carotenoid to Y203 and W290 (shown as sticks) are also shown (green dashes).

Photoactivation of the Orange Carotenoid Protein: structural insights

In order to perform its energy quenching function OCP needs to be photoactivated. Absorption of blue-green light triggers the conversion from the inactive dark-adapted orange state (OCP O ) to an active light-adapted red state (OCP R ). The OCP O state displays an orange color and is characterized by a specific absorbance signature presenting two absorbance peaks at 465 and 495 nm, whereas OCP R has a unique yet broad absorbance peak at ~ 510 nm and thus displays a red color [START_REF] Wilson | A photoactive carotenoid protein acting as light intensity sensor[END_REF] . The OCP O to OCP R photoconversion is characterized by an extremely low quantum yield (~0.3%), sustaining the assumption that in the cyanobacterial cell the OCP-related photoprotective mechanism is induced only in high light conditions. Furthermore, in vivo and in vitro studies [START_REF] Wilson | A photoactive carotenoid protein acting as light intensity sensor[END_REF] have reported on the effect of light intensities and temperature on the rate of photoconversion. High light intensities and low temperature favor the formation of OCP R . The recovery of the latter occurs spontaneously in darkness and it is also temperature-dependent.

Raman and Fourier-transform infrared spectroscopy (FTIR) studies demonstrated that absorption of an actinic photon induces structural changes not only in the ketocarotenoid pigment but also in the protein [START_REF] Wilson | A photoactive carotenoid protein acting as light intensity sensor[END_REF] . Raman spectra showed that in the OCP O state, the hECN adopts a bent, all-trans conformation [START_REF] Kerfeld | The crystal structure of a cyanobacterial water-soluble carotenoid binding protein[END_REF][START_REF] Polívka | Spectroscopic properties of the carotenoid 3'-hydroxyechinenone in the orange carotenoid protein from the cyanobacterium Arthrospira maxima[END_REF] , while upon illumination the conjugation length of the polyene chain increases and the carotenoid adopts a less distorted and more planar structure, that yet remains in an alltrans conformation [START_REF] Wilson | A photoactive carotenoid protein acting as light intensity sensor[END_REF] . Additionally, FTIR spectra showed that upon photoactivation conformational changes also occur within the protein scaffold, i.e. an increase in the flexibility of the helical structures and in the compactness of the β-sheet regions. These results furthermore suggested the possible occurrence of domain rearrangement upon formation of OCP R .

The first insights regarding the structural changes that occur within the protein upon its photoactivation were presented in two key studies. In 2012, the study of Wilson et al. reported that upon photoactivation, the salt bridge between Arg155 and Glu244 localized in the NTD and the CTD, respectively, is broken enabling domain dissociation in OCP R , which therefore would be characterized by an opened conformation. In 2015, by solving the structure of the isolated holo-NTD binding canthaxanthin as functional ketocarotenoid (PDB id: 4XB4), Leverenz et al. proposed a model whereby OCP photoactivation is accompanied by a dissociation of the two domains and a 12 Å migration of the carotenoid into the NTD domain. Indeed, the position of the carotenoid in the holo-NTD structure strongly differs from that in OCP O . The holo-NTD presents all molecular features of the OCP R, i.e. a red-shifted absorption spectrum, the ability to bind to the PBS and fluorescence quenching activity. The two-domain structural rearrangement of OCP R has also been observed in solution in mass spectrometry [START_REF] Liu | Mass spectrometry footprinting reveals the structural rearrangements of cyanobacterial orange carotenoid protein upon light activation[END_REF] and X-ray scattering experiments [START_REF] Gupta | Local and global structural drivers for the photoactivation of the orange carotenoid protein[END_REF][START_REF] Golub | Solution Structure and Conformational Flexibility in the Active State of the Orange Carotenoid Protein: Part I. Small-Angle Scattering[END_REF] . Altogether, these results indicate that following the breakage of the two H-bonds in the CTD, i.e. between the ketocarotenoid β1-ring and the Tyr201 and Trp288 occurs, respectively, the carotenoid migrate into the NTD resulting in domain dissociation after rupture of H-bonds between the NTD and the CTD. More recent studies reported on additional steps that used to occur before domain dissociation takes place, namely the dissociation of the NTE and the CTT from the CTD. Upon release, the C-terminal tail may alter its position and close the carotenoid tunnel which remains empty in OCP R 68,69,71,72 . Despite all these findings, the structure of the native OCP R state remained elusive until recently.

However, cryo-electron microscopy method allowed to obtain structural insights into the OCP R structure from quenched OCP-PBS complex from Synechocystis PCC 6803 at 2.7 Å resolution [START_REF] Dominguez-Martin | Structure of the Quenched Cyanobacterial OCP-Phycobilisome Complex[END_REF] .

The observed OCP R structure fully agrees with previous findings, i.e. it features separated domains and the carotenoid is found in the NTD. It also reveals that full quenching on the PBS requires binding of a 2 OCP R dimer.

The OCP photocycle: carotenoid and protein excited-state dynamics

Despite multiple functional and structural studies conducted with aim to characterize: (i) the photoactivation mechanism of OCP; (ii) the consequential accumulation of OCP R state; and (ii) the related energy quenching function in vivo and in vitro, the precise mechanisms of OCP photoactivation, PBS binding and fluorescence quenching remain unclear. The main challenge stems from the extremely low quantum yield of OCP, which makes it impossible to accumulate OCP R and preceding intermediates in pulsed experiments.

Over the last years, numerous spectroscopic approaches have been used to study the structural and spectral changes undergone by the protein and the carotenoid upon photoactivation. Among those, ultrafast spectroscopy studies have allowed to identify the intermediate states formed by the carotenoid. When solubilized in polar solvent (e.g. acetone), photoexcited ketocarotenoids first transition from the ground state (S0) to the optically allowed excited state (S2), from which they relax within femtoseconds to the singlet excited state (S1), characterized by a lifetime of tens of picoseconds [START_REF] Polívka | Ultrafast dynamics of carotenoid excited States-from solution to natural and artificial systems[END_REF] . The behavior of protein-bound ketocarotenoids is more complex because of a possible transition from the S2 state to an intra-charge transfer state (ICT) which displays a redshifted spectrum compared to S1 and a shorter lifetime. The relaxation kinetics of the S1 and ICT have yet been shown to vary depending on the surrounding buffer [START_REF] Polívka | Molecular factors controlling photosynthetic light harvesting by carotenoids[END_REF] .

Transient absorption spectroscopy confirmed that in OCP, the ICT and S1 states follow different relaxation kinetics, exhibiting lifetimes in the order of 0.5 and 1 to 5 ps, respectively depending on experimental conditions. In addition, a femtosecond time-resolved spectroscopy study on OCP and RCP binding different carotenoids, e.g. echinenone or canthaxanthin, has been conducted with aim to compare the magnitude of the transient signal of the ICT state [START_REF] Šlouf | Ultrafast spectroscopy tracks carotenoid configurations in the orange and red carotenoid proteins from cyanobacteria[END_REF] . Because of the twotime shorter lifetime of the ICT/S1 states compared to those of the S1 bilin lifetime, a mechanism of energy transfer was proposed in which the ECN in OCP R accepts energy from the excited bilin via ICT. The intramolecular charge transfer state can either directly decay to the S0 ground state or overtake the S1 → S0 decay [START_REF] Berera | The photophysics of the orange carotenoid protein, a light-powered molecular switch[END_REF] . Studies demonstrated that after the ps decay of the hECN, a primary red-shifted photoproduct forms at very low amplitude. The spectral properties of this photoproduct differ from those of the red final OCP R state indicating that these are not identical and that OCP R relaxation happens on a longer than time scale (ms -s).

Most recently, steady-state fluorescence spectroscopy was used to gain additional insights into the protein structural changes that may take place upon OCP photoactivation. By measuring the fluorescence signals of both Trp residues within the protein and the hydrophobic stain Nile red bound to OCP, Maksimov et al. proposed that upon photon absorption, local structural rearrangements occur in the vicinity of the carotenoid before a global change takes place within the protein matrix, which leads to an increased structural flexibility and the loss of tertiary structure. This hypothesis is in full agreement with previously published structural results from Leverenz et al. [START_REF] Leverenz | A 12 Å carotenoid translocation in a photoswitch associated with cyanobacterial photoprotection[END_REF] ,which showed that photoactivation triggers carotenoid migration followed by domain dissociation.

The most complete and comprehensive description of the sequence of events occurring along the OCP photoactivation pathway, has been proposed based on a combination of multiple transition spectroscopy techniques (UV-Vis, mid-IR, FTIR)(Figure 7) [START_REF] Konold | Photoactivation Mechanism, Timing of Protein Secondary Structure Dynamics and Carotenoid Translocation in the Orange Carotenoid Protein[END_REF] . Konold et al. have suggested that upon photon absorption, the carotenoid transits through three ps-lived intermediate states formed following the sub-ps decay of the initial S2 state: an S1, and an ICT excited states, and an additional state, coined S*. It is believed that in the S* state, the conformation of the carotenoid is distorted, effectively leading to H-bond elongation in the CTD. According to this hypothesis, S* would be the precursor of the OCP primary photoproduct (P1) which forms within tens of ps and presents an absorption maximum at 563 nm. In P1, recovery of a planar conformation would induce H-bonds breakage. Within 50 ns, about half of the molecules in the P1 state will progress towards formation of the second photoproduct P2, characterized by an absorption maximum at 556 nm and by a repositioning of the β1-ring in the CTD, at distances from Y201 and W280 that are incompatible with H-bonding. It has been suggested that full carotenoid translocation must be first preceded with reorientation of 𝛼-helices in the NTD. Such structural changes of helical elements occur on a time-scale of 0.5 -1 µs and correspond to the formation of P2' state. The third photoproduct P3 forms within 10 µs and presents a similar spectrum to OCP R and would be characterized by a complete migration of the carotenoid across the NTD and by dissociation of the NTE from the CTD. Still, in the study of Konold et al., the formation of the final signaling OCP R state could not be probed, indicating that OCP R forms on a longer time-scale (ms-s) (Figure 8). The OCP photocycle was recently complemented with the proposal that two additional intermediate states exist, named PM and PX, both forming in the tens to hundreds of ms and suggested to be associated with dissociation of the NTE and CTT [START_REF] Maksimov | Probing of carotenoid-tryptophan hydrogen bonding dynamics in the single-tryptophan photoactive Orange Carotenoid Protein[END_REF] . While in the two previously cited studies, OCP photoactivation was triggered with a pulsed photoexcitation, the OCP photocycle was also studied under continuous illumination by static and time-resolved FTIR difference spectroscopy [START_REF] Mezzetti | Two-Step Structural Changes in Orange Carotenoid Protein Photoactivation Revealed by Time-Resolved Fourier Transform Infrared Spectroscopy[END_REF] . Specifically, it was demonstrated that the photoinduced conformational changes involve two asynchronous steps on the ps-ms timescale. The first step would correspond to a reorganization of a non-solvent exposed α-helix in the NTD, and the second one to a partial reorganization of a solvent exposed α-helix, i.e. NTE and/or CTT. During my thesis, we combined time-resolved (TR) visible spectroscopy, with static and TR X-ray scattering enabled to complement the already existing knowledge on OCP photoactivation mechanism. By using pulsed illumination, we obtained evidence that a 'red'-shifted state can form and decays within ~10 μs and ~10-200 ms, respectively. Intriguingly, this state differed from the OCP R state accumulated under strong continuous illumination, which forms and decays within ~1 ms and ~1-30 min. Additionally, our study also reported on the formation of a non-photoexcitable structural state which lives for at least ~ 0.5 μs. The latter state presumably features a spectrum characteristic of the OCP O state, since it is not observed in spectroscopic pump probe (vide infra) experiments.

Insights into OCP O and OCP R oligomerization states

In cells, one third of the proteins are present as oligomers [START_REF] Ali | Protein oligomerization: how and why[END_REF] . Besides, factors such as local changes of e.g. temperature, concentration, pH etc. within the cell and the environment, and/or protein composition can affect the equilibrium between oligomeric states allowing coexistence of multiple forms of the protein. It is strongly believed that oligomerization processes are important to confer higher stability to proteins and could favor their function. This is exampled by domain swapped proteins whose functional state can only be reached upon dimerization. Oligomeric proteins can be divided in two major classes depending on whether they form homo-oligomers proteins, wherein all subunits are equivalent, or hetero-oligomers, wherein different subunits associate. An important number of studies, have been carried out over the past few decades with the aim of understanding the mode of interaction of oligomeric proteins [START_REF] Monod | ALLOSTERIC TRANSITIONS: A PLAUSIBLE MODEL[END_REF] , the nature and specificity of oligomerization interfaces [START_REF] Jones | Protein-protein interactions: a review of protein dimer structures[END_REF] , and oligomeric protein folding [START_REF] Zitzewitz | Probing the folding mechanism of a leucine zipper peptide by stopped-flow circular dichroism spectroscopy[END_REF] .

The OCP oligomerization state is also the matter of debate. All published crystal structures of the dark-adapted OCP state [START_REF] Kerfeld | The crystal structure of a cyanobacterial water-soluble carotenoid binding protein[END_REF][START_REF] Wilson | Structural Determinants Underlying Photoprotection in the Photoactive Orange Carotenoid Protein of Cyanobacteria[END_REF] revealed presence of a dimer wherein the dimerization interface is mainly supported by H-bonds and a salt bridge between structural elements belonging to the NTD.

Notably, the salt bridge involves the three most conserved residues among the 22 making up the dimerization interface, i.e. Asp19, Arg27 and Asn134. Nonetheless, because the high concentration of OCP in crystals (in the range of 20 mM) is likely never reached in the cells, many investigators envisage OCP dimerization as an artefact. Hence, in vitro techniques such as sizeexclusion chromatography (SEC), dynamic light scattering (DLS), mass spectrometry (MS) and X-ray and neutron scattering (SAXS/SANS) were used in order to investigate OCP oligomerization in both the dark inactive and the light active states. For instance, static SAXS measurements supported the hypothesis that OCP O is present as a dimer, at least at concentration between 1 -1.5 mg/mL [START_REF] Gupta | Local and global structural drivers for the photoactivation of the orange carotenoid protein[END_REF][START_REF] Golub | Solution Structure and Conformational Flexibility in the Active State of the Orange Carotenoid Protein: Part I. Small-Angle Scattering[END_REF][START_REF] Maksimov | The Unique Protein-to-Protein Carotenoid Transfer Mechanism[END_REF] . In contrast, native MS spectrometry analysis pointed to the presence of OCP O as both monomer and dimer, where the abundance of dimers increases with the increase of concentration and can form at concentrations as low as 0.01 mg/ml [START_REF] Lu | Native Mass Spectrometry Analysis of Oligomerization States of Fluorescence Recovery Protein and Orange Carotenoid Protein: Two Proteins Involved in the Cyanobacterial Photoprotection Cycle[END_REF] . The presence of OCP darkadapted dimers was furthermore observed in SEC and X-ray scattering experiments [START_REF] Golub | Solution Structure and Conformational Flexibility in the Active State of the Orange Carotenoid Protein: Part I. Small-Angle Scattering[END_REF] .

Interestingly, a recent study has reported the simple mutation of the highly conserved Arg27 to a Leu completely abolishes dimer formation, advocating for a possible role for OCP dimers [START_REF] Muzzopappa | Interdomain interactions reveal the molecular evolution of the orange carotenoid protein[END_REF] and offering the possibility to the effect of OCP monomerization in vivo.

Further adding to this complexity is whether or not OCP R can also oligomerize. Aside from OCP oligomerization in its dark-adapted state, in solution studies have also been used to investigate OCP R oligomeric state and follow monomer/dimer distribution upon photoactivation. Native mass spectrometry [START_REF] Zhang | Molecular mechanism of photoactivation and structural location of the cyanobacterial orange carotenoid protein[END_REF] as well as SEC-SAXS data [START_REF] Gupta | Local and global structural drivers for the photoactivation of the orange carotenoid protein[END_REF] revealed that upon accumulation of the OCP R state, it may undergo a monomer to dimer transition with the OCP R dimer presenting a radii of gyration that is 30% higher than that of OCP O dimers. In other words, the OCP light dimer would feature a more elongated/expanded structure than the OCP O counterpart. Recent SAXS/SANS data combined with simulation modeling performed on OCP W288A mutant [START_REF] Maksimov | The photocycle of orange carotenoid protein conceals distinct intermediates and asynchronous changes in the carotenoid and protein components[END_REF] , in which the carotenoid is not entirely branched to the CTD thus allowing to mimic the active OCP state structure, confirmed that OCP R is characterized by an elongated structure wherein both NTD and CTD domains are separated while the NTE is detached from the latter [START_REF] Golub | Solution Structure and Conformational Flexibility in the Active State of the Orange Carotenoid Protein: Part I. Small-Angle Scattering[END_REF] . The data showed that OCP R oligomerizes and form light dimers in which the dimer interface is ensured by the CTD domains. The used modeling approach for OCP R was based on previously published scattering studies of Apo-OCP 82 .

As an important part of my thesis work, the oligomerization of both OCP O and OCP R was investigated by conducting static X-ray scattering experiments on two OCP variants: a wild-type OCP and a monomeric mutant [START_REF] Muzzopappa | Interdomain interactions reveal the molecular evolution of the orange carotenoid protein[END_REF] . The collection and analysis of the X-ray scattering profiles demonstrated that in solution, the two variants can oligomerize upon photoactivation.

Furthermore, the reconstructed ab initio envelopes, clearly showed formation of OCP R dimers and OCP R tetramers. The publication referred to this study is presented in the result section.

OCP clades and paralogs

In 2017, Bao et al. [START_REF] Bao | Additional families of orange carotenoid proteins in the photoprotective system of cyanobacteria[END_REF] , conducted a thorough phylogenetic study based on all available cyanobacterial genomes. Their results pointed to the existence of more than one copy/variant of the full-length OCP encoding gene in some cyanobacterial species. For instance, while Synechocystis encodes a single copy of OCP, the cyanobacteria Tolypothrix sp. PCC 7601 encodes for two diverging sequences. This phylogenetic comparison of OCP paralogues allowed to classify them in three distinct clades (families). The first clade OCP1 include OCP variants that are constitutively expressed in the cells, form dimers in the OCP O state and can interact with FRP, resulting in accelerated recovery. This clade comprises the well-studied FRP-regulated OCP of Synechocystis, as well as those from Anabaena, Arthrospira and Tolypothrix.

The second clade, OCP2, presents different characteristics from OCP1 in terms of photoactivation, PBS quenching and oligomerization state. For example, OCPs from clade 2 are present only as monomers in the dark state and they display faster photoactivation and recovery kinetics, however their expression is not constitutive but induced in case of high light irradiance.

The third OCP clade, called OCPX was showed to rarely co-occurs with OCP1 and OCP2 clades.

Recently, two OCPXs were functionally characterized, i.e. one from the cyanobacteria Scytonema hofmanni and another from Synechocystis PCC7509 [START_REF] Muzzopappa | Interdomain interactions reveal the molecular evolution of the orange carotenoid protein[END_REF] . Native gel and size-exclusion chromatography experiments have reported that members of OCPX clade are present as dimers.

Interestingly, in OCPX the R27, involved in stabilization of the dimer, is absent. This strongly suggests that the monomer-monomer interaction within an OCPX dimer involves other residues.

Regarding OCP2 clade, the R27 is replaced by serine (S) residue that furthermore explains the presence of OCP2 as a monomer.

My thesis focused on a third member of the OCP1 clade, the OCP from the cyanobacteria Planktothrix PCC780, hereafter referred to as OCP Planktothrix. Indeed, it was shown that the OCP based PBS quenching mechanism is more efficient in Planktothrix than in Synechocystis 87 , raising questions on the structural features that could underlie this difference. Since no structural information was available on this variant, a main goal of my thesis was to obtain insights into the structure and dynamics of OCP Planktothrix using static, kinetic and time-resolved X-ray crystallography.

Besides the existence of ocp gene paralogs, many cyanobacteria present homologs encoding OCP domains. For instance, the homologues of the OCP N-terminal domain, called helical carotenoid proteins (HCP) are all 𝛼-helical, monomeric proteins, which presents the same absorption properties as OCP-NTD, thus signing for a similar carotenoid positioning. To date, three different structures of HCPs were solved [START_REF] Melnicki | Structure, Diversity, and Evolution of a New Family of Soluble Carotenoid-Binding Proteins in Cyanobacteria[END_REF][START_REF] Dominguez-Martin | Structural and spectroscopic characterization of HCP2[END_REF] , all featuring a structure similar to the NTD of OCP. The two main functions of HCP are believed to be energy and singlet oxygen quenching, which they are capable of performing regardless of photoactivation.

The paralogs of the C-terminal domain of OCP are the CTD-like proteins and are referred to as CTDH [START_REF] Muzzopappa | Paralogs of the C-Terminal Domain of the Cyanobacterial Orange Carotenoid Protein Are Carotenoid Donors to Helical Carotenoid Proteins[END_REF] . CTDHs are homodimer proteins in which a carotenoid molecule is shared between the two monomers. It was shown that in the apo-state CTDH can form two types of dimers: (i) a "headto-head dimer (F-type) where the dimerization interface is stabilized by an S-S bond (Cys103), thus resulting in the formation of a carotenoid tunnel in which could lodge the ketocarotenoid pigment, and (ii) a "back-to-back" dimer (A-type) in which the dimerization interface is stabilized by β-sheets between two monomers 91 (Figure 8B). Recent studies demonstrated that the F-type is the main dimer formed in biological context, whereas the A-type dimers form mostly in solution.

Structural comparison of CTDH and CTD of OCP O showed that the two types mainly differ in the position of the CTT. In the CTDH, the CTT adopts a close position (Figure 8C), and partially covers the carotenoid tunnel. However, in CTD of OCP O , the CTT adopts more open conformation, as it interacts with the CTD β-sheet. The principal role of CTDHs is to ensure the carotenoid uptake from the membranes and to efficiently deliver them to the HCPs, the latter presenting the inability to perform carotenoid uptake. Thus, CTDH encoding genes are present in all HCP-containing strains.

In the present thesis work, we used the apo-Anabaena CTDH crystal structure, as well as the three available crystal structures of HCPs, as templates for building the dimerization interfaces for OCP R dimers and tetramers observed in solution. (PDB id: 6FEJ 90 ). Two dimer interfaces are be observed, i.e. a "back-to back "dimer, also called A-type, and a "head to head" dimer or F-type. In the A-type, dimerization occurs through the CTD interface buried between domains in the OCP O monomer. This interface was proposed to underlie dimerization of the isolated OCP-CTD. In the F-type dimer, the residues involved in the formation of the carotenoid tunnel in OCP-CTD, face one another at the dimerization interface. This interface was proposed to yield the functional CTDH dimer involved in carotenoid transport. (C) Structural alignment of CTDH monomer (coloured in wheat) and Synechocystis OCP-CTD (coloured in pale cyan) (PDB id: 3MG1 [START_REF] Poddar | A guide to time-resolved structural analysis of light-activated proteins[END_REF] ). Both 3. Structural methods used for the study of OCP structure and dynamics: X-ray scattering and X-ray crystallography Despite the notable progress in characterizing and understanding the photoactivation mechanisms of OCP, important aspect of the protein light-induced structural changes remains to be addressed. Especially, knowledge is missing on how the energy absorbed by the carotenoid is funneled into the protein scaffold and how the protein structure varies during photoactivation. This can only be understood by conducting a global structural study of OCP. Hence, in order to attain better knowledge of OCP photoactivation mechanism from a structural point of view, two different methods of structural biology were used during my PhD, i.e. X-ray solution scattering and X-ray crystallography, in their static and time-resolved flavors.

In particular, static and time-resolved solution X-ray scattering enabled to investigate the role played by oligomerization in the photoactivation mechanism of OCP, as well as to determine the time-scale on which large scale structural changes occur in the protein. By using conventional crystallography, we obtained structural insights into the previously uncharacterized OCP from Planktothrix agardhii. Time-resolved crystallography allowed to characterize the first intermediate leading to the OCP R photoactive state, as well as to elucidate the structures of the excited states, formed ps after absorption of the actinic photon.

In this section, the basic principles of these structural approaches will be described, as well as the requirements that they imply.

X-ray scattering from proteins in solution

X-ray scattering is an analytical method used to obtain low-resolution structural information on biomolecules, e.g. proteins, in solution [START_REF] Doniach | Changes in biomolecular conformation seen by small angle X-ray scattering[END_REF][START_REF] Koch | Small-angle scattering: a view on the properties, structures and structural changes of biological macromolecules in solution[END_REF] . Back in the 1950s, data collection of X-ray scattering profiles on protein solution was possible but impractical because of the extended data acquisition time (ranging from many hours to even days) imposed by the X-ray producing sources 95 . Owing to the development of powerful synchrotron radiation sources, it has become possible to collect X-ray scattering data in solution with reduced exposure time, down to few milliseconds, and the method emerged as a possible mean to obtain insights into protein structures. Scattering may be referred to as small-angle X-ray scattering (SAXS), when information of the low-scattering angle (q) region is analyzed, or to wide-angle X-ray scattering (WAXS) when information on higher-q region is obtained. The larger the scattering angle, the higher the resolution of the collected structural information. While SAXS gives an information on molecular weight (MW), size, oligomerization state and low-resolution shape of the macromolecule, WAXS informs about their internal structure, i.e. distribution and relative orientation of structural elements such as α-helices and β-sheets. Since the X-ray scattering method can only provide low space-resolution on biomolecular structures, it is generally used as a complementary approach to other structural techniques, such as X-ray crystallography, Nuclear Magnetic Resonance (NMR) and Electron Microscopy (EM).

In this PhD, the rationale for using small and wide-angle scattering (SAXS/WAXS) was in the ability of this approach to provide information on the large-scale conformational changes underlying OCP photoactivation. Specifically, static SAXS studies provided an important information on the role played by oligomerization in the formation of both OCP O and OCP R states, whereas time-resolved SAXS/WAXS experiments allowed to determine the time-scale on which large structural changes occur within protein upon its photoactivation.

Basic principle of X-ray scattering

The scattering of X-rays results from the interaction of incident photons with the electrons within a sample, e.g. macromolecules, nanocomposite, etc. According to the energy difference between incident and scattered photons, X-ray scattering can be divided into elastic (energy difference = 0) or inelastic (energy difference ≠ 0). In the present work we only refer to elastic scattering, as it is this type of scattering that enables extraction of structural information.

In X-ray scattering experiments (Figure 9), the particles within the sample are illuminated by a monochromatic (single wavelength) and collimated X-ray beam, and the scattered photons are recorded on a detector, e.g. 2D pixel or CCD (charge-coupled device). The obtained scattering pattern is averaged radially to obtain the scattered intensity (I) as a function of the scattering vector (q). The scattering vector can be deduced from the scattering angle 2θ between the incident and the scattered beam, and the incident beam wavelength (λ) using equation ( 1): q = '( ) × sinθ (Equation 1)

Because particles are surrounded by solvent, which is often in great excess, most of the scattering signal originates from the solvent. Hence, the scattering signals from the particle solution and from the solvent are recorded separately, and the latter is subtracted from the former in order to remove the contributions from the solvent and the sample environment to the scattering profile. The shell of solvent surrounding the particle does not feature the same electron density as the bulk solvent, and contributes to the scattering profile of the particle [START_REF] Grishaev | Sample preparation, data collection, and preliminary data analysis in biomolecular solution X-ray scattering[END_REF] . It is worth stressing that the composition of the buffer surrounding the protein has to be identical to that used as a blank, to avoid any distortion of the protein scattering signal, which would subsequently result in errors in the reconstruction of its molecular envelop. For instance, the presence of detergents or buffer components presenting a high scattering factor must be accounted for as exactly as possibly achievable. Other parameters, such as pH and salt concentration, also need to be carefully adjusted since they can affect electrostatic repulsion between the particles, thus resulting in interparticle interference [START_REF] Doniach | Changes in biomolecular conformation seen by small angle X-ray scattering[END_REF][START_REF] Doniach | Small and wide angle X-ray scattering from biological macromolecules and their complexes in solution[END_REF] . Additionally, great care must be given to ensure monodispersity of the sample, i.e. presence of a single state of the protein in solution, otherwise reconstruction of the molecular envelope will again be compromised. The buffer may affect the oligomerization state of a protein or the equilibrium between folded and unfolded states.

Extraction of structural parameters from SAXS data

The obtained experimental scattering profile provides information on various sample parameters, including MW, radius of gyration (Rg), maximum dimension (Dmax) and excluded particle volume.

While for monodisperse systems, these parameters directly inform on the characteristics of the particle, for polydisperse systems the experimentally determined values refer to the average

! k i k s

Incident beam S ca tte re d be am 2θ q X-rays Sample characteristics of an entire ensemble [START_REF] Kikhney | A practical guide to small angle X-ray scattering (SAXS) of flexible and intrinsically disordered proteins[END_REF] . The SAXS scattering profile obtained from a monodisperse solution, is best visualized as log I(q) vs. (q) or log I(q) vs. log(q), and it presents three distinct regions providing information about the particle size, shape and surface. These regions refer to Guinier, Fourier and Porod, respectively.

From the Guinier region, one can extract the parameter Rg that provides direct information about the size of the particle. In the Guinier approximation, valid for q x Rg < 1.3, the scattering intensity is expressed using Equation (2).

ln(I (q)) = ln(I(0)) - D E ×F G E H (Equation 2)
As shown in Equation 2, the Guinier approximation stipulates that the scattering intensity plotted as ln I(q) vs. q 2 should be a linear function for a particle of any shape, and from its slope Rg can be estimated. Additionally, the interception of the slope with the ordinate provides the scattering intensity at zero angle, i.e. I(0). Further normalization of the latter with the sample concentration allows to properly determine the MW of the particle. Because the scattering intensity at zero angle must be proportional to the concentration of the solute, the proportionality constant is determined experimentally from collecting data on a sample of already known MW. Several standard samples can be used for this purpose, such as lysozyme or bovine serum albumin (BSA).

The Guinier region (Figure 10A) is highly sensitive to the presence of large particles in solution.

For this reason, any particle aggregation would results in an increase of the scattering intensity at low q and wrong estimation of the Rg and I(0) values. In the Fourier region, the pair distribution function P(r) can be determined by applying indirect Fourier transformation of the experimental form factor (Equation ( 3)). 3)

I(q) = 4π ∫ P(r) NOP(DQ) DQ dr STUV W (Equation
The P(r) measures the distribution of pairwise distances within a particle with maximum dimension (Dmax), i.e. the longest interatomic distance in the molecule (Figure 10B). The P(r) function informs on the overall particle shape, e.g. globular, rod-like or flat, and enables estimation of the Dmax. P(r) also allows to estimate Rg and I (0) parameters.

The Porod region provides information about the surface to volume ratio for compact particles. In addition, the Porod plot offers the possibility to gain insights into the MW of the particles.

The most commonly used program software to process experimental SAXS data is ATSAS [START_REF] Manalastas-Cantos | ATSAS 3.0: expanded functionality and new tools for smallangle scattering data analysis[END_REF] . The latter is best suited for analysis of SAXS data of biological macromolecules. Automatic data processing, including averaging, buffer subtraction, curve fitting and Rg estimation from Guinier and Porod plots, can be performed using PRIMUS software 100 , whereas calculation of the P(r) function is carried out using GNOM 101 . Additionally, the ATSAS package also proposes a program called OLIGOMER 100 , that enables estimation of the percentage of volume fractions in case of polydisperse systems.

Ab initio shape determination from SAXS data

Ab initio shape reconstruction from the scattering data offers the possibility to obtain a 3D lowresolution model generally by using a bead-modeling approach. The principle of the bead modeling method is to use densely packed beads (called dummy atoms) that fit the search particle volume according to the P(r) distribution. The upper limit of the particle size is defined by Dmax from the scattering profile (Figure 11A). An important requirement is that the sphere radius of the bead must be inferior to Dmax. The reconstructed bead model (Figure 11B) is then validated by comparison with the experimental scattering profile (Figure 11C). Two ab initio modelling programs are commonly used: DAMMIF 102 where the search volume is unlimited, and DAMMIN 103 , which restricts the search in a confined volume. A recently published algorithm not using a bead modeling approach, called DENSS 104 has allowed ab initio reconstruction of the 3D electron density of a particle directly from the 1D solution scattering profile.

Wide-angle X-ray Scattering (WAXS) of proteins

While small-angle X-ray scattering is a low-resolution technique providing information on the overall particle shape but unable to track smaller conformational changes, wide-angle X-ray scattering (WAXS) potentially enables monitoring of secondary, tertiary and quaternary changes within proteins. While the low-resolution limit of structural information provided by SAXS generally does not exceed 10 Å -1 (q ~0.3 Å -1 ), WAXS data, collected at larger scattering angles up to q ~2.5

Å -1 , enable to monitor more detailed structural features in the protein, such as internal motions related to movement or unfolding of helices and sheets. Scattering signals in the wide-angle region are significantly weaker (by a factor ~ 100) than those measured in the SAXS region, requiring to use higher concentration of the protein of interest to collect useful data. As in standard solution scattering experiments, solvent contribution needs to be subtracted to obtain the WAXS signal arising from the protein. In general, WAXS measurements include data at 2 Å -1 where the presence of the water scattering peak informs on sample/buffer mismatches but can also obscures the extent of structural information derived from the experimental data at high q values.

Time-resolved X-ray solution scattering (TR-XSS)

In living organisms, the 3-dimensional (3D) structure and function of proteins is governed by the sequence of its constituent amino acids. In order to perform its function, the protein undergoes structural changes in order to adopt specific functional conformation 106 . The functional conformation passes through formation of structural intermediate(s) that generally occur on different time-and length-scales, e.g. from fs to min and from sub-Å to tens of Å, respectively. TR-X-ray scattering (TR-XSS) remains a powerful technique that allows to monitor global conformational changes occurring within proteins in solution as a function of time. In this regard, TR-XSS can be considered as complementary technique to other methods, such as TRcrystallography, TR-spectroscopy and nuclear magnetic resonance (NMR), as it gives the possibility to gain insights into protein dynamics and formation of structural intermediate states occurring over a broad range of time-scales 107 . The strength of this method resides on its ability to obtain such information in solution, i.e. freed from crystal packing interaction. This can be particularly useful in cases where no crystals are available of the protein of interest or if largescale conformational changes are involved that would not be compatible with preservation of crystal packing interactions. Nevertheless, a major drawback of TR-XSS remains its low resolution which prevent obtaining structural information on local conformational changes within proteins.

Time-resolved studies of protein dynamics are generally performed using the "pump-probe" data collection scheme. As shown in Figure 12, the method employs the use of ( 1) an optical laser pulse (pump), that will initiate structural transitions in the system, ( 2) and an X-ray pulse (probe) that will detect the structural evolution of the system after a certain time-delay (Δt). The timeresolution of a pump-probe experiment depends on the convolution of the laser and X-ray pulse lengths 108 . In order to achieve high time-resolution, the use of short (fs-ns) laser pulses is required.

The laser pulse can directly photo-excite the system, (for example in OCP, photoexcitation will initiate photoactivation of the protein), but also may trigger a photoreaction in photochemical compounds (so-called caged compounds) or generate a temperature-jump which will then propagate into the structure. In light triggered reactions, a compromise between an optimal photoactivation, i.e. the need for efficient laser penetration through the sample, and a good signal to noise (S/N) ratio, i.e. the use of high sample concentration, has to be found. This is a kinetic analysis which enable to extract the minimum number of components that portray the time-evolution of the signals. Despite the significant advantage of TR-XSS to probe conformational changes in solution as a function of time, its main weakness remains that the low spatial resolution of the structural information does not allows building a 3D structure at the atomic level. Combination of TR-XSS data with computational approaches, e.g. molecular dynamics simulations, can facilitate the structural interpretation of the data. However, X-ray crystallography remains the method of choice for obtaining high-resolution structural information on biomolecules and thus allow their use as a starting point for solution scattering studies.

X-ray crystallography

Macromolecular X-ray crystallography (MX): protein crystallization

Macromolecular X-ray crystallography (MX) is the most commonly used technique to determine the atomic-resolution of 3D structure of macromolecules (proteins and nucleic acids) and assemblies thereof (viruses and ribosomes) in structural biology. These structures offer a base to understand how the macromolecule carries out its function. The success of X-ray crystallography lives in the advent of third generation X-ray sources, as well as development of new tools, methodology and automation approaches over the last fifty years. These have enabled MX to allow determination of more than 120 000 structures among a total of 194 311 deposited in the Protein Data Bank (PDB -November 2021). Most of the structures have been determined at cryotemperature (100K) with view to mitigate the progression of radiation damage during data collection.

Principle of protein crystallization

The crystallization of biological macromolecules, such as proteins, is a phase transition phenomenon, by which molecules pass from a disordered liquid state to an ordered solid state.

This phase transition pass by the formation of partially ordered structural intermediates called nuclei which consist in relatively small non-specific aggregates 110 . In this regard, crystallization can be considered as two-step process comprising nucleation and crystal growth. Both steps are dependent of supersaturation which is a variable that drives both processes and is compulsory their occurrence. Supersaturation refer to a state in which some of the molecules are still present in solution, i.e. in excess of the solubility limit. The formation of a solid state (crystal) is reached once the saturation limit is attained. In order to generate a supersaturated solution, one must modify two parameters: (i) the ability of the medium to solubilize the macromolecule needs to be reduced; (ii) the property of the macromolecules must be changed so that their solubility is reduced, thus leading to an increase of the attraction between them. The crystallization process is illustrated in Figure 13 by means of a crystallization phase diagram in which four distinct areas can be distinguished. The first area refers to undersaturation, where neither crystal nucleation nor growth occurs because the protein is completely dissolved. The second area is that of low supersaturation (below nucleation) where crystals will grow but no nucleation will occur. This area is also referred to as metastable zone. The third area corresponds to that within which spontaneous nucleation occurs but growth is less favored. The fourth area (precipitation zone)

refers to an area of high supersaturation characterized with protein precipitation.

Crystallization methods

Various methods have been developed to grow protein crystals in vitro. These include batch crystallization, dialysis, vapour-diffusion, and free-interface or counter-diffusion 112 . All of them will be briefly introduced in this section.

Batch crystallization is in appearance the easiest method for obtaining protein crystals as it consists of mixing a macromolecular solution with a crystallizing agent (precipitant reagent). In this method, one generally needs sample volumes ranging between few µL up to tens of mL. In batch crystallization, the nucleation zone is excessive because crystallization starts directly in the supersaturated region on the phase diagram. As a consequence, formation of small crystals is favored, however the increase of crystal size can be achieved by reducing the supersaturation so as to be as close as possible to the metastable region of the phase diagram. Because temperature variations generally affect crystal formation, batch crystallization can also be conducted using temperature gradients which enable to define an optimal range of temperatures to grow high quality crystals. Screening of conditions and further optimization of the batch crystallization method are possible and can be performed using an automatized microbatch. The main advantage of this approach resides in the possibility to investigate various crystallization conditions with small sample quantity, less than 1-2 µl of sample per condition.

The second method used for protein crystallization is dialysis. The general principle of this method consists in the separation of protein samples from a large volume of solvent by the use of semipermeable membrane, the latter allowing the passage of small molecules while preventing that of the protein of interest. The equilibration kinetics depend on various parameters that must be carefully considered, including the MW exclusion size of the semi-permeable membrane, the concentration ratio of precipitants inside and outside the protein sample chamber, the temperature, and the geometry of the dialysis cell. In this crystallization method, dialysis bags are often employed, however their use often requires important protein volumes per assay (~ 20-50 mL). For smaller volumes (< 10 mL), crystallization by dialysis requires the use of microdialysis cells which are made of capillary tubes closed by dialysis membranes.

The third and most widely used method is the crystallization by vapour-diffusion (Figure 14). A major advantage of this method is the possibility to work with small protein volumes, i.e. 1 mL or less. In this method the protein to be crystallized is presented in a drop together with its buffer and crystallizing agents and equilibrates against a reservoir containing a solution of precipitant agents at a higher concentration than that in the drop. Hence, the equilibration process occurs by water vapour exchange. In case of high initial concentration of the precipitant in the reservoir, the water exchange will occur from the drop to the reservoir and will lead to volume decrease of the drop.

The concentration of all constituents in the drop will increase and the protein will reach its oversaturation zone where the nucleation is thermodynamically favorable. When a stable nucleation zone is reached, crystal growth can start. When a final equilibrium is reached (drop vs. reservoir) the crystal growth ceases. In the case where the initial concentration of the precipitants in the reservoir is lower than that in the drop, the exchange will be reversed, i.e. from the reservoir to the drop. The most commonly used method to obtain protein crystals by vapour-diffusion is the "hanging drop" method, illustrated in Figure 14A. In this method, the drop (volumes range between 100 nl and 10 µl) is placed on a glass cover slip which is inverted over the reservoir. The hanging drop crystallization is often performed using Linbro plates of usually 24 wells, wherein each well is covered by a coverslip tightly closed with vacuum grease. Another way to achieve crystallization by vapour-diffusion is the "sitting drop" method (Figure 14B). In this case, each well features a central post which is elevated above the reservoir. The protein drops (up to ~40 µl) are deposited in the center of the post and reservoir is filled with reagent (up to ~1200 µl).

The fourth crystallization technique allowing to obtain protein crystals is the free-interface or counter-diffusion methods. The principle of these consists in the diffusion of the crystallizing agent into the protein solution. To perform such experiments, one needs to prepare an agarose or silica gel the surface of which is covered by concentrated solution of reagent. The latter diffuse in the gel and forms a gradient. The protein sample is then inserted into the gel by the use of capillaries in which the crystallization agent can enter, creating ascending gradient that can subsequently lead to crystal formation. This crystallization method has been used for crystal formation of various macromolecules with varying sizes. Its major advantage remains the crystalline growth at low supersaturation at the end of the capillary.

Figure 14. Principle of vapor diffusion method. (A) Hanging drop technique in which the drop (protein + reagent) is placed in a siliconized cover slide used to close the well and it is inverted over the reservoir (B) Sitting drop

technique where the drop is placed on a platform inside the well. For both geometries, equilibration of water vapor from the drop to the well solution enables reaching the supersaturation level of the protein solution, leading to crystal growth (orange square inside the drop).

Seeding

The reproduction of previously established crystallization conditions can be of a major problem. This is generally due to the difficulty to obtain stable nuclei of protein crystals, or to the formation of spontaneous nucleation at high supersaturation zone characterized with poor crystal growth.

To surpass this problem, crystal growth can be induced at low levels of supersaturation but this requires to provide the crystal nuclei. This approach is referred to as seeding. The principle of seeding consists in using fragments of previously obtained crystals of the protein of interest 113 which are subsequently mixed with a supersaturated protein solution. Two seeding techniques can be used 114 . While the first one consists in using microcrystals for seeds, the second one use large macroseeds. Both methods require fresh supersaturated protein solution, so that the crystal growth can be accomplished in a controlled and slow fashion.

A main disadvantage of the first approach is the introduction of many nuclei into the fresh solution, may result in the formation of non-homogenous masses of crystals unsuited for diffraction analysis. To avoid such issue, a commonly used approach is the preparation of series of diluted microseeds solutions which are each tested so as to identify the best condition for forming nice crystals. Each diluted sample is then added to the fresh protein sample in the crystallization trial, thus allowing to identify the optimal seeding conditions that yield only one or a small number of single crystals. Solutions containing too low concentration of seeds will not yield crystals. A good optimum is ≃ 1 µseed/µL.

The second approach implies the same requirements as the first one, i.e. the elimination of the excess of microcrystals and the avoidance of transfer of too many seeds. Often microcrystals adhere on the surface of large crystals. In order to avoid this, the macroseed solution can be extensively washed in transfer buffer solutions. The advantage of this washing steps resides in the freshening of the crystal surfaces, thus promoting an optimal growth of the supersaturated protein solution.

Cryoprotection of protein crystal

Within a protein crystal, the proteins present an ordered arrangement and interact with each other through non-covalent interactions 115 (e.g. Van de Waals, electrostatic, hydrophobic interactions, and hydrogen bonding). In order to mitigate radiation damage caused by the high intensities of Xrays, diffraction data are generally collected at cryogenic temperatures, i.e. 100 K corresponding to -173 °C. Because one third to a one half of the protein crystal mass is occupied by water, care must be taken to avoid ice formation, which would result in destruction of the crystal and loss of its diffraction power. Direct flash-cooling of the crystals in a nitrogen stream (i.e. 100 K) may be sufficient to avoid ice formation, given the limited volume of the crystal and of the surrounding solvent, and presence of precipitants in the latter. However, a cryoprotectant is generally needed, which is introduced in the crystal first before the flash-cooling.

Various types of cryoprotectants exist, such as sugars (e.g. sucrose, glycerol), polyol sugars (e.g. polyethylene glycol (PEG)) and alcohols (e.g. methanol, isopropanol). While PEGs with a low MW (e.g. PEG 200, PEG 400) can penetrate in the crystal lattice and cause crystal dehydration, higher molecular weight PEGs (e.g. PEG 3350, PEG 4000) only coat the crystal lattice. In practice, to be cryoprotected, a protein crystal is immersed in the cryosolution, containing the cryoprotectant, for a very short time (~ 10-20 s). Since some cryoprotectants can dissolve the protein crystals, the immersion time need to be performed in a controlled fashion.

Macromolecular X-ray crystallography (MX): diffraction and data processing

Diffraction of X-rays by protein crystal: Principle

In order to solve a protein structure, a single crystal of adequate size is subjected to an X-ray beam which interacts with the electrons inside the crystal, thus producing diffracted X-rays recorded on detector. To determine a 3D structure, data must be collected on a various orientation of the crystal. Within a crystal the atoms are arranged in a regular and repeated pattern (lattice)

where the smallest repeating element is the unit cell. The unit cell mirrors the symmetry and the structure of the crystal. The crystal is thus built up by repetitive translation, i.e. without change in orientation and rotation, of the unit cell in the three-dimensions of the space. The unit cells are characterized by three axes, i.e. a, b, c and three angles α, β and γ. The smallest fraction of a unit cell is the asymmetric unit (arrangement of macromolecules) from which the unit cell is generated through symmetry operations defined by the space group.

In an X-ray diffraction experiment, the X-rays scattered in phase by different lattice planes interfere constructively according to the Bragg's law expressed in Equation ( 4): nλ = 2d sinθ (Equation 4), where λ is the X-ray incident wavelength, n is an integer (order of reflection or diffraction), d is the lattice plane spacing and θ is the angle between incident and diffracted X-ray. Thus, the Bragg's law relates the distance between two parallel planes in a crystal and the angle of reflection to the X-ray wavelength. The diffracted X-rays are characterized with a pattern composed of diffraction spots, also called reflections, each corresponding to a set of parallel lattice planes. The crystal is usually rotated with respect to different axes, so that diffraction patterns from different parallel lattice planes can be recorded in sequence, on different diffraction patterns. A specific set of parallel lattice planes is characterized in the reciprocal space (generated by Fourier transform of the crystal lattice) by the Miller indices, (h, k, l) assigned to a given reflection.

The first steps in the analysis of diffraction data, necessary to go from the diffracted signal to the structure factor (from which the electron density is calculated, as will be detailed below), are indexing, integration and merging. The indexing operation consists in assigning a set of Miller indices (h, k, l) to each reflection and deriving from these unit cell parameters and space group.

The subsequent integration procedure consists in determining the intensity of each reflection, i.e.

summing the measured intensity on each pixel of the detector to which it contributes. The intensities of all the reflections deriving from the same lattice planes are then averaged together (merging step) to give the final set of unique reflection intensities. All these steps are usually iterated until important experimental parameters (e.g. the beam center, the sample-detector distance) have been refined and an optimized geometry/methodology has been obtained. In conventional (per say, oscillation based) crystallography, indexing, integration and merging can be performed using programs such as XDS, MOSFLM or HKL-2000 [116][117][118] . These programs feature several routines each dedicated to the different steps mentioned above. The outcome of XDS processing is a file that contain all unique reflections and can be further used to solve or refine the macromolecular structure by means of software suites such as CCP4 and Phenix 119,120 .

Diffraction of X-rays by protein crystal: Structure solution and refinement

The Fourier transform is a mathematical operation that enables to describe the relationship between an integrable function in the real space (a signal) and another function in the reciprocal space (a spectrum). In imaging it is used to link the information encoded in the form of a distance, in a physical object, to that encoded in the form of a scattering angle, in the scattering space. Specific to crystallography the Fourier transform can be used to link the electron density of a molecule, in the real space, to the diffraction pattern produced by the sampling of this crystal under various orientations, with different families of planes slicing the crystal, in the real space, each giving rise to a reflection in the reciprocal space. The Fourier transform implies a dilatation/contraction relationship that explains that the sampling of small distances in crystal gives rise to large scattering angle in the reciprocal (scattering) space. The families of planes are denoted "hkl" depending on the number of times they can "slice" unit cell parameters a, b and c, respectively. In the reciprocal space, the hkl values are the Miller indices of each reflection. While on the diffraction pattern, we measure the intensity of a reflection, the corresponding structure factor can in first approximation be envisaged as proportional to the square root of this intensity.

In order to calculate the electron density of the macromolecule in the crystal, a Fourier transform of the so-called structure factor F(hkl) (defined by its amplitude and phase) has to be performed, using Equation ( 5):

ρ (x, y, z) = _ ` ∑ |F cde | cde • e 4(Oh ijk • e [m4(O(cVndonep)]
(Equation 5), where (αhkl) is the phase angle of the reflection (hkl), |Fhkl| is the structure factor amplitude, (x, y,z) are the atomic coordinates in the unit cell, and V is the volume of the unit cell. While the structure factor amplitude is directly related to the measured intensities of each reflection, the information on the phase in F(hkl) is loss and has to be retrieved independently by either direct determination (using phasing compounds) or iteratively using the structure factors of a search model, i.e. model protein which presents sequence similarity to the target protein. In this context, there are three main methods that allows to overcome the loss of the phase information. These methods are: (i) single-or multi-anomalous dispersion, SAD and MAD, respectively; (ii) multiple isomorphous replacement (MIR); and (iii) molecular replacement (MR). In my thesis, the molecular replacement and single-anomalous dispersion methods were used in order to solve the structure of OCP from Planktothrix agardhii and the mosquitocidal Cry11Aa toxin, respectively. To date, MR remains the most commonly used method to enable phasing of the crystallographic information and overcome the so-called phase problem. This method provides initial estimates of the phases of the new structure using previously solved structure (search model). However, MR requires that both structures are similar. The similarity is evaluated by alignment and comparison of the primary sequences, which generally needs to be higher than 30% for phasing by MR to succeed. In this method, the potentially similar search model is used to locate the protein of interest inside the unit cell of the protein of interest. This is computationally performed in two steps by the calculation of Patterson maps which correspond to the self-convolution of the observed diffraction data. The first step relies on a rotational search, enabling to find the orientation of the molecule in the unit cell. During the second step a translation search, throughout the unit cell, allows to find the position of the previously rotated model. The feasibility of MR relies on the fact that the orientation of a molecule in a cell strongly affect the phases but not the structure factor amplitude and thus the intensities. Hence, the orientation of the molecule can be easily retrieved by just comparing observed and calculated Patterson maps. Once a correct orientation is found, translation will strongly affect the phases, but at this stage these can be calculated from the model at different translated position in the crystals without affecting much the structure factor amplitudes. Hence, by dividing the problem in two steps, phasing can be achieved rapidly by MR 121 . The principle of single anomalous dispersion method (SAD) relies on the anomalous difference in diffraction of X-rays absorbed by the inner electrons of heavy atoms in the crystal. Following absorption, X-rays are reemitted inducing a phase shift in all of the reflections, which can be detected by comparing datasets. The detected differences allow locating the heavy atoms and the atom positions serve as initial approximation of the phases. The nature of the heavy atoms can be either intrinsic to the protein (e.g. metalloproteins), or the atom can be introduced by the use of soaking compounds.

Diffraction of X-rays by protein crystal: Structure validation

Finding the correct initial phases allows to calculate an initial electron density map. The electron density map, also called 2Fo-Fc, is generated from the observed (Fo) and calculated (Fc) structure factors. The electron density map is nowadays usually displayed using the program called Coot 122 . The model building can be performed manually or by the use of automated software.

Generally, model building involves skeletonizing the polypeptide chain along the peaks of the electron density and conversion of this skeleton in a polypeptide chain that complies with the stereochemistry of molecules. At this stage, a knowledge of the primary protein sequence is essential as it allows to identify and build the protein structure in the electron density. After this step, the model can be refined, i.e. adjustment of the atomic positions, displacement parameters (B factors), and occupancies. Through the progression of model building and refinement various validation criteria should be considered. These criteria are important as they prevent adding to the model more features than can be refined on the basis of the diffraction data, i.e. "over-fitting". The progression of the refinement is monitored by a Rwork factor. This factor compares the Fc from the model, with Fo, measured in the diffraction experiment. To avoid over-fitting, usually a small fraction of the reflections (between 5%-10%) are removed before generating the electron density maps and are furthermore used to calculate the Rfree factor. Through the refinement process the difference between the Rfree and Rwork should remain in the range of 3-6%.

Radiation damage

A major drawback in X-ray crystallography is the radiation damage caused by prolonged exposure of a single crystal to the X-rays 123 . In conventional (per say, oscillation crystallography) this damage is cumulated during rotation of the crystal to acquire complete 3D data.

Global radiation damage is manifested in a reduction of the quality of the diffraction data, but specific damage may also occur that will lead to to irreversible structural changes which could compromise the biological interpretation of the structure (e.g. breaking of chemical bonds). It has been reported that radiation damage is related to two components, i.e. a dose-dependent and time-dependent component. The dose-dependent component, also termed primary radiation damage, is associated with the deposited X-ray dose on the crystal, whereby X-ray photons cause atom ionization. This primary absorption event therefore induces formation of secondary electrons which are characterized with low energy and become thermalized. The progression of the heat inside the crystal triggers chemical reactions (e.g. formation of radicals) between the radiationinduced moieties and the crystal components. As between 20 and 70 % of a protein crystal volume is occupied by the solvent, the energy deposited in water molecules, present in and around the crystal, triggers formation of radicals (e.g. hydroxyl and hydrated electrons), which spread and furthermore interact with the protein. The events of heat progression and radical formation, arising from the primary radiation damage process, are related to a time-dependent component and are referred to as secondary radiation damage processes. Additionally, radiation damage can be either direct, i.e. primary absorption of an atom, or indirect in cases where the radiation is absorbed by the solvent and furthermore propagated in the crystal. Both direct and indirect radiation damage cause global damage which is manifested with a diminution of the crystalline order and therefore the resolution of the data.

The most effective way to reduce radiation damage is to collect data on crystals at cryogenic temperature as it slows down the heat progression and the diffusion of free radicals. However, even at cryo-temperatures primary radiation damage still occurs and cannot be completely avoided as incident photons directly cause the appearance of ionization events. A possible strategy, proposed in the past for highly radiosensitive proteins, is to collect data using partial rotation on several crystals instead of a complete rotation on a single crystal, thus reducing the absorbed dose on each crystal. Alternatively, a single large crystal can be used that is impinged at different position for the collection of partial datasets overlapping in a rotational series 124 .

Serial crystallography (SX)

The "serial" approach for data collection, originally introduced to minimize radiation damage during X-ray crystallography experiments on highly sensitive virus crystals, was revised and improved with the advent of X-ray free electron lasers (XFELs). When the extension of free electron lasers to the hard X-ray regime became realistic, computer simulation studies on the possible applications in structural biology were performed 125 and predicted that the ultra-short (femtoseconds) and bright XFEL pulses could be used to obtain damage-free structural information from single molecules or crystals, at room-temperature provided that the X-ray pulse was shorter than 50 fs in length. The concept was termed as "diffraction-before-destruction". Indeed, due to the high brilliance of the X-ray pulses, the sample is eventually destroyed, and thus for a complete dataset to be recorded, a continuous supply of fresh crystals is needed. This approach using the fs X-ray pulses produced by the XFEL sources has been coined "serial femtosecond crystallography (SFX)" 126 .

The serial nature of SFX experiments, in which single diffraction pattern is collected from an individual crystal, required the development of new data processing tools to recover the integrated X-ray intensities from the recorded partial reflections. This new data analysis software paved the way for the implementation of serial crystallography at synchrotrons, under the acronym SSX for serial synchrotron crystallography. However, a major challenge for both SSX and SFX approaches remains the efficiency of sample delivery, thus various methods have been developed. These will be described in the following section.

Sample delivery methods for serial crystallography experiments

Sample delivery methods for serial crystallography experiments utilize either injection or fixedtarget techniques.

The first-established and most commonly used injection method is the gas-dynamic virtual nozzle (GDVN) 127 . A GDVN is composed of two concentric capillaries wherein the crystal slurry continuously flows in the inner capillary while a sheath gas (e.g. helium or nitrogen) is flowed through the outer one. The pressure of the flowing gas enables to micro-focus the liquid stream as well as it governs his speed 128,129 . The use of sheath gas also prevents ice formation at the nozzle tip in case where data collection is performed in vacuum. GDVN jets present a typical size of 1-5 µm, and a minimum flow rate of 10 µL/min is required to ensure their stability 130 . A major bottleneck of GDVN method, however remains the poor efficiency of protein sample use. While performing a serial crystallography experiment using GDVN injection at an XFEL facility, for instance the Linac Coherent Light Source (LCLS) where the data acquisition rate is 120 Hz, i.e. 120 pulses/s, an estimation shows that the crystal hit rates are between 0.01% and 0.1% 131 .

To overcome this issue and furthermore increase the efficiency of data collection, a possibility is to decrease the sample flow-rate. This can be attained by use of electrical current in place of the sheath gas, although at the price of stability and need for cryoprotectant in the crystal slurry. Such electrokinetic injectors enable to increase the sample utilization to around 5%, due to the relatively low flow rates, e.g. 0.1-3 µL/min associated with the use of a ≃ 100 nm jet 132 . However, settling of microcrystals is a risk, that again can be overcome by the use of viscous solvent such as PEG and glycerol (also acting as cryoprotectants).

As GDVN, the electrokinetic injection method also consists of delivering the slurry of crystals in a continuous way hence both methods can be classified as inefficient in sample utilization. A delivery method called on-demand droplet delivery has emerged as alternative strategy, which enables an increase of the crystals hit rate up to 90%. This approach uses acoustic droplet injectors, wherein the slurry of crystals is loaded on a microplate and crystals are ejected as droplets with a volume of 0,002 to 0,01 µL. The ejection of the droplets is under the control of radiation pressure associated to focused ultrasound energy 133 . However, a main disadvantage of this method remains the large diameter of the sample droplets that subsequently increase the background signal and make the method most adapted to experiment conducted on large crystals.

The easiest way to improve the sample efficiency is to increase the viscosity of the stream, thus slowing down the overall flowrate. A variety of high viscosity materials such as lipidic cubic phase (LCP), agarose, and hyaluronic acid have been used as carrier media. The choice of a particular matrice can be challenging as it strongly depends on the protein system and crystallization conditions. When using the high-viscosity extrusion (HVE) method, the crystal slurry is indeed mixed with the high viscosity component. It is worth stressing that a transfer of crystals in the viscous medium can alter crystalline order. In addition, the diameter of the extruded material (crystals + viscosity component) can impact the level of background scattering and induce a significant loss in signal to noise ratio. To minimize the background problem, an alternative viscous media must be used, e.g. cellulose-based media 134 .

Additionally to injection techniques, solid supports 135 (called fixed-targets) have also been used to present samples to the X-ray beam. Fixed-targets are generally used during serial crystallography experiments at synchrotrons. They are unsuited for experiments at XFELs, as the generated X-ray pulses can destroy the material of the mounts and compromise sample utilization.

Sample requirements for serial crystallography experiments

As mentioned above, during SFX experiments, the peak brilliance of the XFEL beam destroys the sample upon its interaction, thus a main requirement is the delivery of a steady stream of a myriad of randomly oriented crystals. The large volumes of microcrystalline sample required for the serial crystallography approach of data collection involves different crystallization strategies, as compared to those used for the growth of large crystals 137 , the batch crystallization method being generally used to achieve this. The principle of this method consists in rapidly mixing the protein solution with the reservoir solution, which will trigger the formation of a supersaturated protein solution, nucleating instantly. A drawback of batch crystallization methods is the inability to control the crystal size. Indeed, the size of the microcrystals obtained by this method usually vary between 1 µm to 300 µm 138 .

Microcrystals for serial crystallography experiments can be also obtained by free interface diffusion method (FID) in which a concentration gradient allows to obtain small (5-20 µm) well-ordered crystals. Two additional methods are also applied, i.e. lipidic cubic phase (LCP) and in vivo crystallization. LCP is a biscontinious mesophase that acts in mimicking biological membranes, and it is commonly used for the crystallization of membrane proteins, thus enabling to use detergents. In vivo crystallization is a powerful method as it offers the possibility to obtain nano-to micrometer-sized crystals in physiological conditions. The main advantage of the in vivo methods relies on the possibility to control the crystal size, which is imposed by the cell's dimensions.

Time-resolved crystallography (TRX) at synchrotrons and XFELs

Time-resolved crystallography enables to gain insights into the relationships between protein structure, dynamics and function. Protein crystals generally exhibit important solvent content and present large solvent channels, offering to proteins the possibility to move and perform their function. Additionally, these channels enable the diffusion of a substrate to an active site in case of enzymatic reactions. By performing X-ray crystallography in a time-resolved fashion, one can understand the mechanism of biological processes.

Similar to TR-XSS, in time-resolved crystallography the initiation of a dynamic reaction inside crystals can be achieved by the use of several methods, such as excitation by laser pulse, mix and inject, by the use of caged substrates, temperature jumps and electric field. Light-activation and mix and inject are the two most commonly used methods since they can be easily applied.

When studying light activated processes, the initiation of the reaction is done by laser pulse (pump) at t = 0. The induced reaction is probed by an X-ray pulse (probe) at a defined and variable timedelay (Δt) after the optical pump. This approach is called pump-probe. In order to obtain an entire structural data set, probing conformational changes occurring in a protein along a reaction pathway, the pump-probe procedure needs to be repeated for the various delays of interest.

Figure 18: Illustration of the experimental setup for a pump-probe time-resolved crystallography experiment. Firstly, crystallographic data are collected on protein crystals at t=0 and in absence of laser pump. The collected data set corresponds to the resting-state ("dark") structure (showed in orange), the latter serving as a reference model. Within crystals, the reaction is induced by a laser pulse (pump, in blue) and probed by the X-ray probe (in grey) after a defined time-delay (Δt). For instance, data are collected for Δt1, Δt2 and Δt3, allowing to collect structures of different structure intermediates, viz. structures in pink, red and dark red. These are then used as frames and allows to build a molecular movie.

TR-crystallography at synchrotron sources

Historically, time-resolved structural studies have been initially performed on large single crystals using Laue crystallography at synchrotrons. Laue diffraction uses a polychromatic X-ray beam which enables to increase the incident photon flux by a factor of ~100 compared to a monochromatic beam 139 . Indeed, the number of photons contained in a single 100 ps monochromatic pulse is not sufficient for obtaining a good S/N ratio of the diffraction pattern.

Furthermore, the use of polychromatic beam enables collection of more structural information (Bragg peaks) per frame, given that the Bragg law is verified for multiple wavelengths at the same time. The Laue technique, first implemented at synchrotrons, has enabled to follow the structural dynamics of proteins on atomic scale with up to 100 ps time-resolution. However, this approach presents several limitations among which: (i) limited temporal resolution imposed by the X-ray pulse length; (ii) inability to study irreversible reactions, as the induction of the reaction would trigger long-term changes in the crystal and data collection would require multiple large crystals for each time point ; (iii) only studies on photoactivated reactions are possible since activation of chemical reactions necessitates the diffusion of a substrate in the crystal, thus limiting the reaction on longer time-scales; (iv) the study of light-induced structural changes is generally limited by the crystal size. Indeed, in TR-crystallography the crystal size is the main parameter to be considered as it governs the homogeneity of the triggered reaction. Absorption by optically dense protein crystals, e.g. OCP, limits light penetration at outer layers in large crystals (more than ten microns in size) 140 . In small crystals, i.e. micro-to nanometer-sized, the pump pulse penetration is more efficient. In OCP, for instance, the penetration depth at 475 nm, i.e. the distance at which the half of the photons have been absorbed is ~ 3-4 μm, thus structural dynamics studies of OCP using TR-crystallography necessarily require the use of micrometer-sized crystals.

TR-serial femtosecond crystallography (TR-SFX) at XFELs

The advent of XFELs and of the SFX data collection approach enabled to overcome the limitations imposed by Laue crystallography and furthermore offered the possibility of studying non-reversible reactions. The femtosecond pulse duration improves drastically the temporal resolution, and the high-intensity of the pulses reduces the requirements on crystal size in a way that submicron crystals can be used. Time-resolved SFX (TR-SFX) has allowed to trap structural intermediates occurring over the reaction and to provide snapshots of these transient intermediates. These snapshots are used to build a "molecular movie" which unravel how biological molecules exert their function.

While performing a pump-probe TR-SFX experiment on photosensitive proteins, microcrystals are excited by the laser and probed by X-ray pulses. When fast reactions and short-lived intermediates are studied, the duration of both pump and probe needs to be shorter than the lifetime of the transient intermediate. In order to achieve a femtosecond time resolution, the reaction needs to be initiated by femtosecond pump pulse, whereby the time resolution depends on the duration of the pump and the probe pulses. Data collection on ultrafast time scales necessitates that the time delay for each X-ray pulse is determined by using a timing tool that allows to measure the temporal distance between the laser and the X-ray pulses. An important requirement for an optimal initiation of the light-induced reaction is the applied laser power on the crystals. The power energy must be carefully adjusted since too high levels of the latter can provoke severe damage of the crystal or elicit dynamics that are not representative of those at play in the physiological context. Conversely, too low power energies lead to suboptimal reaction initiation. Hence, the optimal laser power to be used in a pump-probe experiment has to be assessed by spectroscopic methods, and the diffraction experiment is then performed at an excitation power lying in the linear regime.

In vivo protein crystallization in Bacillus thuringiensis

In structural biology, the use of in vivo-grown protein crystals has been for a long time overlooked as these are limited in size (nm to µm), imposed by the outer's cellular dimensions. In vivo-grown crystals are indeed unsuited for conventional X-ray crystallography experiments as they present low diffraction capacities and high sensitivity to radiation damage. Serial crystallography, has allowed to overcome this size limitation, since the collection of complete data sets from myriads of microcrystals is possible using both XFELs and synchrotrons facilities, using the serial data collection approach.

In 2013, a study has reported that in vivo crystallization can be applied for the growth of nonnatively crystalline proteins. Since then, the in vivo crystallization of recombinant proteins has been attempted and observed in insect and bacterial cells. Even if during in vivo crystal growth several difficulties can be encountered, e.g. cell deformation, accumulation of non-homogenous population of microcrystals, increase in crystal solubility and fragility 141,142 , this approach remains less time-consuming compared to in vitro recombinant crystallization, as it avoid the need to extract, purify and crystallize proteins in nonphysiologically conditions. Indeed, formation of native protein crystals in physiological conditions offer the possibility to gain insights into structurefunction relationship occurring directly within living cells.

The spore-forming bacterium, Bacillus thuringiensis (Bt), is a natural in vivo crystal producer of nano-to micrometer-sized (0.5 µm-1µm) toxin crystals characterized with long-term stability in aqueous environment. Hence, Bt appears as perfect candidate and can be used for the development of a recombinant in vivo crystallization method which would enable the production of large volumes of microcrystalline slurry for static and time-resolved SX experiments at both XFELs and synchrotrons. The development of this method is one of the research lines of our laboratory. One of the main goals of my thesis project was the development of an in vivo recombinant crystallization strategy aiming to obtain calibrated micro-to nanometer-sized crystals of OCP in Bt. As previously mentioned, smaller crystals are better suited than large ones for TR crystallography because of the reduced light penetration in macrocrystals of optically dense proteins. They are also well suited for rapid mixing experiments given that their small size is compatible with rapid diffusion of solutes.

In the present section, I first present an overview of Bt, i.e. the mechanism that the bacterium uses to produce in vivo-grown crystal of its native crystal toxins, as well some structural and functional insights into these. I furthermore present the strategy used for in vivo recombinant crystallization of OCP and thereafter that pursued for the crystallization of non-naturally crystalline proteins.

Bacillus thuringiensis: general Introduction

Discovered for the first time in 1901 in Japan, Bacillus thuringiensis (Bt) was described as the principal cause of a disease affecting silkworms. To date, a large number of Bt strains have been identified and isolated from various habitats worldwide, including soils, fresh water, leaves and insects, in which the bacteria multiply. At the beginning of the past century, it was debated and further investigated whether or not Bt, initially presented as a potential risk to the environment, could prove beneficial for microbial insect control. In the early 40s, the first Bt based pesticide was commercialized, which specifically targeted coleopterous and lepidopterous larvae. A few years later it was discovered that the insecticidal activity of the bacterium originates from crystalline inclusions which are made up of toxins, specifically targeting insect larvae.

B. thuringiensis is a ubiquitous, spore-forming Gram-positive bacterium that presents high similarity, in genotype and phenotype, with Bacillus cereus, and for this is classified as B. cereus group in taxonomy. To date, 82 Bt subspecies were identified, the classification of which has been accomplished based on H serotyping, i.e. the immunological reaction related to the flagellar antigens (H-antigen) 143 . The flagellin encoding hag gene is responsible for eliciting the immunological reaction in H serotyping, and furthermore allows to identify specific amino acid sequences of flagellin that are correlated to specific Bt serotypes. Hence, myriads of Bt strains that present activity against various orders of insects and nematodes have been isolated. One among these is the Bacillus thuringiensis subsp. israelensis (Bti) that was first discovered in a pond in Israel, and has been shown to present a specific insecticidal activity against mosquito and blackfly larvae 144 . The formation of parasporal crystals is of the pivotal aspects of Bt development. These crystals comprise two diverse groups of protein families, namely the Cry and Cyt toxins, also called δendotoxins. Both Cry (crystal) and Cyt (cytolytic) proteins occur as parasporal inclusions in Bacillus thuringiensis and are active against a wide range of insect species from different orders, e.g. Lepidoptera, Diptera, Coleoptera, etc., as well as other organisms like mites and nematodes.

Bt

Upon ingestion by insect larvae, the Bt crystal is solubilized in the midgut, leading to the release of one or more protoxins (Figure 19A). These solubilized protoxins are activated through proteolytic cleavage by midgut proteases, which enables them to bind to their receptors (Cry) or lipids (Cyt) in the cell membrane. The binding and insertion of toxins in the membrane triggers the formation of pores that consequently leads to cell death 145 . The Bt crystals, formed by a combination of δ-endotoxins, present different forms (bipyramidal, spherical, etc.) and sizes (smaller, equal to, or greater than the size of the spore). Briefly, crystals are ingested by an insect larva, and dissolve in the insect gut upon the increase of pH. Upon dissolution, the crystals release protoxins (inactive form of the toxin). Protoxins are activated through proteolytic cleavage by midgut digestive enzymes, which consists in scission of the toxin propeptide located either in the N-and/or C-terminal part of the protein. Then the activated toxin binds to protein receptors (Cry) or lipids at the surface of the gut cell membranes or directly interact with lipids (Cyt). The binding and insertion of toxins in the membrane leads to gut cell disruption and subsequently to cell death. Figure from Tetreau G., Andreeva E.A. et al., 2021 146 . (B) Schematic representation of the nomenclature system used for Bt toxins, (adapted from Palma L. et al., 2014 147 ) To date, several hundred of individual Bt δ-endotoxin sequences have been identified, including 74 classes of Cry and 3 classes of Cyt toxins. Because of the growing number of identified toxins, a nomenclature system for Bt toxins has been developed. The ranking code (Figure 19B) has been first adopted for Cry proteins and furthermore applied to Cyt proteins. According to the nomenclature, the first rank is the toxin class (e.g. Cry), followed by a first arabic number that is different for proteins sharing less than 45% of amino acid identity, i.e., Cry1 and Cry2. The secondary rank consists in adding an uppercase letter which is different for proteins having less than 78% identity, e.g. Cry1A and Cry1B. The tertiary and quaternary ranks involve adding a lowercase letter for less than 95% of identity (e.g. Cry1Ab and Cry1Ba) and a further arabic number that distinguish proteins with identity more than 95% (e.g. Cry1Ab7, Cry1Ab8), respectively 148 . The same ranking nomenclature applies for the two groups of Bt non-crystalline vegetative insecticidal proteins (Vip) 149 and the secreted insecticid1Aal protein (Sip) 150 . Vip and Sip display insecticidal activity against coleopteran and lepidopteran pests.

Control of insecticidal genes expression in Bt during sporulation.

The genes coding for Bt proteins are located in native mega plasmids 151 . The size of these plasmids varies from one strain to another. The production of one or a cocktail of δ-endotoxin(s) implies a single plasmid. The high-level of Cry and Cyt synthesis are tightly controlled by a variety of mechanisms occurring at transcriptional, post-transcriptional and post-translational levels 152 .

Bt sporulation process is controlled by transcriptional regulatory factors, called sigma factors (σ) the activation of which triggers their binding to the RNA polymerase and sporulation-specific promoters. Five sporulation-specific transcription factors, i.e. σ H , σ F , σ E , σ G and σ K operate in fixed order and regulate different stages. For instance, the activity of σ H factor occurs in the predivisional cell during the transition from the vegetative growth to the sporulation phase. The activity of σE and σK occurs in the mother cell during sporulation, and that of σF and σG in the forespore.

The expression of most cry and cyt genes (encoding for Cry and Cyt proteins) is regulated by σE and σK factors. For instance, expression of Cry1Aa toxin encoding gene (cry1Aa) is regulated by two overlapping promoters, i.e. the downstream BtI and the upstream BtII 153 , which respond to σE and σK, respectively. However, few exceptions exist. For example, the cry3Aa gene, encoding for Cry3Aa toxin, is expressed during the vegetative growth phase as its promoter is recognized by the sigma factor of vegetative cells (σA). While σ factors regulate the transcription of most Bt toxin genes, additional mechanisms exist that have been identified for specific Bt strains and/or toxin genes 146 .

The initiation of gene transcription in Bt depend on the presence of ribosome binding sites (RBS) that are situated upstream of the 'start' codon of the target gene and downstream of the promoter.

The length and the sequence of the translation initiation region (TIR), encompassing the RBS region, modulates gene transcription levels and subsequently affect secondary structures formation. In addition to RBS, two additional mechanisms exist that tailor mRNA stability at its 5′ and 3′ ends. In prokaryotes, mRNA indeed undergoes catalytic degradation from both ends under the activation of diverse ribonucleases (RNase). To prevent transcripts cleavage at the 3′-end, mRNAs are normally stabilized by repeated inverted sequences, called stem-loops, forming secondary structures that block the mRNA degradation by 3′-exonucleases. At the 5′-end, stabilization is modulated by Shine Dalgarno (SD)-like (GAAAGGAGG) sequence, positioned between -125 and -117 in the 5′ untranslated transcribed region (5' UTR).

While all together these mechanisms allow the increase of the transcripts half-life and toxin synthesis from a single mRNA, the formation of stable parasporal crystals, comprised of Cry and/or Cyt proteins, relies also on posttranslational regulatory mechanisms, which preserve crystalline toxins from proteolytic degradation. Various studies have reported the presence of additional proteins acting as folding chaperones in the stabilization of the nascent molecules and further crystallization 154 . Examples of such are the molecular chaperones P19 (19 kDa) and P20 (20 kDa), that are involved in Cry2A and Cry11 toxins stability and crystal formation, respectively.

In vitro studies have also reported that P19 and P20 could play a crucial role in the crystallization and cytolytic activity of these toxins. Interestingly, a specific organization and clustering of cry genes in operons has been demonstrated in various Bt strains and some of them, e.g. cry1Ac, cry2Aa, cry2Ac and cry11Aa are constituted of operons.

Bt crystal toxins: structure and mode of action

As mentioned above, Bt Cry and Cyt accumulate as parasporal crystalline inclusions during the stationary phase of growth of Bt. According to their structure, Cry proteins can be sub-divided into two independent groups: (i) the group of the three domain Cry toxins (3d-Cry) 155 , and (ii) the group of the non-three domain toxins comprising the family of Etx_Mtx2 proteins, Toxins_10 and the alpha helical toxins 145,156 .

In the present section, the structures and mode of action of both Cry and Cyt families of toxins will be briefly described. Thereafter, the rationale for using these as chaperones for recombinant in vivo crystallization will be explained.

Structural insights into the 3d-Cry toxins.

The toxins of this group are divided into two groups: (i) Cry toxins with a protoxin form of ~120-140 kDa and (ii) Cry toxins with a protoxin form of 65-70 kDa. The latter differ from the former in the absence of a large ≃ 60 kDa domain at the C-terminus (Figure 20). Even if members of the 3d-Cry toxin family display differences in their amino acid sequence identity, they share similar fold featuring conserved three-domain architecture. Domain I of these toxins, also called the perforating domain, corresponds to the N-terminal region of the toxin, and is composed of a bundle of seven α-helices (α1-α7). A central amphipathic α helix (α5), that is structurally conserved among all described 3d-Cry toxins, stands at the center of the bundle (Figure 20A). Previously published studies 157 have reported that domain I is involved in toxin oligomerization, membrane insertion and formation of ion channels in the cell membrane of the host organism. It was also hypothesized that the orientation of hydrophobic motifs (α4-α5) within this domain could play a role in Cry toxicity. Nevertheless, the lack of in vivo evidences does not allow to support this hypothesis.

Domain II (central domain) is involved in toxin-cell membrane receptors interactions. This domain consists of three antiparallel β-sheets connected by exposed loop regions. The beta sheets are packed around a central hydrophobic core forming a so-called beta-prism structure. It was demonstrated that in several toxins the exposed loop regions could facilitate interaction with some insect gut proteins.

Lastly, Domain III is composed of two twisted antiparallel β-sheets and presents a "jelly-roll" architecture (Figure 20A) 155,158 . Site-directed mutagenesis studies have reported that Domains II and III are both involved in cell membrane receptor binding and can modulate insecticidal activity

Structural insights into the non 3d-Cry toxins.

Multiple other Cry proteins that do not present the three-domain structure are classified in the non-3d Cry family. These are the Etx_Mtx2 superfamily of proteins, Toxin_10 proteins and alpha helical toxins 162 . The overall structure presented by these non-3d Cry family consist of a mixed 𝛼helical/β-sheet head region, and a tail region that features long β-sheet strands.

The main difference between toxins of Etx_Mtx2 family and those from the Toxin_10 family resides on the secondary structure composition of their head domains. While in Toxin_10 the head domain contains beta trefoil motifs formed exclusively from the N-terminal end of the proteins, in Etx_Mtx2 this motif is lacking. It has been suggested that the presence of beta trefoil domain is essential as it ensures interactions between the toxin and glycoproteins and lipids composing cell membranes.

The two Cry toxins that most closely resembles the Etx_Mtx2 family are Cry45 and Cry46 produced by B. thuringiensis. Up to date, there is no available information regarding the activity of these two toxins activity against invertebrates. On the contrary, studies have reported that they present activity against mammalian cancer cells 163 . There is more available information on Toxin_10 family members. The majority of these proteins act with a specific partner protein that leads to the formation of binary toxins. Examples of these are the BinA and BinB 164 , the aerolysinlike Cry34 and Cry35 165 166 , and the Cry48 and Tpp49Aa1 (Cry49) 167 . Some Cry toxins of the Etx/Mtx2 superfamily also require a binary partner to fully perform their toxicity. For instance, Cry23Aa need the binary partnership of Cry37 to enforce its activity against coleopteran insects.

Mode of action of the 3d-Cry toxins

The mode of action of the three-domain Cry toxins has always been and it is still of great interest to the scientific community. The past few years, multiple studies have allowed to gain insights into the precise mechanism of Cry toxicity, as well as the different steps involving receptor binding and membrane insertion.

The mode of action of Cry toxins involves the following steps. First, the Cry inclusions are ingested by the larvae, and subsequently dissolved in the alkaline mid-gut lumen upon pH elevation (varying between 9.5 and 12). Second, the solubilized protoxin (native protein) undergoes proteolytic digestion by midgut proteases, such as serine proteases, trypsin or chymotrypsin, thus leading to activation of the toxin. The conversion of protoxins into active toxins allows the generation of smaller protease-resistant polypeptides. For instance, a trypsin digestion of the ~70-kDa protoxin of Cry11Aa generates two active Cry11Aa fragments of ~32 and ~36 kDa. A third step involves binding of the active toxin on specific membrane receptors in the epithelial midgut of the insect 168,169 . After binding to the receptor, the activated toxin undergoes conformational changes which allow its insertion into the membrane. This last step involves toxin oligomerization and culminates with the formation of a non-selective ion-channel transmembrane pore occurs.

The formation of the pore subsequently leads to cell lysis and insect death.

Functional studies performed on the Bt Cry1A have allowed to identify three distinct receptors involved in its toxicity in lepidopterans. These receptors include: (i) the cadherin-like protein receptor (CADR), (ii) the glycosyl phosphatidyl-inositol (GPI)-anchored aminopeptidase N (APN) and (iii) the GPI-anchored alkaline phosphatase (ALP). In lepidopterans, the binding interaction of activated Cry1A toxins to ALP and APN receptors is of a low affinity, e.g. Kd = 100 nM and 267 nM, respectively. Because of the high abundance of GPI-anchored APN and ALP on the membrane, interaction with these two receptors concentrate the active toxin on the mid-gut cell membrane, thus leading to its high-affinity interaction with CAD receptors, with dissociation constant (Kd) of 1nM 170,171 To interact with APN and ALP receptors, Cry1A toxin use epitopes that encompass a loop of domain II and β-strand of domain III, respectively. The toxin interaction with CADR takes place through loops and 𝛼-helix of domain II, the latter promoting a proteolytic cleavage of the first helix on domain I. In solution studies aiming to identify a possible interaction between Cry1Ab oligomers and CAD receptors, have reported that the latter advocates for Cry toxin oligomerization. Shortly thereafter, it was demonstrated that Cry1Ab oligomers present 200-fold increased binding affinity to ALP and APN receptors with Kd in the order of 0.6 nM. In line with this result, a mechanism was proposed for the mode of action of Cry toxins at the molecular level in which ALP and APN bind Cry toxins and trigger their insertion into the membrane, followed by pore formation and cell lysis.

An additional mechanism of Cry mode of action was proposed in which Cry interaction with CADR will initiate a cascade of events related to the stimulation of G protein intracellular pathway. As a consequence, the insect death would be triggered by protein kinase A, without involvement of receptor binding and toxin pore formation. Cyt1, Cyt2 and Cyt3, that share high level of sequence identity. Among members of Cyt1 family, the Cyt1Aa toxin from Bti has been the most widely studied it was shown that it displays activity against larvae of various dipteran species 172 .

Structural insights into Cyt toxins

Proteins of the Cyt2 family have also been identified and characterized in several Bt serovars, e.g. the Cyt2Aa Bt ser. kyushensis, Cyt2Ba from Bti and Cyt2Bb from Bt ser. Jegathesan 173 . The three-dimensional structures of activated Cyt1Aa (PDB id: 3RON) 174 and Cyt2Ba (PDB id: 2RCI) 175 monomeric toxins have been solved by X-ray crystallography from in vitro grown crystals. Their structures showed that these are single domain proteins composed of a central β-sheet, the latter surrounded by two α-helical layers. The β-sheet comprises six antiparallel β-strands (β1, β4, β5, β6, β7 and β8), whereas the α-helix layer is composed of four helices (α1, α2, α3, α5). In the structure of Cyt1Aa an insertion of β-hairpin (β2-β3) between α1 and α2 has been observed that is common to all members of Cyt1 family. However, a β-hairpin insertion is absent in the Cyt2

Modes of action of Cyt toxins

Similar to Cry toxins, the mode of action of Cyt proteins is a multi-stage process. Cyt proteins are synthesized in Bt as protoxins. Following ingestion by the mosquito larvae, Cyt crystals dissolve under the effect of alkaline environment of the mid-gut. Thus, crystal dissolution is followed by the release of inactive protoxins that are activated through proteolytic cleavage of gut proteases. The active toxins are characterized by a MW of 25 kDa 176 . While Cry toxins require interaction with membrane-bound receptors, Cyt toxins do not rely on this principle, as they directly interact with non-saturated membrane lipids, for e.g. phosphatidylcholine, phosphatidylethanolamine and sphingomyelin. Each activated Cyt toxin eventually self-assembles into cytolytic oligomers that perforate gut cells, leading to mosquito larvae death. Most research endeavors have reported that activated Cyt toxins can also serve as a receptor for other Cry toxins from various Bt serovars including Cry11Aa which is co-expressed in Bti. To date, two contrasting models have been proposed regarding insertion of Cyt toxins into midgut cells of Dipterans 177,178 .

The first model, also called the pore formation model, proposes that Cyt monomers assemble into cation-selective channels in the membrane, which will subsequently lead to osmotic lysis of the cell. The pore forming model postulates that the four α-helices that are present in Cyt toxins are not long enough to span the membrane bilayer, whereas the strands β5, β6 and β7 present a sufficient length to insert into the membrane. Oligomerization follows, leading to the formation of a pore, after membrane insertion, provoking colloid osmotic lysis and cell death 179 . It has been proposed based on density gradient centrifugation, that the formed oligomers present a molecular weight of 400 kDa (i.e. 16 monomers) and their weight remains constant even at high toxin concentrations.

In the second proposed model, the so-called detergent effect model 180 , it was proposed activated Cyt toxins do not fix lipids, but instead aggregate on the membrane leading to defects in the lipid packing and bursting of the cell 177 . In other words, membrane-adsorbed Cyt toxins, would disorganize lipids by favoring formation of protein-lipid complexes, hence the reference to a detergent-like mode of action.

It has been proposed that both the pore-forming and detergent models could coexist, whereby each one can operate on different time scales and at various toxin concentrations. At low toxin concentrations, formation of oligomeric pores would be promoted, whereas high toxin concentrations could induce weakening of the membrane, as the latter becomes unable to accommodate the increasing number of associated molecules and breaks up 177 .

An important part of my thesis work was devoted to the elucidation of the mode of action of Cyt1Aa toxin from Bti. Our study allowed to propose a model that united all available information on Cyt1Aa pore formation. We have identified the formation a membrane bound Cyt1Aa conformers, called membrane-bound aggregates (MBA) that form upon toxin-membrane contact. We have shown that upon contact between adjacent MBA pore formation is triggered but the pore avec very large and eventually to membrane breakage. This study is presented in the Result part (Chapter 3.1).

Synergy mechanism of Bt toxins

In various Bt strains different toxins present synergistic activity. This synergy mechanism has been studied in greater details in Bti, whose highly efficient mosquitocidal activity is conferred by four crystalline toxins, i.e. Cyt1Aa, Cry11Aa and the co-crystallizing Cry4Aa and Cry4Ba. For instance, studies have reported that different combinations between the three Cry toxins, e.g. Cry4Aa + Cry4Ba, Cry11Aa + Cry4Aa, and Cry11Aa + Cry4Aa + Cry4Ba, increase toxicity for a large number of mosquito species from the genera Aedes, Anopheles and Culex 181 182,183 . While Cyt1Aa presents less toxicity, it interacts synergistically with Cry11Aa and Cry4Ba and plays an important role in delaying the resistance of some mosquitoes to Cry proteins. It was proposed that Cyt1Aa can exert the function of a receptor for Cry4Ba and Cry11A, following its insertion in the membrane 184 . However, studies have reported that Cyt oligomerization and membrane insertion is not essential for its synergistic activity. Another Cyt toxin, Cyt2Aa2 was proposed to act as a membrane receptor that specifically binds Cry4Ba. Cyt proteins (Cyt1Aa, Cyt1Ab and Cyt2Ba) have also be found capable of synergism with the Tpp1Aa and Tpp2Aa binary toxins of L. sphaericus, thus increasing their toxicity against A. aegypti. Similarly, some Cry toxins have been found to synergize the activity of other proteins from various Bt serovars. Insights into the synergism mechanism between toxins of various Bt serovars could provide new clues to understand resistance of insects to Cry toxins as well as open new avenues for developing new mosquito control strategies.

In vivo recombinant expression of OCP in Bt.

The role of a crystallization chaperone

Chaperone-assisted crystallization is a successfully used 185,186 approach in protein crystallography which enhances the formation of well-ordered crystals of target proteins (or cargo).

The principle of this method consists in using an auxiliary protein (chaperone) that binds either covalently or non-covalently to the cargo protein, thereby triggering its crystallization in two ways, i.e. first, by trapping a single conformation of the target so that the latter can pack more regularly within the crystal, and secondly by offering an additional surface for interaction. The chaperoneassisted crystallization approach requires: (i) the identification of crystal packing interfaces in both chaperone and target proteins; (ii) the design of new scaffolds which will potentially leading to new crystal functionalities; and (iii) the further characterization and adjustment of the cargo protein properties to those of the crystallization chaperone. It has been reported, that ~ 80% of the crystallized in vitro proteins, were expressed as fusion constructs, and 30 % of these were crystallized through fusion with a macromolecular chaperone.

Due to the variety of produced toxins and their stable crystalline framework, Bacillus thuringiensis can be used as crystallization platform and its toxins as crystallization chaperones. A fusion between Bt toxin and a non-natively crystalline cargo protein could facilitate the production of functional crystalline formulations directly in vivo.

Previously published studies have reported the successful use of Cry proteins as crystallization chaperones for the recombinant expression of other toxins and enzymes. These studies focused on the recombinant expression of Cry1Ac toxin fused either with the galactose-binding domain of the nontoxic ricin B chain (BtRB), or with garlic agglutinin. In these two studies, in vivo recombinant expression has been used to enhance insecticidal activity of Cry1Ac toxin and the formation of a functional recombinant crystal has not been visualized. The proof concept arrived in 2015, when Nair et al. 187 , performed a study in which the Bt toxin Cry3Aa toxin has been expressed in fusion with three non-naturally crystalline proteins: GFP, mCherry and luciferase. GFP and mCherry are fluorescent proteins, while luciferase is an enzyme that produces light upon substrate oxidation.

The formation of functional proteins has been manifested by the detection of fluorescence and luminescence in the Bt cells. Moreover, the presence of crystals was also observed. Additional studies demonstrated Cry3Aa is able to crystallize in both in vitro 155 and in vivo 188 with similar crystal packing interfaces, and that crystallization is driven by the toxin itself.

Driven by these previous successes, the in vivo recombinant crystallization method established in our laboratory, focused on the study of two other Bt toxins, the Cry11Aa and Cyt1Aa both naturally produced in Bti. These two are of an important interest not only because of their toxicity against various mosquitoes and their synergistic properties, but also because till recently the structures derived from their native protoxin form were missing. By producing these in vivo and solving their structure using SFX, we identified protein domains involved in crystal contacts.

Strategy of in vivo recombinant expression in Bt

In my thesis project, OCP Planktothrix was central since information regarding its structure and function was missing. With aim to perform TR-SFX experiments, large volumes of calibrated, functionalized holo-OCP nanocrystals were needed, hence we endeavored their production in vivo by recombinant expression in Bt.

Previous studies have demonstrated that OCP is unable to form crystals in vitro in absence of its ketocarotenoid pigment. Additional studies have reported, however, that a paralog the CTD domain of OCP (CTDH) forms crystals in vitro and in absence of a ketocarotenoid pigment [START_REF] Muzzopappa | Paralogs of the C-Terminal Domain of the Cyanobacterial Orange Carotenoid Protein Are Carotenoid Donors to Helical Carotenoid Proteins[END_REF] . For this reason, CTDH was also of interest in our in vivo crystallization project, as obtaining crystals of it, but not of OCP, could have pointed to suboptimal carotenoid expression in Bt.

The expression strategy used for the recombinant crystallization of OCP and CTDH was built in three different steps. First, we aimed to test the ability of the two crystallization chaperones, i.e.

Cry11Aa and Cyt1Aa to yield functional nanocrystals in Bti. Second, we seeked to explore if crystal formation of the two OCP variants is possible when their encoding genes were expressed in absence of crystallization chaperones. Third, we designed fusions in which (i) either the Nterminal protoxin domain of Cyt1Aa was fused to the OCP encoding gene, or (ii) the full-length sequences of Cry11Aa and Cyt1Aa were fused to OCP, respectively. In all three cases, we used shuttle vectors designed in our laboratory, the so-called pBT vectors, the construction of which relied initially on the use of the modified expression vectors, i.e. pWF45 and pWF53, designed and kindly provided by Prof. Brian Federici. To ensure optimal expression of OCP and CTDH, the constructed pBT vectors contained: (i) Bt sporulation-dependent promoters; (ii) the p20 gene from the ORF3 of the Bti cry11Aa operon, which encodes for the P20 accessory protein known to ensure Cyt1Aa crystal formation during sporulation; (iii) the STAB-SD (GAAAGGAGG) sequence located in the 5'-UTR of the gene and/or a terminator sequence in the 3' region of the gene which the stability of the transcripts; and (v) the gene-sequence(s) encoding for OCPs and/or the crystallization chaperones (Figure 23). An 6X His-tag or a Strep-tag were added at the N-termini of CTDH and OCP Planktothrix to allow assessment of protein expression by performing Western blot. It was also needed to engineer carotenoid production in Bt cells. As previously published, the reconstruction of a carotenoid biosynthesis pathway in E. coli cells requires the expression of 5 distinct genes 189 . Heterologous expression of functionalized holo-OCP in E. coli cells rely on the use of pBADCrt-CrtO/W/R plasmid(s) that carry genes enabling the production of β-carotene and its metabolization into ketocarotenoids. The pBAD Crt-CrtO vector was kindly provided by Dr. Diana Kirilovsky and was used for the construction of pBADBt Crt-CrtO vector, the latter used for β-carotene and carotenoid production in Bti. The vector was modified and adapted in such a way that it can be recognized by Bti expression machinery, i.e. sporulation promoters, antibiotic resistance genes, etc. The recombinant expression and further crystallization of various other Bt crystallization chaperones and cargo proteins was also studied. The designed constructs are presented in Material and Methods (Chapter 2, Part I).

Figure 23: Strategy for in vivo recombinant crystallization of OCP variants and exogenous proteins in Bt. (A) Schematic of the pBT expression vector used for the synthesis of in vivo grown nanocrystals in Bt. Cry and Cyt toxins were used as crystallization chaperones (in green) and were fused to a cargo protein (in orange). Cyt1A BtI/BtII sporulation promoters were used to control the expression of the cargo-chaperone constructs. pBT expression vector features the 20 kDa accessory protein (folding chaperone), a STAB-SD sequence (5'-UTR) and a terminator sequence in the 3' region of the gene. The addition of an 6X His-tag and a Strep-tag (optional) seeked to follow expression. (B) Illustration of the crystallization strategy used in this project. Fusion of crystallization chaperone to a cargo protein yieds the formation of recombinant crystal.

Objectives of the thesis

The main goal of this thesis project was to attain a better understanding of the photoactivation mechanism of the Orange Carotenoid Protein (OCP) by shedding light on the important steps from a structural point of view. Indeed, a complete understanding of this mechanism may only be attained by the combination of methods -spectroscopic and structural -probing time scales ranging from hundreds of femtoseconds (the life time of the first excited state) to the minute (the life time of the active form OCP R ), and spatial scales ranging from the size of an atom to that of the whole molecule. In the context of my thesis, I have used two structural methods, i.e. X-ray solution scattering and X-ray crystallography, in their steady-state and time-resolved fashion. The project has been structured around two main axes, where each axis covered different problematics.

The first axis, consisted in developing an in vivo recombinant crystallization strategy with the aim to produce in vivo calibrated nano-to micrometer sized crystals of OCP, suited for TR serial crystallography experiments. I seeked to obtain these in the spore-forming bacterium Bacillus thuringiensis (Bt), characterized with the ability to produce in vivo-grown nanocrystals of naturally crystalline toxins. Our in vivo recombinant strategy relied on the design and expression of fusion constructs of OCP with two naturally crystalline Bt toxins, or domains thereof, envisaged to act as crystallization chaperones. In this context, we first characterized the native structure and/or function of these two toxins, i.e. Cyt1Aa and Cry11Aa. To do so, the proteins were expressed in vivo in Bt, and their structure was solved using SFX. To further characterize the structural determinant of Cyt1Aa and Cry11Aa in vivo crystallization, we designed mutants, which afforded insights into crystal formation, stability and solubilization properties. Given the vast complexity of the in vivo project, i.e. starting from the development of well-established recombinant crystallization strategy, then going through an extensive structural and functional studies of the two natively growing in vivo in Bt proteins, and ending in troubleshooting not only the expression and synthesis of OCP but as well the crystallization of their complex, the need to obtain OCP crystals in vitro rapidly became evident and was therefore explored to enable progress of the project, despite delay with the success of the in vivo crystallization strategy.

. Expression vectors

The expression vectors used in this project were pWF45, pWF53, pBT and pBADBt-CrtCrtO (Figure 24). In greater detail, the pWF45 and pWF53 190,191 plasmids were used to express wild type and mutants of Cyt1Aa and Cry11Aa toxins, respectively. The pWF45 and pWF53 feature both an erythromycin resistance gene, which is functional in Bt, and an ampicillin resistance gene, functional in E. coli, thus allowing to amplify them in E. coli Dh5𝛼 chimiocompetent strain (transformation procedure is described below in the chapter) and to carry out expression in Bt cells. To this end, both pWF45 and pWF53 feature cyt1Aa and cry11Aa promoters, respectively, which allowed the expression of cyt1aa and cry11aa genes, inserted downstream of their respective promoters. Both plasmids carried out the 20-kDa protein gene (p20), thus enhancing Cyt1Aa and Cry11Aa crystal production during sporulation 191 . To improve crystal production, the expression of p20 gene was under the control of the crylAc promoters. The promoter for cry1Ac included its ribosome binding site (RBS) and its coding sequence. The shuttle vectors were kindly provided by Prof. Brian Federici.

In order to attempt in vivo recombinant crystallization of OCP from Synechocystis PCC 6803 and Planktothrix agardhii, as well as that of the C-terminal domain homolog (CTDH) of OCP, the pBT plasmid library was used. The pBT shuttle vectors contain: (i) various combinations of folding chaperones, e.g. P20; (ii) Bt sporulation-dependent promoters; (iii) a Shine-Dalgarno (SD) sequence located upstream in the 5'-UTR of the start codon (AUG) of the gene; (iv) a stabilizing 3' stem loop located in the 3'-UTR region of the gene; (v) and a fusion of cargo protein genes with either full-length crystallization chaperones or domains thereof (Bt Cyt1Aa and Cry11Aa toxins).

As cargo proteins, the orange carotenoid protein (OCP) from Synechocystis PCC6803 and from Planktothrix agardhii, as well as the C-terminal domain homolog (CTDH) of OCP from Anabaena were used. The rsFolder, which is a GFP-like protein designed in our laboratory and whose structure has already been published 192 , was also used as a target protein for in vivo recombinant 94 crystallization in Bt, thus allowing to obtain a proof of concept of the method. The expression of cargo protein genes was under the control of their consecutive promoters. OCP and CTDH genes were kindly provided by Dr. Diana Kirilovsky.

As previously published, reconstruction of the carotenoid biosynthesis pathway in E. coli resulting in carotenoid synthesis requires 5 genes coding for 5 distinct enzymes 189 . Normally, in order to synthesize β-carotene in E. coli, the cells are transformed with pAC-BETA 193 or pACCAR16ΔCrtX 194 plasmids. These two plasmids possess the operon of the Crt genes, i.e. crtB, crtE, crtI and crtY the expression of which is under the control of crtE constitutive promoter, thus the β-carotene is synthesized constitutively. Heterologous expression of OCP in E. coli cells allowed to design pBAD plasmids, each one carrying the crtO, crtW or crtR genes, coding for the β-carotene ketolase enabling the conversion of β-carotene to into echinenone, canthaxanthin and zeaxanthin, respectively. In pBAD plasmid, the expression of crtO, crtW and crtR is under the control of arabinose (Ara) promoter, thus the expression is inducible by arabinose. Accordingly, for synthesis of echinenone, canthaxanthin or zeaxanthin in E. coli cells, the latter must be transformed with two plasmids, e.g. pAC-BETA and pBAD-CrtO or pBAD-CrtW or pBADCrtR. Heterologous expression of OCP binding various carotenoids in E. coli have also enabled the design of pBAD Crt-CrtO/W/R plasmids in which β-carotene and carotenoid encoding genes were present in the same vector. The pBAD Crt-CrtO vector was kindly provided by Dr. Diana Kirilovsky and was used as a backbone in the construction of the pBADBt Crt-CrtO vector, the latter used for carotenoid expression in Bt (Figure 24). Before my arrival in the laboratory the vector in question was already built and adapted for further recognition by Bt expression machinery. For this purpose, the DNA replication origin (Ori) of Bt was inserted, as well as the kanamycin resistance gene. 2), a pBT expression vector has been constructed. The β-carotene and ketocarotenoid synthesis in Bt was ensured by the construction of pBADBt Crt-CrtO plasmid.

Figure 24. Schematic of the expression vectors used for the synthesis of in vivo grown nanocrystals in Bacillus thuringiensis. pWF45 and pWF53 shuttle vectors were used for the synthesis of wild type and mutants of Cyt1Aa and Cry11Aa toxins, respectively. For the in vivo recombinant crystallization of non-naturally crystalline proteins, e.g. OCP, CTDH, as well as other exogenous protein candidates (see Table

Bacillus thuringiensis strains

The Bacillus thuringiensis strains used in this project are listed in Table 1. All previously described expression vectors were transformed into the acrystalliferous strain 4Q7 of Bacillus thuringiensis subsp. israelensis (Bti; The Bacillus Genetic Stock Center). To test expression and production quality, other acrystalliferous Bt strains were used. These included Bt 4B5 (subsp. finitimus), 4E5 (subsp. sotto) and 4XX6 (unknown). The strains in Table 1 highlighted in grey were used and have also served for the expression and extraction of additional Bt toxins, some of them subsequently tested as crystallization chaperones for in vivo recombinant fusions with candidate cargo proteins. Table 1. Bacillus thuringiensis strains used in the context of my thesis project. Strains colored in pale orange were used for toxins expression and in vivo crystal growth. In grey are highlighted the wild type strains used in order to study and structurally characterize other toxins, in our search to determine the commonalities of naturally crystalline proteins. These strains produce multiple crystals that were all injected at the same time with aim to challenge whether or not structure determination would be possible from a mix of crystals.

DNA library construction using Gibson assembly

To ease vector construction, the Gibson assembly method was used for the construction of the transfer vectors. The Gibson assembly is a commonly used recombinant cloning method which allows to assemble DNA fragments into a circular plasmid. The reaction is held in vitro and consists in designing fragments (primers) presenting 15-80 bp overlap with adjacent DNA parts. The fragments are mixed with a reaction buffer containing three different types of enzymes, i.e. T5 exonuclease, a DNA polymerase and a DNA ligase. When the DNA fragments and the receptor vector are assembled, the latter is ready to use for transformation in the expression organism. In this project, the linearized expression vectors were obtained by PCR (polymerase chain reaction) using Q5 High-fidelity DNA Polymerase (NEB, England). The DNA fragments were also amplified by PCR in order to produce the desired overlaps. The primers were designed according to the NEB instruction manual. The assembly reaction was carried out in a single-tube where the reaction volume was ~ 20 μl for all experiments. The applied concentration of the assembly fragments was three times greater than that of the vector. To trigger the reaction, the samples were incubated at 50 °C between 60 and 90 min. The constructed plasmids were transformed by heat shock procedure into chimiocompetent E. coli strain. The successful construction of each plasmid was assessed by Sanger sequencing at the Eurofins Genomics sequencing platform. The presence of mutated cyt1aa and cry11aa gene sequence in the transformed Bt colony used for crystal production, was verified by PCR colony. The list of primers used for the design of point mutants of cyt1aa and cry11aa genes is available in the Results Chapter (Part I). The list of completed constructions for the in vivo recombinant expression are presented in Table 2.

Polymerase Chain Reaction (PCR)

In this work, PCR reaction solution contained 1 µL of template DNA (1 pg -1ng of plasmid DNA or 1 ng -1 µg of genomic DNA), forward and reverse oligo (the concentration of each was between 0.1 and 0.5 µM), Taq polymerase (5 U/µL), 200 µM of dNTP and Q5 Start High-Fidelity 10X Master Mix (New England Biolabs). Ultrapure water was used to adjust reaction volume up to 50 µL. Amplified DNA fragments were separated by gel electrophoresis and then purified using a commercial DNA extraction kit (Macherey-Nagel™ DNA Plasma Midi NucleoSpin). For the purifications, the DNA was eluted from the column by using 30 µL of pre-heated nuclease-free water.

Transformation of E. coli cells

Validated plasmids were amplified in E. coli cells by thermal shock transformation procedure. For this purpose, 30 µL of chimiocompetent Dh5𝛼 E. coli cells were incubated for 10 minutes on ice and then mixed with 1 μl of plasmid material. The mixed suspension was then incubated for 20 minutes on ice, followed by thermal shock at 42 °C for 1 minute. In order to recover the transformed cells, these were immersed for 2 minutes on ice and subsequently mixed with 0.5 mL of SOC medium. The transformed cells were incubated for 1 hour at 37°C under agitation. Finally, they were spread and selected for growth on LB Agar containing 50 µg/mL ampicillin. The plates were incubated overnight at 37°C.

Transformation of Bti by electroporation

Electroporation (also termed electropermeabilization) is a commonly used transformation technique in microbiology, which is based on the facilitation of DNA transport across cellular membranes due to the permeabilization effect of short electric impulses 195,196 . Electroporation protocol 197 was used for the transformation of the constructed plasmids, into various Bacillus thuringiensis strains. Electroporation of B. thuringiensis strains were done with a MicroPulser electroporator (Bio-Rad, Richmond, CA, U.S.A.). For electroporation, 0.1 mL of electrocompetent bacterial cells were thawed on ice and mixed with 1µg of the target DNA solution (plasmid). After gentle resuspension the cell solutions were transferred to 0.4 cm sterile electroporation cuvette (Gene Pulser/MicroPulser Electroporation Cuvettes, 0.4 cm gap, BioRad), and submitted to a single electric pulse. For all electroporations, the cuvette was gently tapped, allowing the settlement of the cells at the bottom of the cuvette, and then placed in an electroporation apparatus. The machine was operated at the settings of 2.5 kV, 25 µF, and the parallel resistor at 200 Ohms. After electroporation, the cells were recovered in ~ 0.5 mL of SOC medium, and incubated for 90 -180 min at 37°C with shaking. Afterwards, 0.1 and/or 0.25 mL of the cell suspension were plated on selection LB-agar plates and incubated overnight at 37 °C.

Growth of Bacillus thuringiensis on sporulation media

Isolated colonies were collected from Petri dishes and further suspended in 5 ml of Luria Bertini (LB) broth complemented with 25 µg/mL of erythromycin. The pre-cultures were then incubated in a shaker incubator at 37 °C for 6 h. After incubation, the pre-cultures were spread on T3 sporulation agar media 198 plates and stored for 96 h at 30°C until crystal-spore formation.

Afterwards, the spores-crystals mixture was collected using a cell scraper and crystals were purified by discontinuous sucrose gradient (see below).

Expression & Purification of toxin crystals and/or recombinantly produced in

Bt exogenous proteins

4X buffer (Tris 0.5 M pH 6.6, 20 % SDS, 50 % glycerol) in presence or in absence of DTT (dithiothreitol) at 1 M. DTT is a reducing agent that enables disulfide bridge reduction between cysteines. In most of the cases, the samples were heated for 3 minutes at 95 °C and then deposited on the gel and migrated for 60 min at 140V in a running buffer 10X (Tris-Glycine SDS).

After staining by overnight incubation in Instant Blue (Sigma Aldrich, France), gels were washed twice in ultrapure water and migration results were digitized using a ChemiDoc XRS+ imaging system controlled by Image Lab software version 6.0.0 (Bio-Rad, France).

Western blotting

Between 20 and 40 μg of protein samples (e.g. directly purified or purified and solubilized crystals) were deposited on 12% acrylamide gel. The proteins were migrated for 60 min at 140V constant.

After migration, samples were transferred to the nitrocellulose membrane and the transfer was carried out overnight at 4°C. The day after, the membrane was saturated for 30 min in TBS 5X and 5% (m/v) of powdered milk (Regilait) and then incubated with the primary antibody for 1 hour at RT, under agitation. For this purpose, the primary antibody (1/10000) was diluted in TBS 5X -0.1% Tween 20. After 3 successive washes, 5 min each, in TBS 5X-Tween 0.1%, the membranes were incubated with the secondary antibody (1/20000) coupled to HRP (Horseradish Peroxidase) for 2 hours at RT. The revelation was then performed using chemiluminescent HRP substrates, e.g. luminol and peroxide in 1:1 ratio (Pierce ECL, Thermo Fisher). The membrane was digitized using a ChemiDoc XRS+ imaging system controlled by the Image Lab software version 6.0.0 (Bio-Rad, France). Revelation exposure time was adjusted according to the signal to noise ratio. Molecular masses were determined by comparison to simultaneously migrated molecular standards (Thermo Fisher). The β-carotene and carotenoid synthesis in Bt was followed using thin-layer chromatography.

After transformation of pBADBt Crt-CrtO expression vector in Bti cells, bacterial colonies were suspended in 10 mL of LB media and growth overnight at 37°C. The day after, induction of crtO gene was triggered by adding arabinose into the bacterial precultures. For this purpose, different concentrations of arabinose were tested, ranging from 0.01% to 0.3 % (m/v). Overnight precultures were diluted in fresh LB media and grown at 37°C for 3 to 4 hours up to an optical density at 600nm (OD600) of 0.6. The arabinose was then added and the cultures were grown overnight at 37°C. After overnight growth, 2 ml of Bt cells synthesizing carotenoids were centrifuged for 3 minutes at 16 000 g. The supernatant was discarded and the cells were resuspended and vortexed in 2 ml of water with aim to wash the pellet. The suspensions were then centrifuged again for 3 minutes at 16 000 g. The supernatant was discarded and the cells were resuspended in 0.5 mL of acetone, with the goal of breaking the cells and extracting the hydrophobic carotenoids. Cells were left to gently shake with acetone for 20 minutes at room temperature then the mixture was centrifuged for 5 minutes at 16 000 g. The supernatant containing acetone and carotenoids is recovered in a new clean tube Eppendorf 1.5ml. The supernatant (acetone and carotenoids) was gently dried with pulsed air under a Sorbonne hood for 10 minutes. The carotenoid powder glued to the wall of the Eppendorf tube was resuspended in 0.05 mL of diethyl ether. The solution in which the CCM plate was dipped is composed of 10% acetone + 90% of petroleum ether. The carotenoids contained in the 50 μl of diethyl ether were gently deposited on the CCM plate with a glass capillary and migration took place for about 15 minutes.

Nanocrystals stability assays 1.3.2.1. Nano DSF Thermal Unfolding assays

To monitor crystal stability, differential scanning fluorimetry measurements were performed using a Prometheus NT.48 instrument (NanoTemper Technologies). Experiments were performed using

Cry and Cyt purified inclusions, as well as solubilized crystals at different pHs. The measurements were carried out at concentrations of 0.5 mg/mL and 1.0 mg/mL. For the measurements, 15 μL of each sample was loaded in standard capillaries (NanoTemper Technologies). The temperature gradient was set to 1°C/min and measurements were carried out between 20 to 95 °C. Protein unfolding was monitored by plotting the change in tryptophan fluorescence at the emission wavelengths of 330 and 350 nm, as a function of the temperature. were diluted at 1/1000 and mixed with 25 mM of ammonium acetate. Samples were then coated with 2 nm thick gold layer as described above.

Bacillus thuringiensis crystal preparation and further injection using Gas Dynamic Virtual Nozzle (GDVN) at EuXFEL facility

Diffraction data on Cry11Aa mutants at pH 7.0 as well as on Bt strains LM 1212, 4B1, 4L4, 4AR1

and jegathesan were acquired at the SPB/SFX beamline at EuXFEL, Hambourg. Briefly, crystals of either Cry11Aa, or cocktails of toxins produced naturally by various Bt strains (Table 1) were resuspended in distilled water and the suspensions (2 -12 % (v/v)) were transferred into a stainless-steel syringe that was mounted on an anti-settling device, thermalized at 20°C. In order to collect single anomalous diffraction data used for the phasing of LM 1212, 4AR1 and 4B1 Bt strains, the latter were soaked for 30 hours with 10 mM Tb-Xo4 200 , serving as a phasing agent.

Crystal suspensions were passed through a 20 μm filter. The microcrystal suspension was injected with a gas dynamic virtual nozzle (GDVN) injector 127 using helium as the focusing gas. The GDVN carried a sample capillary of 75 μm inner diameter and sample flow rate was 60 μl/min. The resulting jet had a diameter of 6 μm at a focusing helium pressure of 570 psi.

Transformation of E. coli cells

Transformation of vectors coding for OCP and/or carotenoid-producing enzymes in E. coli cells was performed using the thermal shock transformation procedure. For this purpose, 30 µL of chemocompetent Dh5α E. coli cells were incubated for 10 minutes on ice and then mixed with 1 μl of plasmid material. The mixed suspension was incubated for 20 minutes on ice, followed by a thermal shock at 42 °C for 1 minute. The cells were put back on ice for 2 minutes and subsequently mixed with 0.5 mL of SOC medium and incubated for 1 hour at 37°C under agitation. Finally, transformed cells were spread and selected for growth on LB Agar with appropriate antibiotic(s) depending on the transformation carried out (resistance(s) on the plasmid(s)). The plates were incubated overnight at 37°C. For the expression and production of OCP, E. coli BL21-Gold (DE3) cells (Agilent technologies) were used. For the expression of holo-OCP, the cells were transformed simultaneously with three plasmids: pAC-BETA (β-carotene synthesis), pBAD-crtW (canthaxanthin synthesis) and pCDF-OCP (OCP synthesis). The pCDF-OCP plasmid contained either the wild type OCP gene or a modified sequence in which point mutation was introduced.

The transformed E. coli cells were spread and grown in presence of three antibiotics (ampicillin (50 μg/ml), chloramphenicol (17 μg/ml) and streptomycin (25 μg/ml)).

Carotenoid and OCP gene expression in E. coli

For the expression of the genes coding for carotenoids, carried by the three plasmids, overnight E. coli colonies were selected and grown in LB media at 37°C overnight allowing the expression and synthesis of β-carotene. In the morning, the pre-cultures were transferred in fresh TB media (Terrific Broth, Sigma Aldrich) and grown under agitation at 37 °C for 3 to 4 hours, up to OD600 = 0.6. Then, arabinose (0.03 % (m/v)) was added and the cultures were grown overnight at 37°C.

This step allows the synthesis of carotenoids (e.g. canthaxanthin). The day after, the cells were again diluted with fresh medium supplemented with 0.03 % of arabinose. The cultures were incubated at 37°C and grown until an OD600 of 1-1.2 was reached. To induce OCP gene expression, 0.2 µM of IPTG (isopropyl-β-Thiogalactoside) was added and the cells were thereafter incubated overnight at 28 °C. In the morning, the cultures were harvested and subjected to cell lysis and purification. When the purification step could not be performed right away, the were centrifuged for 15 minutes at 4000 g and the pellets stored at -80 °C.

Purification of wild type OCPs and mutants

In Table 4 are listed the designed point mutants of OCP Planktothrix. After being expressed in E.

coli cells, the mutants were purified using the three purification methods listed below. It is to note that mutants L37T, V158A, I51F, L154M, I40F and L250M were purified in the laboratory of Prof.

Ilme Schlichting (MPI Heidelberg, Germany) by Elisabeth Hartmann and sent to us for performing crystallization trials. The rest of the mutants were expressed and purified in our laboratory.

Purification using immobilized metal affinity chromatography (IMAC)

E. coli cells resuspended in lysis buffer (Tris-Base 40 mM pH 8, glycerol 10% and 300 mM NaCl)

were lysed by five sonication cycles (2 sec ON/10 sec OFF) using between 40% and 90% amplitude depending on the prep. The lysis buffer was supplemented with DNAse (10µg/mL), 1mM MgCl2 and EDTA-free protease inhibitor cocktail (cOmplete, Roche). After sonication, the lysed cells were centrifuged at 39 000 g for 30 minutes at 4 °C in a Beckman JA20 rotor. After centrifugation, the supernatant (containing soluble OCP) was loaded onto a nickel column (Niresin Probond, Invitrogen) which had been previously equilibrated with 5 volumes of Tris-Base 40 mM pH 8, glycerol 10% and 300 mM NaCl. After OCP binding, the column was washed with 2 volumes of the equilibrating buffer supplemented with 15 mM of Imidazole and then OCP was eluted with 200 mM of Imidazole.

Purification and separation of apo-and holo-OCP using hydrophobic interaction chromatography (HIC)

Principle

A commonly used technique allowing to separate molecules according to their intrinsic hydrophobic characteristics is the hydrophobic interaction chromatography (HIC) 201 . This is a type of liquid chromatography in which the stationary phase consists of a resin matrix composed of hydrophobic moieties 202 . Interactions between proteins and the resin matrix are influenced by the running buffer where high salt concentrations are used to enhance the hydrophobic interactions.

Precisely, since high molarity salt solutions promote hydrophobic interactions and precipitation, lowering of the salt concentration weakens the interaction. To attach the proteins on the resin matrix, these are introduced on the column in presence of a binding buffer containing high salt concentrations. The protein with the lowest degree of hydrophobicity is eluted first. However, when the ionic strength of the buffer is reduced (decrease of salt concentration), the interaction is reversed and therefore the most hydrophobic protein elutes last.

Protocol

Separation of apo-and holo-OCP was achieved by hydrophobic chromatography using prepackaged phenyl-sepharose columns (5mL HiTrap Phenyl HP, GE Healthcare). For the purification of each OCP sample, two columns were connected and used simultaneously (Figure 28). Before sample loading, the column was washed with 3 volumes of water and then equilibrated Figure 28). The presence of highly pure holo-OCP was estimated by calculating the absorbance ratio at 495 nm and 280 nm. When the A495nm/A280nm was superior to 1.6, the protein sample was considered to present only as holo-OCP. The holo-OCP was then dialyzed against 50 mM Tris-HCl pH 7.4 at 4°C. The pptimization of the protocol used for the preparation of gram quantities of out agent whereas NaCl is a poor one 203 . In greater detail, addition of high concentrations (80 % of salt saturation must be achieved) of good salting out agents to the protein solution leads to strong interactions between the water molecules of the hydration shell of proteins and the salt ions, competing with those they had in place with the charged amino acid side chains of the proteins. Indeed, at a certain ionic strength, the water molecules are no longer able to support the charges of both salt ions and the proteins, thus resulting in the precipitation of proteins (soluble solutes).

Protocol

The production of highly pure gram-quantities of holo-OCP for serial crystallography experiments at XFELs was a major challenge for our lab and those of collaborators (Prof. Ilme Schlichting at MPI Heidelberg, Germany and Dr. Diana Kirilovsky at I2BC, Gif-sur-Yvette, France). In standard expression conditions, i.e. expression of OCP in 1 L medium, purification of around 80 % of holo-OCP (30 mg) is easily achieved. Structural studies using SFX are highly demanding on protein sample quantities, and production of 5 grams of holo-OCP sample were envisaged as needed to carry out the experiment using GDVN liquid injection, which required to scale up production and purification at to volume of 20 L per week. The scale up had unexpected consequences on protein quality, with the amount of holo-OCP decreasing up to 10%, increasing our need of hydrophobic columns. To cope with this problem, the purification of holo-OCP from Planktothrix was performed using the salting-out procedure which allowed to easily separate apo-OCP from holo-OCP by precipitation in 5M NaCl. The choice of using NaCl as a salting out agent was made after first conducting precipitation tests in presence of ammonium sulfate. However, dialysis of purified OCP against ammonium sulfate caused irreversible precipitation of both holo-and apo-OCP.

Precipitation assays with increasing concentrations of NaCl permitted to successfully perform holo-OCP purification in presence of 5M NaCl. dialysis in 150 mM salt, the holo-OCP was further purified on phenyl-sepharose columns (described previously) affording a ratio A495nm/A280nm > 1.6. The highly pure holo-OCP preparation has been sent to Prof. Ilme Schlichting (MPI Heidelberg, Germany) for microcrystallization using the batch method. The establishment of the described purification protocol allowed the purification of a total quantity of 5 grams of holo-OCP.

Purification of OCP using size-exclusion chromatography (SEC)

SEC was carried out on an analytical HiLoad 16/60 Superdex 75 (HiLoad 16/600 Superdex 75 pg, Sigma Aldrich). The column was equilibrated with 50 mM Tris-HCl pH 7.4 and 150 mM NaCl buffer.

Concentrated samples were injected onto the column and purification was followed by monitoring the UV absorbance at 280 nm, in the various eluted fractions. OCP samples were eluted in equilibration buffer at flow rate of 1 ml/min in. The molecular weight of the eluted species was determined through calibration of the column using the following standard proteins: γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa) and vitamin B12 (1.35 kDa). The experiment was performed at room-temperature.

Calculation of the concentration of holo-OCP (OCP binding carotenoid)

The concentration of apo-OCP was calculated using the absorbance at 280 nm and the theoretical extinction coefficient (𝜀) obtained from https://web.expasy.org. For Synechocystis and Planktothrix

OCPs the calculated extinction coefficients are 34 659 M -1 .cm -1 and 36 737 M -1 .cm -1 respectively.

To determine the concentration of holo-OCP, we used an epsilon equal to 63 000 M -1 cm -1 at 475 nm 204 . The absorbance signal at both wavelengths, e.g. 280 nm and 495 nm were recorded using a NanoDrop™ One (Ozyme) spectrophotometer (Thermo Fisher Scientific).

Absorption spectra measurements and kinetic photoactivity of OCP

Individual dark and light absorption spectra were recorded using a SPECORD S600 spectrophotometer (Analytik Jena) at 20 °C. Thermal recovery kinetics of OCP at RT were measured using a JASCO V-630 UV/Vis spectrophotometer (Easton, USA). Purified OCP samples in 50 mM Tris-HCl pH 7.4, 150 mM NaCl buffer were measured at various concentrations.

Absorption at 467 nm was monitored at room-temperature (22°C) in the dark following 30 minutes of continuous blue light irradiation with a 430 nm LED light collimated to 1 cm (~ 500 mW/cm²).

The measurements were carried using different types of cuvettes. For instance, measurements at low protein concentrations (e.g. 0.1 mg/ml) were carried out in a PMMA cuvette with 1 cm pathlength. For concentrations between 2 and 10 mg/ml, a quartz cuvette with 0.1 cm pathlength was used, and for concentrations superior to 16 mg/ml, the experiments were performed in an

Infrasil cuvette with 0.01 cm pathlength. To facilitate comparison of data collected at various concentrations, the absorption difference at 467 nm was normalized using ANorm = [A(t) -

A(t0)]/[A(tmax) -A(t0)],
where A(t), A(t0) and A(tmax) are the absorption values measured at a generic time t after switching off the 430 nm light, at time t0 i.e. just after illumination at 430 nm was switched off.

Stability assays of OCP

To monitor the stability of soluble wild type OCP from Planktothrix and Synechocystis, differential scanning fluorimetry measurements were performed using a Prometheus NT.48 instrument (NanoTemper Technologies). Experiments were performed using soluble OCP and the measurements were carried out at concentrations 0.5 mg/ml of 1.0 mg/ml. For the measurements, 15 μl of each sample was loaded in UV capillaries (NanoTemper Technologies). Temperature gradient was set at 1°C/min in a range from 20 to 95 °C. Protein unfolding was measured by the detection of change in tryptophan fluorescence at the emission wavelengths of 330 and 350 nm, depending on temperature gradient.

Static SAXS measurements.

Acquisition of static X-ray scattering data was performed at the SWING beamline of the Soleil Synchrotron (Saint-Aubin, France). X-ray solution scattering patterns in the SAXS region (q = 0.02 -0.5 Å -1 ) were collected at RT at various concentrations of the OCP samples listed in Table 4, using a monochromatic X-ray beam at 16 keV and a EigerX 4M (Dectris) detector. For all protein samples, two sets of measurements were carried out, before and after 30 min irradiation of OCP solutions with a 430 nm LED source (~ 500 mW/cm²), allowing collection of X-rays scattering signals in the dark-adapted (OCP O ) and light adapted (OCP R ) states, respectively. For each concentration, 30 X-ray scattering images (each registered after 990 ms of X-ray exposure) were collected with 30 analogous images of the buffer before and after each sample switch. Each 2D image was converted to a 1D scattering pattern by azimuthal integration using the Foxtrot-3.5.2 software available at the SWING beamline. Corresponding scattering patterns were averaged and protein signals were obtained by subtraction of buffer scattering. The distance distribution functions were computed using GNOM 101 and structural parameters reported in Table 4 were determined from the reduced data using relevant programs from the ATSAS suite [START_REF] Manalastas-Cantos | ATSAS 3.0: expanded functionality and new tools for smallangle scattering data analysis[END_REF] . Lowresolution ab initio molecular envelopes were computed using the ATSAS reconstitution tool DAMMIF based on a reverse Monte Carlo minimization approach 102 .

Table 5. OCP samples used for static X-ray scattering measurements.

In vitro crystallization of OCP Planktothrix

The OCP samples used for crystallization trials were purified on Ni-NTA and phenyl-sepharose columns, and were further subjected to size exclusion chromatography under dim red light (HiLoad 16/600 Superdex 75 pg, GE Healthcare). Crystallization conditions were screened manually, using as starting conditions those for the crystallization of Synechocystis OCP 66 and Arthrospira maxima OCP [START_REF] Kerfeld | The crystal structure of a cyanobacterial water-soluble carotenoid binding protein[END_REF] . The samples were used at various concentrations ranging from 1 to 7.0 mg/ml.

Crystallization trials were performed in 24-well Linbro plates using the vapour diffusion method.

Both hanging-and sitting drop approaches were tested. Crystallization assays were set at both 4°C and room temperature and the different mixing ratios were tested between the well solution and the protein sample. The first crystals of OCP Planktothrix binding echinenone appeared in ~5 months. Following this success, the crystallization conditions were optimized at room-temperature (~20 °C) enabling growth of crystals in a few hours to a few days. The optimized crystallization conditions were also used for the crystallization of OCP Planktothrix point mutants. The optimized crystallization conditions were achieved using OCP protein concentration of 1.5 -2 mg/ml in 50 mM Tris-HCl pH 7.4, 150 mM NaCl, a well solution composed of 0.1 M sodium acetate, pH 5 and 10 -20% PEG 4000, and by mixing these at 1:1 ratio in the crystallization drops.

✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ Light mesearuments ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✗ ✓ ✓ ✓ ✓ Instrument Beamline SWING (Synchrotron Soleil,

Illumination of OCP microcrystals from Planktothrix agardhii and mounting on SOS sandwich chip

Small volume (0.1 to 0.5 ml) of OCP microcrystals (5 µm) was deposited on a single-isolated well of 24 well Linbro plate. An equivalent volume of 17% PEG 4000, 0.1 M NaCl was added to the crystals and the crystal/reagent suspension was put under agitation at room temperature to prevent crystals sedimentation. Under the plate an ice tray was positioned preventing crystal heating under illumination. The crystal suspension was illuminated with a continuous blue light for 48 hours on a slowly rotating orbital shaker. Afterwards, the crystal suspension was centrifuged for 10 seconds using a benchtop centrifuge. 2 to 5 µL from the crystal pellet were pipetted and deposited on SOS sandwich 205 . Briefly, the SOS sandwich is composed of two (lower and upper)

2.5 µm Mylar sheets. Between the two membranes, a sample solution is sandwiched and then mounted in a chip holder 206 . On the lower Mylar membrane, the OCP microcrystals are spread using a pipette tip, then the crystals covered with the upper Mylar membrane and the SOS chip closed, allowing to place into contact the two membranes. No drying of the sample was observed when mounting the chip. Before mounting, the absorption spectra of non-and illuminated crystals was recorded on the NanoDrop™ One (Ozyme) spectrophotometer.

OCP microcrystallization was performed by Prof. Ilme Schlichting (MPI Heidelberg, Germany).

SOS chips were designed, constructed and kindly provided by Dr. Bruce Doak (MPI Heidelberg, Germany). The illumination protocol was initially designed by Prof. Ilme Schlichting and further optimized in our laboratory.

Data collection and structure refinement of OCP Planktothrix mutants' crystals

For structure determination of OCP Planktothrix various mutants (listed in Table 4), macrocrystals of each mutant were grown in 0.1 M sodium acetate pH 5, 12% -18 % PEG 4000. A minimum of two macrocrystals per mutant were then harvested with a cryo-loop and soaked for a few seconds occur after in vivo crystallization has completed in Bti cells. As a control for the unicity of the role of C7, we also mutated the second cysteine of Cyt1Aa, viz. C190, introducing a C190V mutation with the aim to not interfere with the hydrophobic environment present at the αC/αD interface with the β-sheet (Fig. 1e and Supplementary Fig. 2). C190V crystals are significantly smaller than the WT but production yields are not affected, suggesting a minor impact of the mutation on protoxin folding and in vivo crystallization (Fig. 3).

E32 and E45 were identified as the cornerstone residues controlling the pH sensitivity of the DS interface (Fig. 2b). With the aim of rendering pH-insensitive the H-bonds that these residues contribute at the DS interface, we mutated both residues into glutamines (E32Q, E45Q). The E45Q mutation was also intended to strengthen the tether between the αC/αD region (Q138) and the βsheet, allowing us to test the hypothesis that opening of this interface is involved in function. E45Q crystals are significantly larger than the WT crystals, while E32Q crystals are produced in higher abundance (Fig. 3). Thus, both mutations, intended to stabilize the DS interface, positively impact crystal growth, albeit in a different fashion.

Our observation that interface 3 changes structurally in response to reducing agent and pH prompted us to test the role of D11 and Q168 in conferring these sensitivities. A D11N mutation was introduced to decrease the pH sensitivity of this interface, and a reverse Q168E mutation to increase it. Crystals of D11N are significantly larger and quasi-cylindrical in shape, evincing that the mutation favours crystal growth (Fig. 3). Crystals of Q168E are marquise-shaped and produced in reduced amounts (Fig. 3), highlighting the negative effect of the mutation on in vivo crystallization; the side chain nitrogen of Q168 is indeed involved in two H-bonds with acidic oxygen atoms at interface #3 (Fig. 2B and Supplementary Figs. 2, 3b,c and 4). Last, we introduced a Y171F mutation, attempting to verify whether the pH-sensitive H-bond between the β5 residue hydroxyl and T146(O) (pKr of tyrosine is 10.5) at the tip of αD plays a role in protein folding or alkaline activation. Indeed, packing relaxation at interface #3 was evident as disorder on the hydrophobic αC/αD face of the β-sheet (Fig. 2b). Both the production yield and the crystal size are negatively impacted (Fig. 3), suggesting that this H-bond plays an important role before crystallization -possibly during folding. Supplementary Fig. 4. Fourier difference maps shed light on the crystal dissolution mechanism. Fourier difference maps computed between datasets, and phased by the pH7 structure, highlight the most striking conformational changes upon DTT soak (Fo 'DTT' -Fo 'pH7' map) and pH elevation (Fo 'pH10' -Fo 'pH7' map). These are overlaid on the pH7 protoxin structure, shown as an orange/slate-coloured ribbon, in the two left panels; and on the DTT/pH10 protoxin structure, shown as a black/white-coloured ribbon, in the two right panels. Symmetry related molecules are all coloured differently, with each molecule having the same colour coding in all panels. From top to bottom, the figure shows the maps contoured at ± 3 sigma around the full DS dimer, at the DS and cysteine interfaces, and at crystal packing interface 3, respectively, with positive and negative peaks shown in green and red Fo DTT,pH7 -Fo pH7 Fo pH10 -Fo pH7

Map overlaid on pH7 structure Map overlaid on soaked structure (pH10/DTT) 1 Domain swapped interface, classified as a stable block by PISA (predicted ∆G= -27.1 kcal/mol). Residues 6, 18-60, 71, 134, 137, 138, 140, 141, 144, 145, 149-160, 181+203, 208-213 and 233-247 (C-terminal propeptide) from both chains are involved. The N-terminal (β1 and β2) and C-terminal (αF) propeptides as well as the αC/αD hairpin are buried at this interface which features 46 H-bonds and two salt-bridges. The salt-bridges involve R30 (NH1) and D45 (OE2) from facing monomers in the DS dimer.

H-bonds previewed to disrupt upon pH elevation include those between facing T23(OG1) and D33(OD1), and N181(ND2) and E32(OE2), respectively, but most importantly, that between E32(OE1) and E45 (OE2) wherein the latter atom is an obligate donor.

Per monomer buried surface area: 3077 Å 2

2

Residues 13-19 (N-terminal propeptide), 103, 106 (β3), 111, 113, 114, 117, 118, 121, 122, 124, 125, 129, 140 (αC/αD) and 212 (αE) from the asymmetric unit monomer interact with residues 33, 36, 37 (β1-β2 loop), 53, 54, 57, 63, 64, 66, 68, 71, 75, 77, 83, 86, 87, 90, 92, 93 (αA/αB), 228, 231 (β7), 233, 235, 236, 237, 240, 248 and 249 (Cterminal propeptide) from the symmetry-related mate.

Four H-bonds are found at this interface, of which one involves the side chain oxygen of an acidic residue, viz. that between N15(ND2) and D240(OD2). A salt-bridge is also found at this interface, viz. between K118(NZ) and D75(OD1).

Per monomer buried surface area: 770 Å Six H-bonds are found at this interface, of which a single involves the side chain oxygen of an acidic residues, viz. that between Q168(NE2) and D11(OD1). A salt-bridge is also found, viz. between K198(NZ) and D11(OD1).

Per monomer buried surface area: 646 Å 2

4

Residues 89, 94-98 (αB-β3 loop), 130 (αC/αD hairpin), 182-189 (β5-β6 loop), and 227-229 (β7), from both chains are involved.

There is no H-bond or salt-bridge at this interface.

Per monomer buried surface area: 411 Å 2 155, 164 (αD-β4 loop), 204, 207, 208 (αE) from the symmetryrelated mate.

The three H-bonds found at this interface involve side chain oxygens of acidic residues, viz. E36(OE2), which interacts with N151(ND2); and E204(OE1) and (OE2), which interact with T248(OG) and N35(ND2), respectively.

Per monomer buried surface area: 225 Å 2

6

Residues 48-50 (β2-αA loop), 232, 234 and 235 (C-terminal loop) from the asymmetric unit monomer interact with residues 164-166 (β4-β5 loop) and 202, 204, 205 (αE) from the symmetryrelated mate.

There is no H-bond or salt-bridge at this interface, which nonetheless participates in reducing access to Q168.

Per monomer buried surface area: 224 Å 2

9

Residues 9 and 10 (N-terminal propeptide) from the asymmetric unit monomer interact with residues 71 (tip of the αA/αB hairpin) and 163 (β4-β5 loop) from the symmetry-related mate.

There is no H-bond or salt-bridge at this interface.

Per monomer buried surface area: 27 Å 2 Supplementary Fig. Supplementary Fig. 8. Native mass spectrometry confirms that Cry11Aa is solubilized as a full-size monomer. a, Profile of soluble Cry11Aa in native MS shows three peaks corresponding to a full-size toxin of 72.345 kDa (expected mass: 72.349 kDa) and three peaks for a ~1 kDa smaller form much less abundant, likely resulting from a cleavage of the first 9 amino acids in N-terminal part of the toxin. b, Incubation at room temperature leads to an increase in the smaller form, reaching one fourth of abundance of full size one, suggesting a targeted cleavage of few amino acids in the N-and/or C-terminal extremity. c, When isolating the most abundant peak (i.e., at m/z = 4256, corresponding to Cry11Aa with a charge of 17) in the same condition as in panel a, the peak is sharp and well defined. d, Increasing the collision energy leads to a fragmentation into species with different sizes. If the protein is an oligomer with multiple monomers through non-covalent bound, specific monomers would be easily separated and identified on the MS spectrum. This rather supports that Cry11Aa solubilizes as a full-sized monomer prone to degradation under certain conditions. Relative intensity All Cry11Aa mutants with one and two point-mutations (D507N-D514N) produced crystals that could be properly purified by sucrose gradient coupled with ultracentrifugation. In contrast, the triple mutant Y272Q-D507N-D514N showed a different purification profile. Most of proteins were contained in the upper phase of the sucrose gradient but no clear band at the ~70 kDa size could be seen. This is confirmed by environmental SEM (eSEM) experiments showing that this phase mostly contains empty parasporal bodies, suggesting that this mutation is deleterious for the proper production and/or crystallization of the toxin in the bacterium during its sporulation.

T) (U) (M) (L) (P) (T) (U) (M) (L) (P) (T) (U) (M) (L) (P) (T) (U) (M) (L) (P) (T) (U) (M) (L) (P) (T) (U) (M) (L) (P) (T) (U) (M) (L) (P) (
Y272Q-D507N-D514N

Supplementary Table 2. Effects of Cry11Aa and Cry11Ba point mutations on toxicity described in literature

Comment / Forward GCCGCAGTGTTATCACTCATGG TTA TGGC Amp_F1 was used with each reverse primer of Cry11Aa mutant construction. For each mutation primer, the inserted mutation is indicated as a capital bold letter. To generate the vector containing the mutation, the two fragments generated for the same mutations were assembled by Gibson assembly following the procedure described in the manuscript. Amp_R1 was used with each forward primer of Cry11Aa mutant construction. For each mutation primer, the inserted mutation is indicated as a capital bold letter. To generate the vector containing the mutation, the two fragments generated for the same mutations were assembled by Gibson assembly following the procedure described in the manuscript. andLR30) and a rod-core linker (LRC). Nevertheless, the principal Lcm band in Planktothrix PBS was found to migrate at a lower molecular weight than that of Synechocystis PBS, and was accompanied by a second band at 75 kDa, suggesting partial proteolytic degradation. (B) Absorbance spectra of purified phycobilisomes from Planktothrix (fuchsia), Synechocystis (black) and Arthrospira (blue). The absorption spectrum of Synecocystis PBS features a sharp band characterized by a maximum at 620 nm and a shoulder at 650 nm. A broader band with a maximum at 615-618 nm and no 650 nm shoulder is seen for Planktothrix and Arthrospira PBS (Jallet et al., 2014). In the case of Arthrospira, the observation was rationalized by the presence of phycobiliviolin instead of phycocyanobilin in some phycocyanin (PC) units of the PBS (Babu et al, 1991). This could also be the case for Planktothrix PBS. (C) Florescence spectra at 77K of purified phycobilisomes from Planktothrix (fuchsia) and Synechocystis (black). The intense peak at 685 nm (last acceptor of PBS) and very low peaks at 645-660 nm (PC and APC) in the 77 K fluorescence spectra demonstrate that energy-transfer from the PC to the final emitters (ApcE and ApcD) is as efficient in Planktothrix PBSs as it is in Synechocystis PBS. The plot shows the time evolution of the integrated intensity in the 0.05-0.5 Å -1 region of the X-ray difference profile ΔI(q) = I(q)t -I(q)t0, with I(q)t and I(q)t0 the scattering intensities at a generic time t after switching off the 430 nm light and before the 430 nm irradiation procedure, respectively. The data establish that after accumulation of the OCP R state at 23 mg/ml, recovery of the starting structural state of the OCPwt-Ctag sample spans a time-scale of at least 2.5 hours.

L Q A D A V P A L T A R F N Q L S A E D Q L AWTWF A F L EMGK T I T V A A P G A A S MQ F A E G I L K Q I K EM T F E E Q T Q V MC D L A N H T D T P I C R T Y A T WS P N I K L G FWN Q L G EWME QG A V A P I P A G Y Q L S A N A N A V L E T L K S L DQ GQQ I T V L R S S V V DMG F D A A K L D G Y T R V A E P L V A P K D I SQ R V Q V T I E G I N N S T V L N YMN N L N A N D F D E L I K L F V E D G A L Q P P F Q R P I VGKD A I L R F F R E E CQN L N L L P E RG V A E P A D D G Y T Q V K V T GK V Q T PWF GA A V GMNMAWR F L L N P QG K I F F V A I D L L A S P K E L L N L V R

M P F T I D S A R G I F P E T L T A D S V P A T I A R F NQ L S A E DQ L A L I WF A Y L EMGK T I T I AA PGAANMV

F A E N T L N E L K QM S F Q E Q T Q V MC D L A N R A D T P I C R T Y A I WS V N I K L G FWY R L A EWM E QG I V A P I P Q G Y R L S A N A A A V L Q A I R E L D A GQQ I T V L R N S V V DMG Y D P S K L G S Y T K V S E P V A P P K EMA K R T P V T I E G I D N P T I L S Y MD N L N A N D F D A L I E L F T P D G A L Q P P F Q K P I A G K E A V Y R F F R E D C Q N L K L I P Q R G V S E A V E DG Y T Q I K V T G T V Q T PWF G A S V
V L R N C V V DMG F D T S K L G S Y Q R V A E P V V P P Q E M S Q R T K V Q I EGV T N S T V L Q Y MD N L N A N D F D N L I S L F A E D G A L Q P P F Q K P I V G K E N T L R F F R E E CQN L K L I P E RG V S E P T E DG Y T Q I K V T GK V Q T PWF GG N V GMN I AWR F L L N P E N K V F F V A

M S F T V D S A RG I F P N T L T A D V V P A T I A R F N Q L N A E DQ L A L I WF A Y L EMGK T I T

V A A L G A A NMQ F A E I T MN Q I R QM S F Q E Q T Q V MC D L A N R T D T P I S R A Y G SWT A N I K L G FWYQ L G EWMA QG I V A P I P S G Y K L S A N A A S V L Q A I QG L E S GQQ I T V L R N C V V DMG F D T S K L D S S Q R V S E P V V V P R DMA Q R T Q V T I E G I D N P T V L N YMN N M N A N D F E V L I E L F T P D G A L Q P P F Q K P I V G K D A V L R F F R E E CQN L K L I P E RG V T E P A D GG Y T Q I K V T G K V Q T PWF G A G V GMNMAWR

Figure S6

. The illumination procedure used to investigate OCP photoactivation and recovery does not significantly damage the protein sample. Absorption spectra recorded before (black) and after (green) 30 minutes illumination plus 16 hours of thermal recovery for (A) OCPwt-Ntag at 3 mg/ml, (B) OCPR27L-Ntag at 1.7 mg/ml and (D) OCPwt-Ctag at 1.7 mg/ml. The OCP concentration and optical pathlength were 3 mg/ml and 0.1 cm, respectively. S6 andS7.

Figure S8

. Kinetic analysis of OCP thermal recovery data at various concentrations suggests that oligomerized OCP R reverts more slowly to the dark-adapted state. Plots of the pre-exponential factors retrieved from fits of thermal recovery kinetics (see also Fig. S7, Table S6 andTable S7) when four exponential components are used to account for the change in absorbance at 467 nm of OCPR27L-Ntag (A) and OCPwt-Ntag (B) data, and three exponential components for that of OCPwt-Ctag (C). The apparent lifetime of OCP R state is increased in dimers and higher-order oligomers, suggesting that oligomerization stabilizes the light-adapted state. Table S8: Fitted parameters extracted from nanosecond transient absorption data. Percentage contributions in the table are calculated as a given pre-exponential factor divided by a 1 + a 2 + a 3 + a 4 . Measurement of C-tag at 22 °C was repeated twice to confirm repeatability (note replicated row in the table).

In vivo protein crystallization in Bacillus thuringiensis

Despite all the research progress aiming to understand the precise toxicity mechanism of Bt Cry and Cyt toxins, many details concerning their mode of action remain poorly understood, and among questions remaining open are the following: (i) What are the factors driving crystal formation, stability and dissolution in vivo? (ii) By which mechanism do toxins assemble on the membrane surface? (iii) What is the structure of the toxic pore responsible for larvae death?

A major bottleneck towards understanding of Bt Cyt and Cry mode of actions remains the lack of structural information on these. Solving structure from in vivo-grown in Bt toxin crystals remains difficult in conventional X-ray crystallography, as the minute size of the crystals make them unsuited for conventional crystallography and most toxins do not recrystallize after dissolution of the natural crystals.

5.1.A. In vivo crystallization of Cyt1Aa protoxin: the role of domain-swapping (DS)

In the past, it was demonstrated that Cyt1Aa is unable to form crystals of its native protoxin form when expressed in E. coli cells 174 . The growth of in vitro crystals became possible only after the toxin has been subjected to a proteolytical cleavage of its N-terminal propeptide. Thus, only the protease-resistant core protein (active toxin) was crystallized.

In this work, we studied the features that govern in vivo crystallization of Cyt1Aa in Bt, and whether or not the N-terminal propeptide plays a role in Cyt1Aa protoxin crystal formation and stability. To do so, we produced in vivo-grown nanocrystals of the native Cyt1Aa in Bt, and these were used for structure determination using SFX. Our SFX structures demonstrated the presence of 37 residues in the N-terminus of the toxin, corresponding to the N-terminal propeptide, involved in the crystallization of Cyt1Aa as a domain-swapped (DS) dimer. The structure revealed, that the N-terminal propeptide buries ~70% of its surface area across interactions which involve H-bonds, salt-bridges and a disulfide bridge at position C7. These interaction zones are contributed from residues absent in the activated toxin structure, and point to the DS dimer being a key element for Cyt1Aa crystallization in Bt.

To date, various DS proteins have been identified 211 . For some of them domain swapping appears to be physiologically relevant as it enhances specific biological functions (e.g. facilitates receptor/ligand binding, membrane insertion, etc.). For Bt, the production of toxins in the form of crystals ensure a long-term storage of its main virulence factors so that they remain functional until they are ingested together with the spores by the insect host. Intriguingly, some Bt toxins can crystallize their protoxin form under non-physiological conditions, i.e. in vitro, such as the Cyt2Ba protoxin which presents a similar to Cyt1Aa 3D structure (~30% of sequence identity). Structural studies have already showed that the crystal lattice of in vitro Cyt2Ba crystals is driven by a DS interface (contributed by its N-terminal propeptide), however its biological function remains yet unascertained 212 . Presumably, the difference in the crystallization properties of both toxins, i.e.

Cyt1Aa and Cyt2Ba, involves the presence of the folding chaperone P20, being absent in Cyt2Ba.

As structural modelling studies 212 showed that P20 shares structural homology with the activated form of Cyt1Aa, one can hypothesize that P20 may serve as a scaffold for the formation of the DS interface in Cyt1Aa protoxin crystals.

5.1.B. Cyt1Aa crystal dissolution: the role of the disulfide bridge and the DS interface

In the mosquito larvae midgut, the release of toxins is controlled by crystal dissolution, the latter occurring in highly alkaline conditions (pH ~ 10-11). Our study demonstrated that a disulfidebridge, contributed by C7 of the N-terminal propeptide, and an interface #3, are both involved in crystal dissolution of Cyt1Aa under alkaline conditions. We used a reducing agent solution (pH 7) and buffering agent allowing to increase the pH up to 10, thus mimicking the midgut alkaline environment. The pH elevation up to 10.3 has prompted breakage of the C7-C7 disulfide bridge associating two protoxin monomers in the dimer, and destabilized the DS interface through loss of H-bonds between the two monomers. The evidence that the packing crystal interface #3 play a role in crystal dissolution, was supported by site-directed mutagenesis of Q168 residue (buried at this interface) and Q168E crystals presented pH sensitivity to lower pHs. The observed mechanism of crystal dissolution in Cyt1Aa crystals, differs from that in naturally occurring nanocrystals of BinAB, where no cysteine interface is involved, but still the crystals dissolution is induced by the increase of pH. In BinAB, the crystal interface is stabilized by Tyr residues, thus the increase of pH induces electrostatic repulsion between Tyr residues 213 .

Our study also showed that upon crystal dissolution an active protoxin dimer unit is released, however the presence of a monomer and decreasing amounts of trimers, tetramers and pentamers were also observed. The protoxin dimer is disulfide-bridged at position C7 and dissociates into monomers in presence of reducing agents. Intriguingly, we observed that even at the highest employed concentration of reducing agents, some residual dimers persist, furthermore suggesting a presence of higher-order oligomers. As it remains poorly understood whether or not the mosquito larvae midgut presents a reducing environment, questions about the precise form of released protoxin upon crystal dissolution in physiological conditions. However, we demonstrated that the presence of proteases allows the activation of both disulfide-bridged dimer and protoxin monomer and a 23 kDa toxin monomer forms.

5.1.C. Bioactivation pathway of Cyt1Aa

To date, two opposing models have been proposed as for how Cyt1Aa exerts its toxicity, i.e. the pore formation and the detergent models. The pore formation model suggests that active Cyt toxins assemble on and therefore bind to the cell membrane, where Cyt1Aa oligomerizes in cationselective channels leading to colloid-osmotic lysis of the cell and further death 176 . The second model suggests a detergent effect, whereby the toxin aggregates on the membrane surface and triggers lipid bilayer disassembly 177 . While the detergent effect requires neither stoichiometric toxin assemblies nor stable insertion of the toxin throughout the cell membrane, pore-formation requires partial unfolding of the toxin following conformational changes 145 . Whereas some poreforming toxins used against mammalian cells unfold upon low pH conditions 214 , in lepidopteran and dipteran insects unfolding of the toxins is triggered by the increase of pH (alkaline pH in the midgut lumen) 159 . In such cases, pre-pore and membrane inserted oligomers adopt a more flexible structural conformation compared to the monomeric toxin.

With the aim to investigate the possible oligomerization and/or aggregation of both Cyt1Aa protoxin monomer and proteolytically activated toxin, we combined mutagenesis, structural and functional studies. Our results showed that both protoxin monomer and proteolytically activated toxins form membrane-bound oligomers (MBO). In the monomeric protoxin, the N-terminal propeptide does not abrogate MBO formation, however it masks the lipid binding interface in the oligomers. This was furthermore supported by the observation that less MBO form when the protoxin was administered to liposomes, compared to the activated toxins.

Additionally, when in presence of lipid membranes, the protoxin undergoes conformational changes, which involve an opening of the structure at a αC/αD hydrophobic interface. The emitted by us hypothesis that an opening of the protein at this interface is required for MBO formation, was supported by the introduction of a E45Q mutation. Since this mutation completely abrogated the toxicity of the protoxin, we suggested that opening of the structure in at this interface is a crucial step for membrane insertion and subsequent MBO formation. Furthermore, dynamic structure studies on MBO demonstrated that these can assemble into membrane-bound aggregates (MBA), where side-by-side contact between adjacent MBA protomers would enable the transition to the porous conformation. Since our study has demonstrated that full membrane perforation is visible for holes of ~54 nm diameter (, corresponding to the assembly of ~56 monomers), only a fraction of the porous oligomers presents sufficient depth to fully perforate the membrane. This observation furthermore suggested that the structural transition between MBA and porous oligomers is independent of MBA size and involves formation of a pre-pore.

Characterization of the porous oligomers by cryo-EM demonstrated pointed on the presence of arciform oligomers, presumably resulting from from the breakdown of larger cytolytic oligomers, observed by AFM. However, our study has leaved open the question regarding the exact structure of Cyt1Aa porous oligomers. For future perspective, obtaining high resolution cryoEM structures of these, i.e. porous oligomer and/or MBA conformers could of an important interest as it can offer a better comprehension on how Cyt1Aa oligomers, may serve as substitutes for mosquito receptors of Cry toxins. In 2005, Perez et al. 184 demonstrated that Cyt1Aa can interact with Cry toxins and serves as membrane receptor for the latter with the aim to increase its toxicity 184 . The manuscript reported that synergistic interaction between Cry11Aa and Cyt1Aa triggers oligomerization of the Cry11Aa toxin on the cell membrane and formation of a 250 kDa pore-like structure. Presumably, the observed by us MBA conformers enable Cry11 toxins to dock on mosquito midgut cell membranes and subsequently assemble into toxic pores, even in absence of their specific membrane-bound receptors.

5.1.D. De novo structural insights into Cry1Aa and Cry11Ba toxins

The Cry11 family of δ-endotoxins is comprised of dipteran-active proteins including the 72 kDa Cry11Aa from Bti, the 81 kDa Cry11Ba from Bt subsp. jegathesan, and the 94 kDa Cry11Bb from Bt subsp. medelin. The Cry11 family also belongs to the large group of the three-domain toxins, whereby each domain is characterized with specific function and contributes to the stability of the protein. The domain I is a seven-𝛼-helix bundle involved in pore formation in target membranes.

Domain II forms a β-prism with exposed loops regions and it is implicated into the recognition and binding mosquito specific receptors. Domain III is a β-sandwich of two antiparallel β-sheets that are involved in oligomerization and insect specificity 161,168,169 . Among the Cry11 proteins, Cry11Aa has been extensively studied as it is the most active toxin of Bti against Aedes aegypti larvae, the transmission vectors of dengue and malaria. Cry11Ba toxin from Bt jegathesan has a mosquitocidal activity as high as the Cry11Aa and it is considered as an alternative use for mosquito control. Despite the large use of Bti as an effective biological control agent, the understanding of how Cry11Aa exerts its toxicity remains elusive, partially due to the lack of information regarding its 3D structure. Likewise, Cry11Ba structure is unknown.

In this study we obtained insights into the structure of Cry11Aa and Cry11Ba by carrying out SFX studies on in vivo-grown in the acrystalliferous 4Q7 Bti strain crystals. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. Our study showed that the building block of Cry11Aa and Cry11Ba crystals the main building block is a tetramer consisting of two dimers interacting to each other via domain II of the toxin. The dimers are themselves composed of two monomers, interacting via domains II and III. Cry 11 crystal packing is formed from brick-wall piling of tetramers, whereby domain I of symmetry-related tetramers contribute to the interaction. Thus, the Cry11Aa and Cry11Ba structures featured a yet unobserved in vivo crystallization mechanism, wherein all domains of the toxin are involved in the packing of the crystals. Intriguingly, this finding is in contrast with previously determined structures from in vivo grown crystals, in which the main contributor to the crystallization is either a specific portion of the protein, i.e. either a N-terminal and/or C-terminal propeptide 213,215 , or a specific domain 188 .

Additionally, our study has demonstrated that both Cry11Aa and Cry11Ba structures present an insertion of 36-38 residue in their domain II resulting in an extra β-strand, the β-pin, which is absent in the canonical organization observed in other Cry δ-endotoxins. The β-pin contributes to the interface that assembles dimers into stable tetramers and it is enclosed on each side, i.e. N-and C-terminal, by two flexible loops, the position of which match the mosquito gut proteases cleavage site. This suggests that the β-pin is removed upon proteolytic cleavage, thus triggering the activation of the Cry11 toxins, the latter monitored by the generation of two protein fragments, i.e. ~32 and ~36 kDa. Our finding suggested that the βpin is likely involved in in vivo crystallization of Cry11 toxins and its removal may result in the dissociation of tetramers into dimers and into monomers.

Most likely, the increase of pH in the mosquito larvae midgut will favor electrostatic repulsions between residues in this region and will lead to an increased disorder in the latter, thus facilitating the access of midgut proteases and the activation of the toxins. Our study furthermore questioned about the crystal dissolution pathways of Cry11Aa and Cry11Ba. As structure determination of Cry11Aa and Cry11Ba at alkaline pHs was unsuccessful, due to the increased sensitivity of crystals diffraction quality at high pHs, we opted for site-specific mutagenesis to explore this phenomenon. Our results showed that the crystal interface enabling the pilling of Cry11 tetramers is the one which is more affected by the increase of pH, whereas the dimerization and tetramerization interfaces, as well as the three-domain interface within the Cry11 monomer are less pH-sensitives.

As in this study, we focused solely on the structural study of Cry toxins, toxicity tests on Cry11Aa and Cry11Ba were not performed. Going forward, an additional study which could be of interest is that of the synergistic interaction between Cyt1Aa and Cry11 toxins.

5.1.E. Attempts of an in vivo recombinant crystallization of OCP in Bacillus thuringiensis

In structural biology, the use of in vivo-grown protein crystals is of particular interest not only because crystals contain the native protein, formed in physiological conditions, but also because this approach suppresses the need for protein extraction, optimization of protein purification protocols, and in vitro crystallization screening assays 216 . An often-asked question however is whether in vivo crystallization is reserved to few proteins, or if by developing appropriate experimentally-designed conditions, it could be used for the production of crystals of other proteins. In vivo recombinant crystallization of non-naturally crystalline proteins has already been successfully achieved in plant, animal, insect, and bacterial cells, among which Bacillus thuringiensis (Bt). Previously published studies as well as recently ours, have demonstrated that this spore-forming bacterium has the ability to produce a large array of protein toxins characterized with contrasting size and shape, and the crystallization of each of these toxins is achieved through finely regulated crystallization pathways 162,217 .

In this context, a significant part of my work was devoted to the development and further optimization of an in vivo recombinant crystallization strategy, enabling the production of calibrated, nanometer-sized crystals of OCP. In the context of these developments, other cargo proteins were tested (see Materials and Methods, Table 2).

To facilitate OCP crystallization in Bt, a co-crystallization approach was used, wherein Cyt1Aa and Cry11Aa were used as crystallization chaperones. At a first glance, the only promising result was obtained when the first 50 residues of Cyt1Aa (referring to the N-terminal propeptide) were fused to the full-length sequence encoding-gene for OCP Planktothrix. While expression of recombinant fusion protein was confirmed by Western blot (Figure 32A), scanning electron microscopy imaging reported the absence of crystal formation. Instead, presence of aggregates and heterogeneous population of both spores and bacteria were observed. The absence of recombinant crystals can be interpreted in least two possible ways. i.e. either the presence of an exogenous protein affects the sporulation mechanism of Bt during which the bacterium form crystals, or the inability of OCP to fold, in absence of a ketocarotenoid pigment (Figure 32B)). This last hypothesis can be furthermore supported by previously reported studies where apo-OCP was produced as aggregate and could not form crystals in vitro. The reconstitution of the carotenoid synthesis was thereafter solely studied before proceeding to further attempts of holo-OCP expression in Bt. The β-carotene and carotenoid encoding genes were successfully inserted in the pBADBt-Crt-CrtO cloning vector designed in our laboratory, wherein carotenoid synthesis was under the control of the arabinose dependent pBAD promoter.

Thus, reconversion of carotene to carotenoid was triggered by adding arabinose in the culture media. Carotenoid extraction, followed by migration on thin-layer chromatography showed the expression (even low) of β-carotene (Figure 32C), however no visible band corresponding to ketocarotenoids expression was observed. Intriguingly, previous study 218 reported that arabinose induced regulation in Bt is not possible, and have suggested that pBAD arabinose promoters can be replaced by either an "hyper-spank" IPTG inducible promoter or a Xylose promoter "Xyl", both found in B. subtilis. The idea of replacing pBAD promoter with hyper-spank or Xyl promoter was envisaged and strongly encouraged, however due to delayed progress of the project, no further experiments were held and the effect of the promoter on carotenoid expression was not studied.

It has been previously suggested that carotenoid synthesis may occur simultaneously with Bt toxin synthesis, in cyanobacteria. This hypothesis was furthermore supported by the successful recombinant expression of Cry4Ba in Synechococcus PCC 7002 cells. Intriguingly, the cry4Ba gene expression was controlled by a cyanophycocyanin (CPC) promoter 219 , the latter encoding for a pigment-protein gene complex from the light-harvesting phycobiliprotein family. This finding, as well as others [220][221][222] , may bring important information and subsequently help in the identification and optimization of suitable promoters for OCP or OCP-like protein expression in Bt.

In addition to OCP, we studied the recombinant expression and the in vivo crystallization of the paralog of the CTD domain of OCP, the CTDH. As previously published, CTDH can form crystals in vitro, and in absence of a bound ketocarotenoid pigment. This finding has furthermore suggested, that in CTDH carotenoid binding is not required to prompt protein stability. As for wt OCP, recombinant expression of CTDH as a fusion construct with the N-terminal propeptide of Cyt1Aa, did not yielded crystal formation. In line with this result, we have suggested that both the recombinant expression crystallization of OCP in Bt, does not solely depends on the presence of functional carotenoid biosynthesis in Bt cells, but most likely relies on the Bt gene expression machinery.

Despite the extensive efforts and investment that were made in order to succeed the in vivo OCP crystallization in Bt, the current strategy was not successful. Yet it has allowed us: (i) to identify other Bt toxins that can be potentially used as crystallization chaperones, (ii) to select and carefully identify other protein sequences that can be recombinantly crystallized in Bt, (iii) to combine genetic elements from various toxin genes and operons, thus enhancing the expression of new fusion constructs, as well as (iii) to identify the role played by some facultative folding chaperones in crystal formation and stability. Our strategy was furthermore optimized based on these criteria and successfully allowed the construction of 76 fusion vectors. Cyt1Aa, Cry11Aa as well as the Cry1Ae toxin from Bt alestii were used as crystallization chaperones and fused to different protein sequences (Material and Methods, Table 2). In particular, the Bt Cry1Ae toxin presents a protoxin region in its C-terminus, which has been previously proposed to be responsible for toxin crystallization 223 .

To date, all 76 fusions were successfully constructed and their sequences validated, and some of them were tested for in vivo crystallization in Bt. At this stage of the study, a successful crystallization was not yet achieved, however preliminary results strongly suggest that another Bt protein, called Bti152, could enhance the crystallization of recombinantly expressed proteins in Bt.

The Bti152 is essential for preserving the integrity of the parasporal body in which the crystals are harbored 224 .

Structural and functional insights into the photoactivation and recovery of OCP

5.2.A. Structural characterization of OCP from Planktothrix agardhii

With the aim to characterize the excited state structure of OCP, formed ps after absorption of the photon, and as well as to elucidate the conformational changes that permit and/or forbid productive photoactivation, a main part of my thesis project was to first structurally characterize the OCP from Planktothrix agardhii using conventional crystallography. In this context, we solved the darkadapted (OCP O ) ground state structure, by collecting diffraction data using conventional crystallography at 100K and phasing those data by molecular replacement, using as a starting model the previously published structure from the cyanobacteria Synechocystis PCC6803. We crystallized the structures of OCP O featuring either echinenone or canthaxanthin as functionalized pigment (in collaboration with the Prof. Ilme Schlichting (MPI Heidelberg, Germany)).

The structure of OCP Planktothrix was solved in two space groups, i.e. P21 and C2, with a dimer and a monomer in the asymmetric unit, respectively. Since the C2 symmetry allows the formation of a crystallographic dimer, in both cases OCP O was present as a dimer. This finding is in line with the presently available crystal structures of three other members of OCP1 clade, i.e. the OCPs from Synechocystis PCC6803, Arthrospira maxima and Tolypothrix.

In the P21 structures of OCP, irrespective of the bound carotenoid, the dimerization interface buries ~70 % of the BSA and it is supported by the contribution of N-terminal helices αA (NTE) and αB.

Intriguingly the structural data have shown that the contributions of NTE and αB to the to the dimerization interface depend on the bound ketocarotenoid. Indeed, when functionalized with canthaxanthin a more stable contribution was observed as compared to echinenone. The dimerization interface is contributed by conserved interaction, among which the previously described salt-bridge between D19-R27. This observation is in line with all previously published structures of OCP members from clade 1. In all these, the salt-bridge is preserved, suggesting its role in in the naturally-occurring OCP dimer. Intriguingly, in OCP-CAN structure, two additional H-bonds are observed (R9 -Q30 and R9 -L31) which fix αA and αB helices between two facing monomers in the dimer. Since, these are suppressed in the structure of OCP-ECN dimer, the NTE is less constrained at the dimerization interface in the OCP-ECN structure than in the OCP-CAN structure. Comparison between OCP and with other available OCP crystal structures, pointed out that the extent of the biological dimerization interface is overall preserved, with a mean BSA of 1051 ± 90 Å 2 .

Intriguingly, our structural data demonstrated the presence of an additional large interface which was absent in previously characterized OCP structures. This additional interface, that we coined interface X, involves multiple secondary structure elements, among which the αC-αD loop (which contributes with ~ 20 and 36 % of the overall BSA) and αD (10% -20%). In P21 crystals these two structural elements contribute H-bonds at interface X, whereas in C2 all structural elements are involved in this interface. Thus, the changes in crystalline packing are related to changes in the opening-angles at the dimerization and NTD/CTD interfaces. The P21 structures have demonstrated that within the dimer, the two monomers adopt different structures, which differ in the opening angle at the NTD/CTD interface, in the orientation of the carotenoid in the tunnel. In the C2 structures, however the positioning of helices around the carotenoid and the conformation displayed by the αC-αD and αG-αH loops were different as compared to P21. Changes in the conformations of these two loops result in the opening and closing of water-channels from the protein surface to the carotenoid tunnel in the NTD. In biological context, the αC-αD loop is central as for the interaction between OCP R and PBS (Wilson et al., 2021). With all these features the structure of OCP Planktothrix offer a peek into the molecular breathing motions that animate the protein at monomer and dimer level.

whereas in presence of ECN up to 50% of ICT was formed. As CAN is fully symmetric compared to ECN, the absence of ICT state was proposed to be a consequence of the symmetry nature of the CAN pigment. Our study reported that irrespective of the species, CAN-functionalized OCPs are characterized with an increased photoactivation rate, but a reduced recovery rate, binding to PBS and energy-quenching activity. Differences have also been observed regarding the formation yields of the excited states, without clear changes on their characteristic lifetimes. Intriguingly, in CAN functionalized OCPs, an increase of the S* yield has been observed, which was not mirrored by an increase in the P1 yield. This finding suggests that S* is not the precursor of the first photoproduct P1, which opposes previously published results (ref). Differences in the P1 yield were observed when data were recorded on a longer time-scale (ns-s). Indeed, the yield of P1 was higher for CAN-functionalized OCPs, which could be related to a decreased recovery of the OCP O state. However, the decreased absorbance signal observed at 565nm, i.e. monitoring formation of OCP R , for OCP Planktothrix on the ns-µs time-scale could sign for a sub-optimal translocation of ECN and CAN into the NTD, as compared to the NTD of Synechocystis.

Altogether, the results from our study suggest that: (i) carotenoid translocation and NTE/CTT detachments are affected by the change in protein scaffold, whereas it is domain dissociation that is most affected by a change in the functionalizing carotenoid. The domain dissociation is faster, as well as more efficient when OCP is functionalized by CAN. In fact, OCP Planktothrix functionalized with CAN is characterized by faster domain separation and 100% conversion from P3 to final OCP R on the µs-s time scale. Altogether, these results could explain the previously observed high-efficiency of OCP Planktothrix in the photoprotection, i.e. NPQ-related mechanism.

5.2.C. High-throughput purification of OCP: preparation of gram-quantities for TR-SFX experiments

The most critical step in the accomplishment of a successful crystallographic experiment resides in the availability of highly pure protein samples. In serial crystallography experiments, i.e. SSX and SFX, data collection call for the development and/or optimization of new and already existing crystallization methods, respectively, to tailor a particular sample delivery approach. Most SFX experiments use injector-based sample delivery systems, e.g. GDVN injectors, which call for the production of large volumes of homogeneous sample with an optimal crystal size to ensure delivery with high crystal hit rates. This is usually challenged by the need of high sample consumption. Typical amount of protein required for full data collection ranges from hundreds of milligrams up to few grams depending on crystal size and injection method. The production of gram amounts protein as required, e.g. to conduct a TR-SFX experiment on microcrystals injected by a GDVN, makes protein expression and purification steps defiant, especially in the case of pigment-binding proteins like OCP, in which pigment and protein synthesis are a multi-stage process.

As our attempts to produce calibrated in vivo grown microcrystals of holo-OCP Planktothrix were unsuccessful, we opted for an in vitro crystallization approach. In this context, several months of expression and purification were necessary to produce the required protein quantity, i.e. ~5 grams.

OCP Planktothrix binding canthaxanthin was produced recombinantly in E. coli using already published experimental protocols 189 , which generally enable the production of 30 mg of holo-OCP from 1L of bacterial culture. However, the production of large quantities of OCP required to increase drastically the amount of bacterial culture volumes, which had a negative impact on the protein preparation quality. Indeed, only 10-20% of the protein yielded the holo-OCP form, whereas the remaining 80-90% corresponded to the apo-form of the protein (apo-OCP form aggregates). Since apo-and holo-OCP can interact to form mixed dimers, they must be separated, otherwise residual apo-form can influence crystal formation of holo-OCP. The generally used approach which allows the removal of apo-OCP is the hydrophobic interaction chromatography (HIC), the principle of which is to separate proteins according to their hydrophobicity. By the addition of salt and subsequently increasing its concentration, hydrophobic interactions between the protein and the resin matrix are enhanced. During standard HIC purification of OCP, small prepacked columns are generally used, whereby adding 2M salt (NaCl) strengthen apo-OCP binding onto the resin matrix. Subsequent decrease of the salt concentration (0.5M) enables the separation of both OCP forms, with only holo-OCP being eluted. This standard protocol, however is not optimal in case where hundreds of milliliters of protein solution are produced, as it implies the use of up to hundreds of hydrophobic columns at high cost. Indeed, the produced amount of apo-OCP is so high so that it also triggers an irreversible precipitation of apo-OCP on the column, rendering the column unusable at 2 or 3 utilizations.

We overcame this problem, by developing a purification approach which allowed to remove in total more than 90 % of the apo-OCP and produce highly pure holo-OCP. The purity was assessed by measuring the holo-to apo-OCP ratio, i.e. when the ratio was superior to 1.6 the protein solution was considered as highly pure. In this regard, after elution on Ni-NTA, large volumes of OCP protein solution were recovered and first dialyzed against 40 liters of buffer containing 5M salt, enabling precipitation of apo-OCP. For this step, we used black garbage cans as these were the largest suitable receptacles we could find. Usage of these allowed preserving the protein from the ambient light. After dialysis, the supernatant corresponding to holo-OCP was recovered and subsequently dialyzed against 70L of 150 mM NaCl. At this stage, still around 10-20 % of apo-OCP was present, thus the protein solution was further purified using HIC. Because of the comparatively low amount of apo-OCP, only few columns were needed for the complete purification.

Our purification approach using salt induced precipitation is a commonly used method for protein purification [225][226][227] . Nevertheless, as protein solubility can vary, there is no 'one size fits all' strategy for developing this approach. Indeed, various parameters, e.g. pH, hydrophobicity, salt type and concentration can govern protein solubility.

5.2.D. Light-adapted OCP forms dimers and higher-order oligomers in solution

Whether or not OCP oligomerizes in its dark-and light-adapted states was until recently largely overlooked. Within crystals, OCP features a dimer, however it remained elusive if the crystallographic dimerization interface was functional or artificial, due to the high protein concentration in crystals (~20-30 mM). Recently published results reported that a mutation of a single conserved residue (R27) among the 22 making up the dimerization interface abolishes protein dimerization in vivo. This finding suggests that oligomerization could play an important role in biological context. Previous studies have already reported on the oligomerization state of darkadapted OCP (OCP O ) and confirmed that dimers can form at concentrations as low as 3 µM in vitro. The light-adapted OCP R state, however, demonstrates significantly different oligomerization behavior, as previously published results showed presence of both monomers and dimers, in solution.

In this context, our study aimed to elucidate the oligomerization states of both OCP O and OCP R in solution, using static X-ray scattering. The data were collected from two OCP variants, a wild-type and a monomeric mutant, whereby a concentration-dependent study was performed for each in the dark and in the light-adapted states. Our results confirmed the presence of an OCP O dimer in vitro, previously suggested in the literature, and indicate that it becomes predominant at concentrations ≳ 28 µM. Moreover, we demonstrated that OCP R accumulated by continuous illumination also tends to oligomerize as the scattering intensity at zero angle I(0) indicated clear difference in the molecular weight of both protein variants compared to their OCP O states.

Remarkably, both proteins in the OCP R and OCP R state displayed similar radius of gyration and maximal dimension, suggesting that they form the same OCP R state(s).

Changes in the scattering patterns of OCP R at high concentration could have stemmed from structural changes, e.g. domain dissociation, and not necessarily oligomeric state transitions [START_REF] Gupta | Local and global structural drivers for the photoactivation of the orange carotenoid protein[END_REF] .

The molecular envelopes of OCP R reconstructed using ab initio bead modeling, demonstrated the presence of OCP R dimers and higher-order oligomers, i.e. either OCP R trimers or OCP R tetramers or a mixture if both, forming at 20 µM and 300 µM, respectively. The observed result, was not in full agreement with previously reported studies proposing that OCP R resides in the same oligomerization state, i.e. a dimer, at both 1.8 mM and 65 mM 70 . Intriguingly, the monomeric mutant also formed OCP R dimers and tetramers, thus strongly suggesting that the dimerization interfaces in OCP O and OCP R differ and do not overlap. According to this hypothesis, and based on prior knowledge from the literature, we built models for OCP R dimers and higher-order oligomers, i.e. trimers or tetramers, assuming that: (i) OCP R higher-order oligomers are formed either through association of two coalescent dimers (tetramers) or one OCP R monomer and one OCP R dimer (yielding trimers), (ii) the interactions of monomers in the dimer and of dimers and/or monomers in the higher-order oligomers involve the same oligomerization interface, (iii) the residues involved in OCP R dimers and higher-order oligomers assembly are not the same as in the dark OCP O dimer, and iv) that OCP R dimers assemble via their CTDs, so that their two NTDs are available for interaction with PBS via R155. Since possible interactions between two NTDs and two CTDs have been already observed in the crystal structures of HCP and CTDH homologues, respectively, we furthermore suggested that OCP R higher-order oligomers form from an interaction between two NTDs using an interface similar to that observed available crystal structures of OCP-NTD homologous. The proposed model of OCP R dimer was supported by a recently published structure of the quenched cyanobacterial OCP-PBS complex [START_REF] Dominguez-Martin | Structure of the Quenched Cyanobacterial OCP-Phycobilisome Complex[END_REF] . The structure showed that OCP R is present as a dimer which assembles through CTD-CTD interaction. The residues involved in the dimerization interface are conserved in OCP, however in OCP O they are hidden from the bulk by interaction with the NTE.

Additionally, we investigated the structural and thermal recovery of OCP O using spectroscopy. Our proposed model, illustrated in Figure 7 (Results Chapter 4, Section 4.2) informs on four molecular steps involved in OCP R to OCP O recovery. The first step refers to the transition from monomeric OCP R to OCP O , characterized by the shortest characteristic time, i.e. 50-100 s. The second step is characterized by longer characteristic time of 60-600 s and a dissociation constant in the order of 30-40 µM, and corresponds to the dissociation of OCP R -dimer into two OCP R -monomers. The dissociation of OCP R -higher-order oligomers into OCP R -dimers occurs on even longer time-scale (640-950 s) and present a dissociation constant in the order of ~ 280 µM. However, the Furthermore, our study suggested that the first step identified in our transient absorption spectroscopy and TR-SAXS/WAXS experiments, i.e. visible signal at 2-3 µs and 15 µs, respectively refers to the P2' to P3 transition. This finding agrees with already published results from Konold et al. [START_REF] Konold | Photoactivation Mechanism, Timing of Protein Secondary Structure Dynamics and Carotenoid Translocation in the Orange Carotenoid Protein[END_REF] , demonstrating partial recovery of OCP O from P2'. Since the next spectroscopically-visible step occurs on the ~1 ms time scale, only limited recovery of the OCP O state occurs on this time-scale. In line with this, we suggested that this spectroscopic step can be referred to a back-migration of the carotenoid into the tunnel, and occurs in a fraction of P3 intermediates. TR-SAXS data suggest that depending on whether the his-tag is present at the Nor C-terminus, the dimeric P3 intermediate reverts to the OCP O state, with characteristic times of ~50 and ~200 ms, respectively (Results Chapter 4, Section 4.2, Figure 8). Thus, structural recovery follows the back-migration of the carotenoid on the 1 ms time scale. Our study showed that the last visible spectroscopic transition occurs on the time-scale of hundreds of ms (200 ms) and is characterized with the same activation energy as that occurring on the ~1 ms. We proposed that this step signs for the back migration of the carotenoid into the carotenoid tunnel, through the formation of an intermediate wherein the NTE and the CTT have detached from the CTD. In our TR-SAXS/WAXS data, large conformational change related to detachment of the NTE and/or CTT were not visible which opposes previous findings suggesting that these occur on the ~ 1 ms time scale. One possible hypothesis is that these structural steps occur either later in the photoactivation mechanism, or that TR/SAXS method is not enough sensitive to detect such small structural changes. As detachment of NTE is a prerequisite to OCP O dimer dissociation, the formation of P3 must occur in each of the two monomers, whereby the dimeric P3 will fall apart into its monomeric counterpart, before the reaction can further proceed. Given the low quantum yield of OCP, and in line with the finding that P3 starts to recover the OCP O state on the time scale of 50 and ~200 ms, an optimal photoactivation of dimers would be achievable if one photon is delivered per chromophore every few ms.

Introduction

La photosynthèse est un processus chimique naturel, au cours duquel l'énergie solaire est convertie en pouvoir réducteur et en énergie chimique. Cependant, lorsque la quantité d'énergie lumineuse excède la quantité d'énergie gérable par la cellule photosynthétique, la production des espèces réactives de l'oxygène (ERO) a lieu au sein de la cellule, qui peuvent endommager l'appareil photosynthétique et provoquer la mort cellulaire. Pour se protéger, les organismes photosynthétiques, i.e. les plantes et les cyanobactéries, ont développé des mécanismes de photoprotection réversibles qui permettent la dissipation de l'excès d'énergie sous forme de chaleur. Chez les cyanobactéries cette fonction, dite d'extinction non-photochimique repose sur l'Orange Caroténoïde Protéine, (OCP), une protéine à deux domaines stabilisés par la présence d'un pigment caroténoïde à leur interface. Dans l'obscurité, la protéine est inactive (OCP O ), mais en cas d'illumination forte par de la lumière bleue-verte, elle sera photo-activée en une forme rouge (OCP R ) seule capable de se lier aux antennes collectrices de lumière chez les cyanobactéries, i.e. les phycobilisomes (PBS), et de dissiper l'excès d'énergie collectée par ceuxci sous forme de chaleur. L'OCP attire une attention toute particulière du fait qu'elle est non seulement impliquée dans la photoprotection des cyanobactéries, mais elle joue aussi un rôle crucial dans la régulation des ERO. Le mécanisme de photo-activation de l'OCP a été extensivement étudié par différentes approches de spectroscopie, mais l'ordre précis dans lequel ont lieu les changements structuraux au sein du caroténoïde et de la matrice protéique, respectivement, reste à ce jour méconnu et débattu. Ces changements ne peuvent être compris que par une étude globale -spectroscopique et structurale -sur des échelles de temps allant de la centaine de femtosecondes (le temps de vie du premier état excité du caroténoïde) à la minute (le temps de vie de la forme active OCP R ). Cette thèse a visé à obtenir une meilleure compréhension du mécanisme de photoactivation de l'OCP d'un point de vue structural. A dessein, nous avons utilisé deux méthodes complémentaires de la biologie structurale : (i) la diffusion des rayons X aux petits (SAXS) et aux grands (WAXS) angles, et (ii) la cristallographie aux rayons X. Chacune de ces méthodes a été utilisée dans son mode statique et résolu en temps. Spécifiquement, la diffusion des rayons X a permis de renseigner sur la structure à basse résolution de l'OCP R (en utilisant le variant produit par la cyanobactérie Synechocystis PCC6803) et sa capacité à oligomériser, ainsi que de déterminer les échelles de temps (µs-min) sur lesquelles ont lieu les changements structuraux larges, permettant d'aboutir à cette forme photoactivée de la protéine. La cryo-cristallographie aux rayons X au synchrotron a permis de résoudre plusieurs structures du variant d'OCP le plus rapide ayant été caractérisé à ce jour, i.e. celui produit par la cyanobactérie Planktothrix agardhii. Pour obtenir ces structures, nous avons utilisé le rayonnement synchrotron de l'ESRF, et la méthode d'oscillation appliquée à des cristaux cryoprotégés par trempe ultra-rapide dans de l'azote liquide.

Nous avons également visé à obtenir des informations à résolution atomique sur les toutes premières étapes de la photoactivation de ce variant de l'OCP, i.e. la formation des états excités et leur devenir. Pour ce faire, nous avons eu recours à la cristallographie sérielle à l'échelle de la femtoseconde, réalisable sur des microcristaux dans des installations de type laser à électrons libres (ou XFELs). En effet, comme les toutes premières étapes de la photoactivation se déroulent sur des échelles de temps très courtes (fs-ps), une étude structurale en utilisant les faisceaux de rayons délivrés par les synchrotrons n'est pas adaptée, ces installations étant limitées à une résolution temporelle de 100 ps. Ainsi, nous avons pu caractériser les intermédiaires structuraux de durée de vie extrêmement courtes qui se forment lors de la photoactivation d'OCP. Nous avons pour cela dû utiliser de très petits cristaux, afin d'assurer une pénétration suffisante de la lumière actinique et donc maximiser l'occupations desdits états intermédiaires. Spécifiquement, dans le cas de l'OCP, la profondeur de pénétration de la lumière à 530 nm (i.e. la distance à laquelle la

Préparation de larges quantités d'OCP pour des expériences de cristallographie sérielle résolue en temps

Quoique nos tentatives de produire des microcristaux de l'OCP in vivo aient échoué, nous n'avons pas abandonné l'idée de contribuer à une meilleure compréhension du mécanisme de photoactivation de l'OCP, par la résolution des structures des états intermédiaires (et notamment les états excités) se formant sous illumination pulsée. Ainsi, nous nous sommes rabattus en milieu de thèse sur la cristallisation in vitro. Dans ce contexte, plusieurs mois ont été consacrés à l'expression et la purification de la quantité de protéine requise, environ 5 grammes. L'OCP Planktothrix a été produite de façon recombinante dans E. coli en utilisant des protocoles expérimentaux déjà publiés. Généralement, ces protocoles permettent la production de ~30 mg de protéine à partir d'un litre de culture bactérienne. Cependant, la production de grandes quantités d'OCP a nécessité une augmentation drastique des volumes de culture, ce qui a eu un impact négatif sur la qualité de l'échantillon. En pratique, seul 10 à 20% de la protéine était produite sous la forme holo-OCP, i.e. complexée avec un caroténoïde, tandis que les 80 à 90% restants correspondaient à l'apo-OCP. Puisque l'apo-et l'holo-OCP peuvent interagir formant des dimères mixtes, ils devaient être séparés pour éviter que l'apo-forme résiduelle n'influence négativement la pureté de nos préparations d'OCP, et donc la qualité de nos cristaux. Pour faire face à ce défi, nous avons revisité une approche de cristallisation ancienne, basée sur la précipitation induite par le sel. Cette méthode a permis d'éliminer plus de 90 % de l'apo-OCP et de produire une holo-OCP extra-pure après chromatographie sur colonne hydrophobe. Cet échantillon a micro-cristallisé sans souci, et des données de cristallographie sérielle à l'échelle de la femtoseconde ont été collectées au SwissFEL, sur des échelles de temps allant de 300 fs à 100 ps, et à une résolution entre 1.6 et 1.8 Å.

Conclusion

Ce manuscrit de thèse présente une de nouvelles informations sur le mécanisme de photoactivation de l'Orange Caroténoïde Protéine. En combinant la spectroscopie et la diffusion des rayons X nous avons montré que l'OCP photoactivée peut oligomériser, et que les processus d'oligomérisation participent à la régulation de la photoactivation de la protéine. Notre étude est la première à offrir une vision des changements structuraux larges qui surviennent lors de la photoactivation d'OCP et à identifier les échelles de temps sur lesquelles ils surviennent. Nos résultats démontrent la faisabilité de telle études même dans des cas où le rendement quantique de photoactivation est faible, typiquement ~ 0.3% dans le cas de l'OCP.

Grâce à la cristallographie aux rayons X, nous avons pu déterminer la structure de l'OCP synthétisé par la cyanobacterie Planktothrix agardhii. Cette structure a permis d'établir que c'est la plus grande flexibilité de cette protéine qui sous-tend sa plus forte vitesse de photoactivation.

Dans le but de produire des cristaux d'OCP de taille nanométrique pour des études de cristallographie sérielle résolue en temps, nous avons investigué les mécanismes de cristallisation in vivo de la bactérie Bacillus thuringiensis. Ce travail nous a permis d'élucider la structure et le mode d'action des toxines Cry11Aa, Cry11Ba and Cyt1Aa.

Grâce à l'optimisation du protocole de purification présenté dans le Chapitre 2 (Matériels & Méthodes) et discuté en plus grand détails dans le Chapitre 5 (Discussion), il a été possible de produire de manière reproductible et en grande quantités l'OCP Planktothrix nécessaires aux expériences de cristallographie résolue en temps. Celles-ci ne sont pas présentées dans ce manuscrit mais feront l'objet de publications futures.

Résumé

Titre : Étude structurale du mécanisme de photoactivation de l'Orange Caroténoïde Protéine (OCP) par la diffusion aux petits angles et la cristallographie aux rayons X en temps résolue.

L'Orange Caroténoïde Protéine (OCP) est une protéine à deux domaines stabilisés par la présence d'un pigment kéto-caroténoïde à leur interface. Chez la cyanobactérie, l'OCP joue un rôle de photoprotection, et est capable de se lier aux antennes collectrices de lumière, i.e. les phycobilisomes (PBS) et dissiper l'excès d'énergie collectée sous forme de chaleur. Pour exercer sa fonction de photoprotection la protéine doit être photoactivée, par absorption d'un photon bleuvert, ce qui déclenche sa transition d'un état orange inactif (OCP O ) vers un état rouge photoactif (OCP R ). Le mécanisme de photoactivation s'étend sur 13 échelles de temps et implique des changements structuraux au sein du caroténoïde et de la matrice protéique. En vue d'investiguer ces changements, au cours de ma thèse j'ai étudié le mécanisme de photoactivation d'OCP en utilisant diverses méthodes de biochimie et de la biologie structurale. Tout d'abord, en combinant la spectroscopie et la diffusion des rayons X en mode stationnaire et résolue en temps (TR), nous avons investigué les réarrangements conformationnels à grande échelle impliqués dans la photoactivation et le retour thermique de l'OCP. Nous avons démontré que l'OCP O et l'OCP R peuvent oligomériser, et que les processus d'oligomérisation participent à la régulation du photocycle de la protéine. Deuxièmement, en utilisant la cristallographie aux rayons X, nous avons déterminé la structure de l'OCP synthétisé chez la cyanobacterie Planktothrix agardhii, auparavant non caractérisée. Notre analyse a renseigné sur comment la flexibilité structurale de cette variante influence son activité d'extinction d'énergie, ainsi que sa vitesse de photoactivation et relaxation. En utilisant la cristallographie résolue en temps, nous avons caractérisé les premières étapes de photoactivation de l'OCP. Nous avons utilisé des méthodes de cristallisation in vitro et in vivo pour produire des cristaux OCP de taille nanométrique. Nous avons investigué la cristallisation in vivo dans la bactérie Bacillus thuringiensis (Bt), à dessein d'obtenir des cristaux de fusion de l'OCP avec deux toxines de Bt naturellement cristallines, la Cyt1Aa et la Cry11Aa. En termes de production de cristaux d'OCP, ce travail a été sans succès, néanmoins il a permis d'étudier la structure et la cascade de bioactivation de ces deux toxines, ainsi que d'élucider les mécanismes moléculaires qui sont à la base de leur cristallisation in vivo.

Mots-clés : Orange Caroténoïde Protéine (OCP), mécanisme de photoactivation, diffusion des rayons X, cristallographie aux rayons X, oligomérisation, in vivo nanocristallographie, Bacillus thuringiensis

Figure 1 :

 1 Figure 1: Three-dimensional scheme of chloroplast and granum organization. (A) Chloroplast (B) Granum.

Figure 2 :

 2 Figure 2: The photosynthetic electron transport chain. In the thylakoid membranes of chloroplasts, photosystem II (PSII) is excited by absorption of a photon light energy. In the reaction center of PSII (P680) a chlorophyll molecule transiently loses an electron, which is transmitted to the plastoquinone pool (PQ). The chlorophyll is a very strong oxidizing agent, thus upon oxidation it is able to accept electrons from water. Water splitting occurs in the reaction centers, situated on the lumen side of the thylakoid membrane and results in generation of oxygen and protons. The reduced plastoquinone (PQ) move through the membrane, from PSII to cytochrome b6f (Cytb6f), where PQ is oxidized and its proton is released in the lumen. This enables transport from the stroma to the lumen. Electrons are further transferred to photosystem I (PSI), where the electron transfer allows reduction of the excited chlorophyll localized in the PSI reaction center. Upon excitation, this chlorophyll transfers its electrons to the stromal ferredoxin (Fd) which can reduce NADP + through the ferredoxin NADP reductase (FNR). The photolysis of water photolysis and the proton transport (via PQ) contribute to the establishment of a proton gradient. This gradient is used as an energy source for the generation of ATP via ATP synthase.

Figure 3 :

 3 Figure 3: Phycobilisome structure. (A) "Up-up" conformation of Synechocystis PBS. The PBS features six rod structures, wherein each rod is composed by three phycocyanin trimers (grey), stacked in hexamers and connected by linker polypeptides (orange). The PBS core is composed of three allophycocyanin cylinders, wherein each cylinder consists of four trimer APC discs. The upper cylinder is composed of APC trimers (blue), whereas the two bottom cylinders are composed of either APC trimer containing ApcD (pale violet) or APC trimer with ApcE (fuchsia) and ApcF, respectively. (B) Schematic representation of the maximum absorbance and fluorescence emission of the different phycobiliproteins composing the PBS.

Figure 4 :

 4 Figure 4: Schematic representation of carotenoid excited states. The relaxation process is represented by broken arrows and corresponding time constants. Thick and thin lines show the electronic and vibrational states, respectively. Upon excitation the carotenoid molecule is excited up to the S2 state from which it relaxes to the S1 state either by vibrational relaxation and/or internal conversion (broken arrows). The energy levels (adapted fromHashimoto H. et al., 2018 34 ).

Figure 5 :

 5 Figure 5: Illustration of the OCP-related non-photochemical quenching (NPQ) in cyanobacteria. In darkness and under low irradiance, OCP resides in its inactive dark form (OCP O ). However, high-light triggers the photoactivation of the protein and the latter is photoconverted from OCP O to the photoactive red form (OCP R ). Only the OCP R can bind to the PBS core, thus inducing the dissipation of the excess absorbed energy into heat. The detachment of OCP from the PBS core is driven by FRP. After detachment, OCP R recovers thermally to OCP O .

Figure 6 :

 6 Figure 6: Crystal structure of OCP from Arthrospira maxima. (A) Structure of dark-adapted OCP (OCP O ) dimer from the cyanobacteria Arthrospira maxima (PDB id: 5UI2). OCP crystallizes as a dimer, where the dimerization interface is supported by three conserved H-bonds between D19, R27, N134 (inset) (B)Within each monomer, a ketocarotenoid (hECN shown as spheres and coloured in wheat) pigment lodges in a hydrophobic tunnel. (C) Structures of the three carotenoid pigments, i.e. hECN (hydroxy-echinenone; coloured in wheat), ECN (echinenone, coloured in green) and CAN (cantaxanthin, coloured in ruby). (D) Each OCP monomer features two domains, i.e. an N-terminal domain (NTD; coloured in orange) and a C-terminal domain (CTD; coloured in slate. The N-terminal

Figure 7 :

 7 Figure 7: Photocycle of OCP. The different intermediates formed in the photocycle are indicated with the wavelength of maximum difference-absorbance and timescales of occurrence. The proposed photocycle is based on the spectroscopic signature of the different intermediates.

Figure 8 :

 8 Figure 8: Paralogs of the N-terminal and the C-terminal domains of the Orange Carotenoid Protein. (A) Structure of the HCP1 dimer (PDB id: 5FCX 88 ) from the cyanobacteria Nostoc 7120. Each HCP monomer binds noncovalently a CAN pigment. The two monomers are represented in different coulour for clarity. The bound CAN in each monomer is shown in stick. (B) Structure of CTDH from the cyanobacteria Nostoc 7120 (PDB id: 6FEJ[START_REF] Muzzopappa | Paralogs of the C-Terminal Domain of the Cyanobacterial Orange Carotenoid Protein Are Carotenoid Donors to Helical Carotenoid Proteins[END_REF] ). Two dimer interfaces are be observed, i.e. a "back-to back "dimer, also called A-type, and a "head to head" dimer or F-type. In the A-type, dimerization occurs through the CTD interface buried between domains in the OCP O monomer. This interface was proposed to underlie dimerization of the isolated OCP-CTD. In the F-type dimer, the residues involved in the formation of the carotenoid tunnel in OCP-CTD, face one another at the dimerization interface. This interface was proposed to yield the functional CTDH dimer involved in carotenoid transport. (C) Structural alignment of CTDH monomer (coloured in wheat) and Synechocystis OCP-CTD (coloured in pale cyan) (PDB id: 3MG1[START_REF] Poddar | A guide to time-resolved structural analysis of light-activated proteins[END_REF] ). Both

  the position of their C-terminal tail (CTT). The CTT adopts a close position in CTDH, whereas in OCP O the CTT adopts an open conformation.

Figure 9 :,

 9 Figure 9: Scheme of an X-ray scattering experiment. An incident X-ray beam with wavelength λ and momentum 𝑘 0 111⃗ , where 3𝑘 0 111⃗ 3 = 45 6 , interacts with the sample and is deflected with an angle 2θ and momentum 𝑘 7 1111⃗ . The momentum transfer 𝑞 ⃗ = 𝑘 7 1111⃗ -𝑘 0 111⃗ defines the so-called scattering vector whose amplitude is 𝑞 = |𝑞 ⃗| = 45 6 𝑠𝑖𝑛 𝜃.

Figure 10 :

 10 Figure 10: Molecular parameters calculated from small-angle X-ray scattering data: (A) The radius of gyration Rg, derives from the lowest angle using the Guinier approximation in equation 2. (B) The pair-distribution function P(r) of a molecule is obtained by applying the indirect Fourier transformation on the experimental scattering data. The P(r) yields the maximum dimension (Dmax) of the particle.

Figure 11 :

 11 Figure 11: Ab initio shape reconstruction of monomeric OCP O from SAXS profiles. (A) P(r) profile and Guinier plot (inset). (B) Ab initio dummy atom model of OCP monomer (surface coloured in red) superimposed with the monomeric OCP structure, the latter generated from the crystallographic OCP dimer (PDB id: 3MG1 105 ) using PyMOL. (C) A fit between the experimental data and the obtained bead model informs about the goodness of the fit.

Figure 12 :

 12 Figure12: Schematic of the "pump-probe" data collection approach used in TR-SAXS/WAXS experiments. A protein solution (red) flows through the interaction region, where it interacts with a laser pulse (pump; blue) synchronized with an X-ray pulse train (probe; gray). Both pump and probe are characterized with a certain time-delay between arrivals at the sample. Protein structural changes give raise to changes in the X-ray scattering pattern measured on a CCD detector. (Adapted fromPoddar H. et al., 2021 92 )

Figure 15 :

 15 Figure 15: Illustration of X-ray diffraction experiment.

Figure 16 :

 16 Figure 16: Schematic of the workflow for structure determination by X-ray crystallography

Figure 17 :

 17 Figure 17: Sample delivery methods for serial crystallography experiments (figure from Johansson L.C. et al., 2017 136 ). (A) Gas-dynamic virtual nozzle (GDVN). (B) Lipidic cubic phase injector. (C) Drop-on-demand. (D) Fixed target.

  cell development comprises two different phases: (i) a vegetative phase during which cell division occurs and (ii) spore development or also referred to as the sporulation phase. The vegetative Bt cells are rod-shaped and round-ended, and their length and diameter range from 2-5 µm and 1.0 µm, respectively. During the vegetative phase, the cell divides into two uniform daughter cells following formation of a division septa (DS) that forms along the plasma membrane. Sporulation, however, is characterized by an asymmetric cell division and comprises seven different stages, including: (i) axial filament formation (stage I); (ii) forespore septum formation (stage II); (iii) first appearance of parasporal crystals (stage III) and formation of a forespore; (iv) formation of exosporium primordial cell wall, cortex and spore coats accompanied by transformation of the spore nucleoid (stages IV to VI); and (v) spore maturation and lysis (stage VII). The toxins produced by Bt during sporulation accumulate in the mother cell as parasporal crystals that account for 20 to 30 % of the sporulated cell dry weight.

Figure 19 :

 19 Figure 19: Mode of action of B. thuringiensis (Bt) toxins. (A) Cry and Cyt toxins are produced as parasporal crystals during the sporulation of Bt. Briefly, crystals are ingested by an insect larva, and dissolve in the insect gut upon the increase of pH. Upon dissolution, the crystals release protoxins (inactive form of the toxin). Protoxins are activated through proteolytic cleavage by midgut digestive enzymes, which consists in scission of the toxin propeptide located either in the N-and/or C-terminal part of the protein. Then the activated toxin binds to protein receptors (Cry) or lipids at the surface of the gut cell membranes or directly interact with lipids (Cyt). The binding and insertion of toxins inthe membrane leads to gut cell disruption and subsequently to cell death. Figure fromTetreau G., Andreeva E.A. et al., 2021 146 . (B) Schematic representation of the nomenclature system used forBt toxins, (adapted from Palma L. et al., 2014 147 )

  3D structure of the protoxin form of Cry1Ac (proCry1Ac)(PDB id: 4W8J 160 ) showed that besides the three-domain Cry cores structure, an additional region exists, viz. a C-terminal protoxin region, formed by four domains (Figure20A). Domains IV and VI are composed of 𝛼-helices, whereas domains V and VII are composed β-strands, featuring a beta roll architecture. Other 3d-Cry toxins lack the three-domain lack the extended C-terminal region and are synthesized as shorter protoxins of approximately 70-kDa. Among these are the Cry3, Cry11 and Cry2 toxins. For instance, the structure of Cry2Aa protoxin (PDB id: 1I5P 161 ) (Figure20B) showed an N-terminal region composed of 49 amino acids which are cleaved upon protease activation, triggering exposure of domain II region and receptor binding.

Figure 20 :

 20 Figure 20: Structure organization of the three-domain (3d) Cry toxins. (A) Structure of Cry1Ac protoxin (PDB id: 4W8J 161 ). The structure features the typical domain organization of 3d Cry toxins:(i) domain I (coloured in warmpink) is the perforating domain, composed of a bundle of seven α-helices (α1-α7) with a central amphipathic α helix (α5)(inset); (ii) domain II or central domain (couloured in lightteal) features a β-prism structure and it is involved in toxin-receptor interactions; (iii) domain III (coloured in deepblue) is involved in receptor binding and pore formation. Cry1Ac toxin presents a C-terminal protoxin region, composed of four domains: domain IV (coloured in deeppurple), domain V (coloured in forest), domain VI (coloured in orange) and domain VII (coloured in palecyan). Upon proteolytic activation the C-terminal protoxin region is cleaved, thus yielding the active form of the toxin. (B) Structure of Cry2Aa protoxin (PDB id: 1I5P 161 ) indicates the domain of the 3d-Cry toxins with: domain I (coloured in ruby), domain II (coloured in slate) and domain III (coloured in forest). Additionally, Cry2Aa protoxin features an N-terminal protoxin region, referring to domain IV (coloured insand) which is cleaved upon protease activation. In (A) and (B) the 3D structures are represented in cartoon mode using PyMOL v.2.1.1. (C) After ingestion by the mosquito larva, Cry protoxins are proteolytically activated. As a result, the 3D structure of the toxins changes, resulting in a release of the toxic core comprising domain I, II and III.

Figure 21 :

 21 Figure 21: Mode of action of the 3d Cry toxins. After ingestion of the Cry crystals by mosquito larva, the protoxin crystal dissolve in the larvae mid-gut upon increase of pH. Gut proteases cleave the protoxin region (N-and/or Cterminal of the protein), resulting in the formation of active toxin (grey). (A) The pore-formation model stipulates that the monomeric toxin binds to both APN (yellow) and ALP (red flag) receptors with a low-affinity interaction. Then, the monomeric toxin then binds to Cadherin receptors (blue) and undergoes further proteolysis (cleavage of helix α1), enabling toxin oligomerization. The oligomerized toxin then binds to ALP and APN receptors with a high-affinity. The toxin oligomer inserts into the membrane, thus forming a pore. (B) In the signal transduction model, interaction between monomeric toxin and Cadherin receptor, initiates the activation of G proteins. Activation of G proteins increase the activity of adenylate cyclase (AC, in green), which triggers the increase of cAMP levels and which in turn activate PKA (protein kinase A). PKA triggers the activation of intracellular pathways controlling cell death.

  Cyt (cytolytic) toxins, encoded by cyt genes, represent another insecticidal protein family in Bt. As most of the Cry toxins, Cyt are produced during the sporulation phase and present cytolytic activity against Diptera insects. The Cyt family of toxins presents a different tertiary structure than members of the Cry family. To date three distinct families of Cyt toxins have been identified, i.e.

  family. In the study of Cohen et al., Cyt1Aa was present as a monomer and lacked the first β0 strand corresponding to the N-terminal propeptide, ensuring toxin dimerization. In the present PhD work, the structure of Cyt1Aa has been solved, from in vivo grown in Bti nanocrystals, using SFX. Our study demonstrated that the β0, corresponding to the N-terminal propeptide of the toxin drives the in vivo crystallization of Cyt1Aa (see Result Chapter, Part 3.1).

Figure 22 :

 22 Figure 22: Structural insights into Bt Cyt toxins. (A) 3D structure of Cyt1Aa toxin (PDB id: 3RON 175 ). Αlpha helices (coloured in violetpurple) and β-sheets (coloured in cyan) are represented in cartoon mode using PyMOL v.2.1.1. The β-sheet structure features six antiparallel β-strands. In Cyt1Aa, an insertion of β-hairpin (β2-β3) between α1 and α2 is observed. (B) Structural comparison of Cyt1Aa (coloured in wheat) and Cyt2Ba (PDB id: 2RCI 175 )(coloured in grey). In the structure of Cyt2Ba, the β-hairpin (β2-β3) insertion is missing.

Figure 25 :

 25 Figure 25: Schematic representation of the steps to follow during Gibson assembly cloning. After amplification of the DNA fragments and linearizing the expression vector using PCR (Step1), both fragments and plasmid are mixed in a single tube where exonuclease, polymerase and ligase are subsequently added. An incubation at 50 °C for 60 -90 mins allows the exonuclease to chew the DNA fragments (5' to 3') that leaves complementary single stranded regions that can anneal (Step 2). The polymerase and the ligase allow to fill these gaps and to assemble the DNA fragments into the vector (Step 3).

Figure 27 .

 27 Figure 27. Schematic illustration of hydrophobic interaction chromatography technique. For instance, mixtures of protein samples characterized with differences in intrinsic hydrophobicity are retained on the column under high salt conditions. With the decrease of salt concentration, protein forms are eluted with an order of increasing hydrophobicity.

with 3

 3 volumes of equilibration buffer (40 mM Tris pH 7.4, 2 M NaCl). OCP was diluted in 40 mM Tris pH 7.4, 4 M NaCl buffer and loaded on the column. The flow rate was set at 3 mL/min. After sample binding, OCP was eluted using 0.5 M NaCl in 40-mM Tris-HCl pH 7.4. The holo-OCP was eluted first (red fraction in Step 4, Figure 28) and the apo-OCP at last (violet fraction in Step 4,

Figure 29 :

 29 Figure29: Effect of varying concentrations of sodium chloride (NaCl) on the degree of purification of holo-OCP. Purified on Ni-NTA holo-OCP from Planktothrix agardhii was subjected to salting out precipitation using a dialysis method against 3, 4 or 5 M of NaCl. The absorbance signal at 495 and 280 nm was measured and the holo-to apo-OCP ratio was estimated.

Figure 30 .

 30 Figure 30. Purification steps protocol allows preparation of gram-quantities of highly pure Planktothrix holo-OCP for SFX experiments at XFELs. (A) OCP sample with a RatioA495/A280 of < 1 was subjected to overnight dialysis against 5 M of NaCl. Salting out effects induce apo-OCP precipitation (white clouds) in the dialysis bag. The protein sample is centrifuged for 30 min at 4000 g (B) in order to separate soluble (supernatant corresponding to holo-OCP) fraction from the precipitate (white pellet corresponding to apo-OCP). The supernatant is then subjected to overnight dialysis in presence of low salt concentration (0.15 M) in order to exchange high salt concentration and prevent from protein precipitation (C). As a final step, the dialyzed protein sample is purified using HIC in order to get rid of any residual apo-form or precipitates (D).

Figure 31 :

 31 Figure 31: Illumination of OCP microcrystals. (A) OCP microcrystals were deposited on a single-isolated well of 24 well Linbro plate and illuminated for 48h using a continuous blue-light (emitting at 430 nm). (B) Few µl of microcrystalline slurry were pipetted and deposited on SOS sandwich. (SOS chip image courtesy: Dr. Bruce Doak)

  in a cryo solution composed of either of 0.2 M sodium acetate pH 5, 40 % PEG 4000 or 0.2 M Tris Bis propane pH 7.5, 40 % PEG 4000. X-ray crystallographic oscillation data were collected at 100 K on the MASSIF-1 (ID30A-1) beamline at the European Synchrotron Radiation Facility (ESRF, Grenoble, France). MASSIF-1 is a fully automated beamline operating at fixed X-ray wavelength of 0.969 Å (12.8 keV) 207 with a minimum beam size of 10 x 10 μm 2 . Data were collected with an oscillation range of 0.2 and were recorded with a Pilatus3 2M detector (Dectris). Images were indexed and intensities were integrated and merged using XDS 208 and XSCALE 116 . Molecular replacement was performed with Phaser 209 using as a starting model the cryo-structure of C2 OCP Planktothrix binding canthaxanthin (pdb id: 7qd2) or P21 structure of OCP Planktothrix binding canthaxanthin (pdb id: 7qcZ). Manual model building was performed in Coot 122 and refinement with the Phenix suite 210

Supplementary Fig. 1 .

 1 Serial synchrotron crystallography allowed assessing diffraction quality prior to XFEL experiments. a, Cyt1Aa nanocrystals were deposited between two silicon wafers and flash-cooled in a gaseous nitrogen stream at 100 K. b, Diffraction data were collected serially by raster-scanning with a sub-micron X-ray beam (0.7 µm FWHM), with 80 ms exposure per pattern and 10 µm horizontal and vertical spacing between X-ray shots. In this panel, each pixel represents a diffraction pattern, with the colouring intensity scaling to the summed intensity of Bragg peaks. The figure offers a X-ray microscopic image of the sandwiched crystal suspension presented to the Xray beam. c, Powder rings extend to 4.0 Å resolution on the maximum projection of ~1 million diffraction patterns collected at 100 K using raster-scanning serial synchrotron crystallography at the (sub)-microfocus ESRF-ID13 beamline.

7 .

 7 MALDI-TOF MS analysis confirms that Cry11Aa and Cry11Ba are present as a fullsize monomer in the crystal. a-b,Cry11Aa crystals mixed with SA matrix in absence (a) or presence of 10 mM DTT (b) display the same profile with the most abundant peaks corresponding to the singly-charged molecular ion of a monomeric species of 72.235-72.246 kDa (mass error: ± 100 Da; expected mass: 72.349 kDa). c, Cry11Ba crystals mixed with DHAP showed the presence of full-length monomer of 81.145 kDa in agreement with the predicted mass of 81.344 kDa.

10 . 11 .

 1011 Cry11Aa mutation strategy. a, Hydrogen and salt bonding interaction network between domain I, II and III within a single monomer formed by Y272, D507 and D514. Domain I is shown in blue; domain II is shown in light orange; domain III is shown in pink. For clarity, a part of domain III is not shown in cartoon mode. Hydrogen bonds are shown up until a distance of 3.2 Å. The 2Fobs-Fcalc electron density map contouring the concerned residues and water molecules at 1 σ is shown at the bottom of each panel. b, Hydrogen and salt bonding interaction network between domain I and III within a single monomer involving E583. Color code as in (a). c, Location of F17 and E180 in interface #2 between two adjacent monomers. Monomers are color coded as in Fig. 3. d, Water-mediated hydrogen bonding network around Y449 in interface #1 between two adjacent monomers. For clarity, domain III of each of the monomer is omitted from the figure. Color code as in Fig. 3. e, interaction between E295 and Y349 in the βpin region which connects two adjacent monomers via interface #3. Color code as in Fig. 3. The triple mutation Y272Q-D507N-D514N of Cry11Aa affects crystal formation.

14 . 16 .

 1416 Cry11Aa mutant crystal structures indicating the crystal packing (top), tetramer (middle) and interaction region (bottom). a, Cry11Aa-F17Y; b, Cry11Aa-E583Q; c, Cry11Aa-Y449F. The dashes in (a) indicate the hydrogen bond made between E180(OE1) and Y17(OH) (2.4 Å). Due to the lower resolution as compared to Cry11Aa-WT, no water molecules are observed in the interaction region. Therefore, the specific interaction pattern is not shown in (b) and (c). The 2Fobs-Fcalc electron density map shown at the bottom of each panel is contoured at 1 σ. Structural interpretation of Cry11Aa mutations described in literature. Color code as in Fig. 2. Dashes indicate hydrogen bonds (up to 3.2 Å). The 2Fobs-Fcalc electron density maps on the right of each panel are contoured at 1 σ. a, Position of and interactions formed by residues in Domain I which can explain the suppression of toxicity of the V104E mutation, and reduced toxicity by the E97A, R90E and V142E. b, Hydrophobic pocket in which Y98 resides c, Interactions made by S105 d, Polar and hydrophobic interactions formed by P261, V262, E266 at the interface between α8 and the β1-α8 loop.

  and single water-mediated interactions formed by D515, Y272 and D507

Supplementary Figure 3 :

 3 assembled with the two above-mentioned fragments by Gibson to create the shuffle vector encoding the C11AB chimera. C11AB chimera Reverse CGATCGCCTATTTAGGATAG TTA TTG TTA TACCCTTGG TTA CATG TACAG Supplementary Table 3. Primers used to generate the mutants. Planktothrix and Synechocystis phycobilisomes. (A) Commassie Blue SDS gel analyzing the proteins present in Synechocystis (Syn) and Planktothrix (Pkl) phycobilisomes. Synechocystis and Planktothrix PBS are characterized by a similar phycocyanin (PC) to allophycocyanin (APC) ratio of ~ 3 (compare to ~ 2 in Arthrospira PBS) and feature a comparable protein composition, with an Lcm (ApcE) of about 95 kDa, two rod linkers (LR33

9 :

 9 Sequence and secondary structure alignment of the five OCP variants compared structurally. β-sheets are shown in marine blue, while canonical (3.613-helix) and 310helices are shown in red and yellow, respectively. Starbursts indicates residues whose substitution might play a role in the change of photoactivation rate (green). The lower panel shows a phylogenetic

  T A R S I F P E T L A A D V V P A T I A R F K Q L S A E DQ L A L I WF A Y L EMGK T I T I AA PGAANMQ F A E N T L Q E I R QM T P L QQ T Q A MC D L A N R T D T P I C R T Y A SWS P N I K L G FWY E L GR F M D QG L V A P I P E G Y K L S A N A N A I L V T I QG I D P GQQ I T

  S Q L N A E DQ L A L I WF A Y L EMGK T L T I AA PGAA S MQ L A E N A L K E I Q A MG P L QQ T Q A MC D L A N R A D T P L C R T Y A SWS P N I K L G FWY R L G E L M E QG F V A P I P A G Y Q L S A N A N A V L A T I QG L E S GQQ I T V L R N A V V DMG F T A G K D G . . KR I A E P V V P P Q D T A S R T K V S I EGV T N A T V L N YMD N L N A N D F D T L I E L F T S D G A L Q P P F Q R P I VGK E N V L R F F R E ECQN L K L I P E RG V T E P A E DG F T Q I K V T GK V Q T PWF GG N V GMN I AWR F L L N P E G K I F F V A I D L L A S P K E L L N F A R

FFigure S2 .

 S2 Figure S2. SREFLEX modeling of light-adapted OCP. The red circles show the X-ray scattering profiles of OCP R wt-Ctag and OCP R R27L-Ntag at 3.5 and 10.5 mg/ml. Continuous curves (coloured in green) represent calculated profiles using SREFLEX normal mode analysis with different OCP light-adapted oligomeric forms. The resulting chi-squared of SREFLEX analyses are indicated.

Figure S5 .

 S5 Figure S5. Structural recovery of OCP O wt-Ctag, following accumulation of OCP R wt-Ctag.The plot shows the time evolution of the integrated intensity in the 0.05-0.5 Å -1 region of the X-ray difference profile ΔI(q) = I(q)t -I(q)t0, with I(q)t and I(q)t0 the scattering intensities at a generic time t after switching off the 430 nm light and before the 430 nm irradiation procedure, respectively. The data establish that after accumulation of the OCP R state at 23 mg/ml, recovery of the starting structural state of the OCPwt-Ctag sample spans a time-scale of at least 2.5 hours.

Figure S7 .

 S7 Figure S7. Global fitting of the recovery data recorder for the different OCP variants. (A, B) Thermal recovery was monitored at four concentrations of OCPR27L-Ntag (A, B), OCPwt-Ntag (C, D) and OCPwt-Ctag (E, F), and data were globally fitted for each variant using a model accounting either for three (A, C, E) or four (B, D, F) exponential components. Fitting parameters are reported in TableS6 and S7.

Figure S9 .

 S9 Figure S9. Nanosecond transient absorption kinetics recorded on OCPwt-Ctag at different temperatures. (A-E) Difference absorption was monitored at both 440 and 565 nm, enabling to probe photoactivation and recovery at the blue and red edges of OCP O and OCP R absorption spectra, respectively. (F) Nanosecond transient absorption data recorded at 565 nm for OCPwt-Ctag at various temperatures, from 8 to 36 °C. See TableS4for complementary data.

Figure S10 .

 S10 Figure S10. OCP dimerization increases the yields at shorter time delays and delays the occurrence of the last recovery step in the ns-s spectral evolution. (A) Estimated yields of OCPwt-Ctag red-states at various time delays and concentrations. The plot indicates that dimerization benefits the P1 and P3 yields. The OCP R yield, however, only improves slightly. (B) The lifetime for the third component used to fit the ns-s spectral evolution of OCPwt-Ctag increases as a function of OCP-dimer concentration. The average lifetime extracted from fits of the OCPR27L-Ntag data at various concentrations was used for zero dimer concentration. (C, D) Predicted fraction of OCP O dimers as a function of concentration (C) or optical density (OD) at 490 nm (D) based on a dissociation constant (Kd) of 14 µM for OCP O dimers.

Figure S11 .

 S11 Figure S11. Size exclusion chromatography assesses the purity and dispersity of our OCP samples. (A) At all tested concentration, the OCP O R27L-Ntag sample is monodisperse and features a monomer. (B) Contrastingly, the OCP O wt-Ctag sample features monomers and dimers.

Figure S12 .

 S12 Figure S12. SVD analysis. Python-based script for singular value decomposition (SVD) analysis of time-resolved (TR) difference profiles measured at ID09 (ESRF, Grenoble) in the µs-ms time-scale (See Fig. 6D).

Figure 32 :

 32 Figure 32: Attempts for in vivo recombinant crystallization of OCP in Bt. (A) Western blot shows expression of the recombinant fusion Cyt50-OCP, i.e. corresponding to the fusion of OCP with the N-terminal propeptide of Cyt1Aa (Cyt50). The expression band of 43 kDa corresponds to the fusion between the two proteins. (B) Scanningelectron microscopy (SEM) imaging demonstrate the lack of in vivo OCP crystals. Only aggregates, spores and bacteria are observed. (C) Synthetsis of β-caroten in Bt. .

1 :

 1 moitié des photons ont été absorbés) est de ~ 4 μm, ce qui indique que des cristaux de cette taille au maximum doivent être utilisés pour nos études de dynamique structurales par la cristallographie résolue en temps. Deux méthodes s'offraient à nous pour produire de telle cristaux : la cristallisation in vitro, et la cristallisation in vivo. Cette dernière présente le double avantage de permettre la production dans des conditions physiologiques de cristaux micro-ou nanométriques calibrés (en fonction de la taille de la cellule utilisée) et d'éliminer toutes les étapes d'extraction, purification et cristallisation manuelle inhérentes à la cristallisation in vitro. Dans ce contexte, il faut distinguer les protéines naturellement cristallines, dont la fonction dépend de leur cristallisation et qui ont donc évolué pour former des cristaux de grande qualité, et les protéines non naturellement cristallines, dont la cristallisation recombinante est soit désirée et réussie, soit heureusement accidentelle. Dans un cas comme dans l'autre, cette cristallisation peut survenir dans différents types cellulaires allant des cellules mammifères aux cellules d'insectes, aux virus et aux bactéries. Dans notre laboratoire, nous utilisons à dessein de cristallisation recombinante la bactérie Bacillus thuringiensis (Bt), qui produit naturellement sous la forme de cristaux des toxines spécifiquement actives contre des larves d'insectes. Ces cristaux naturels sont caractérisés par une taille nano-à micrométrique (0,5 µm-1µm) et une stabilité à long terme en milieu aqueux. Ainsi, Bt est un des candidats les plus prometteurs pour qui vise à développer une méthode de cristallisation recombinante in vivo. Dans notre cas, nous voulions utiliser Bt pour faciliter la production de grands volumes de microcristaux d'OCP pour nos expériences de cristallographie résolue en temps. Quoique nos efforts n'aient pas été couronnées du succès espéré, ils nous ont occupés durant une bonne partie de cette thèse, et le chemin parcouru a permis de mieux comprendre les mécanismes de cristallisation au sein de Bt.Objectives de la thèseMa thèse a été structurée autour de deux axes principaux, recouvrant des problématiques différentes. Le premier axe visait la cristallisation recombinante de l'OCP in vivo. Dans ce contexte, nous avons exprimé au sein de Bt des fusions d'OCP avec deux de ses toxines naturellement cristallines, i.e. Cyt1Aa et Cry11Aa. Dans ce contexte, un prérequis était de caractériser leurs structures et leur mode de cristallisation, i.e. d'identifier les interactions permettant leur arrangement régulier au sein d'un cristal. A cette fin, Cyt1Aa et Cry11Aa ont été exprimées in vivo dans Bt, et leurs structures ont été résolues à l'aide de la cristallographie sérielle à l'échelle de la femtoseconde. Pour mieux comprendre les mécanismes de cristallisation in vivo à l'oeuvre au sein de ces deux toxines, nous avons généré des mutations ciblées, qui ont permis de tester différentes hypothèses sur les déterminants moléculaires pour la formation, la stabilisation et la solubilisation des cristaux. Cependant, compte tenu du peu de succès rencontré avec la cristallisation recombinante de l'OCP, il est rapidement devenu évident que nos études structurales sur cette protéine ne seraient possibles qu'à partir de cristaux formés in vitro. Les principaux objectifs du deuxième axe consistaient à fournir une étude complète -de la cristallisation in vitro à la détermination structurale et fonctionnelle -de l'OCP de la cyanobactérie Planktothrix agardhii. Mon travail consistait à: (i) établir des conditions favorables de cristallisation, optimiser de la croissance cristalline et collecter des données structurales par le moyen de la cristallographie aux rayons X aux synchrotrons; (ii) établir et optimiser un protocole expérimental permettant la production de grammes d'holo-OCP, utilisées par la suite pour la croissance des microcristaux in vitro, en vue d'effectuer des expériences de cristallographie résolue en temps; et (iii) d'exprimer, cristalliser et caractériser la structure de ~30 mutants d'OCP Planktothrix, avec pour but de comprendre comment les résidus mutés influent sur les vitesses de photoactivation et de retour thermique. En outre, je me suis consacrée à l'étude structurale en solution des changements conformationnels larges survenant dans les dernières étapes de la photoactivation de l'OCP, avec pour but principal de déterminer sur quelle échelle du temps ils surviennent. Mécanisme de cristallisation in vivo de la toxine Cyt1Aa Cyt1Aa est l'une des quatre protoxines cristallines produites par la bactérie Bacillus thuringiensis subsp. israelensis (Bti). Dans le passé, il a été démontré que Cyt1Aa est incapable de former des cristaux de sa forme native (protoxine) lorsqu'elle est exprimée d'une manière recombinante dans des cellules d'E. coli. L'obtention des cristaux in vitro n'est devenue possible qu'après clivage protéolytique du propeptide N-terminal de la toxine. Le travail présenté dans le Chapitre 3.1, a eu pour le but d'élucider le mécanisme de cristallisation in vivo de Cyt1Aa dans Bt. Nous avons investigué si oui ou non le propeptide N-terminal joue un rôle dans la formation et la stabilité des cristaux naturels de Cyt1Aa. Pour ce faire, nous avons produit ces cristaux in vivo dans Bt, et ceux-ci ont été utilisés pour la détermination structurale par cristallographie sérielle à l'échelle de la femtoseconde au XFEL de Stanford, LCLS. Nos structures SFX ont démontré que le propeptide est impliqué dans sa cristallisation, mais aussi dans sa dissolution. Spécifiquement, des structures déterminées dans des conditions pH/redox différentes ont permis d'éclairer le rôle joué par un pont disulfure dans le propeptide N-terminal dans la formation de cristaux de Cyt1Aa dans Bt, puis leur dissolution de ces cristaux dans l'intestin de larves de moustique. Nous avons effectué des tests biochimiques, biophysiques ainsi que des tests de toxicité pour deconvoluer les étapes clés de la cascade de bioactivation de Cyt1Aa. En utilisant l'approche de mutagénèse dirigée notre étude a montré que la taille, la forme, le taux de production, la sensibilité au pH et la toxicité des cristaux peuvent être contrôlé par substitution d'un atome unique au sein d'une protéine en comptant plus de 2700. Chapitre 3.2. : De novo caractérisation structurale des toxines Cry11Aa et Cry11Ba La famille des toxines Cry11 rassemble plusieurs protéines actives sur des insectes de l'ordre Diptera, y compris Cry11Aa, Cry11Ba et Cry11Bb, respectivement produites par les bactéries mosquitocidal Bacillus thuringiensis subsp. Israelensis, Bacillus thuringiensis jegathesan et Bacillus thuringiensis subsp. medelin. D'un point de vue structural, la famille Cry11 appartient au large groupe des toxines Cry à trois domaines, où chaque domaine est caractérisé par une fonction spécifique. Plus précisément, le domaine I est composé de sept hélices 𝛼, impliquées dans la formation de pores membranaires au niveau de l'intestin du moustique. Le domaine II, en forme de prisme β, est impliqué dans la reconnaissance et la liaison à des récepteurs spécifiques dans l'intestin des larves de moustiques. Le domaine III est un β-sandwich constitué de deux feuillets-β antiparallèles, et est impliqué dans l'oligomérisation. Parmi les toxines Cry11, la toxine Cry11Aa a été la plus étudiée, du fait de sa forte activité sur les larves d'Aedes aegypti (vecteurs du virus de la dengue et de la fièvre jaune), en sus de celles d'Anopheles gambiae (le vecteur de transmission du paludisme) et de Culex quinquefasciatus (vecteur de la fièvre du Nil occidental et de la filariose). La toxine Cry11Ba a également une activité mosquitocidal, et elle est considérée comme candidate pour remplacer Cry11Aa dans des souches recombinantes de Bt, du fait de la largeur des cristaux qu'elle produit et de son activité plus forte sur Aedes aegypti. Malgré leur importance biotechnologique, les structures de ces toxines restent inconnues, ainsi que leur mécanisme de toxicité. Cela est dû à la taille nanométrique des cristaux produites in vivo, qui sont inadaptés pour des collectes de données structurales en utilisant la cristallographie aux rayons X dans les sources synchrotrons, et à l'impossibilité pour cette toxine de recristalliser in vitro après dissolution des cristaux naturels. A nouveau, nous avons utilisé la cristallographie sérielle à l'échelle de la femtoseconde aux XFELs de Stanford (LCLS) et Hambourg (EuXFEL) pour élucider les structures de Cry11Aa et Cry11Ba à partir des nanocristaux produits in vivo. La structure de Cry11Aa a été déterminée de novo, en utilisant la diffusion anomale comme méthode de phasage, puis la structure de Cry11Ba a été résolue par remplacement moléculaire. Les deux structures ont révélé un nouveau mécanisme de cristallisation in vivo des toxines Cry, où chacun des trois domaines est impliqué dans le réseau cristallin. La diversité des modèles de cristallisation in vivo, suggère des approches rationnelles pour améliorer ces toxines pour la lutte contre les moustiques.

Chapitre 4 . 1 :Chapitre 4 . 2 :

 4142 photoexcitation non productive (i.e. pour les protéines qui ne s'engagent pas vers la formation de l'OCPR mais retombent à l'état fondamental après illumination pulsée), un état orange se forme qui reste non-photoexcitable jusqu'à 0.5 µs. Nous avons baptisé cet état OCP NS , pour OCP « numbed-state » ou engourdie.

Table of contents

 of Acknowledgments/Remerciements ............................................................................................. 3 Table of contents ......................................................................................................................... 7

	Chapter 1 -Introduction
	1. Photosynthesis
	1.1. Overview

Table 2 :

 2 Expression constructs. The table lists all fusion constructs used in this project, aiming to succeed in vivo recombinant crystallization in Bt.

Table 3 :

 3 Buffers composition used for Western Blot

	Transfer Buffer 1X at pH 8.3	48 mM Tris base, 39 mM Glycine, 20 % Ethanol
	TBS 5X (Tris Buffered Saline) at pH 7.4	125 mM Tris base, 0.68 M NaCl

1.3. Biochemical & biophysical characterization of in vivo-grown in Bt crystals 1.3.1. Identification of β-carotene and carotenoid synthesis in Bacillus thuringiensis subsp. israelensis by thin-layer chromatography (TLC)

Heat stability tests on wild type Cyt1Aa and mutants' crystals toxins

  and their mutants were compared by calculating SP50 (the pH at which 50% of the crystal is solubilized). The SP50 is calculated by dividing the protein concentration measured at a given pH by that measured at pH 11.8. Statistical analysis was applied to estimate the significance of the results.

	1.3.4. Crystal visualization using scanning electron microscopy (SEM)
	Crystal quality was assessed using scanning electron microscopy (SEM). Briefly, crystals (diluted
	at 1/100 or 1/1000) were resuspended in a 25 mM ammonium acetate solution and deposited on
	microscope circular glass slides and left for drying under a Sorbonne hood for ~ 1 h. Samples
	were coated with a 2 nm thick gold layer with the Leica EM ACE600 sputter coater and imaged
	using the Zeiss LEO 1530 scanning electron microscope (SEM) of the European Synchrotron
	Radiation Facility (ESRF, Grenoble, France). From the recorded images, the length and width of
	crystals of Cyt1Aa WT and all mutants were measured using the software ImageJ v1.51k (N = 40

1.3.1.2. crystals). Purified crystals of wild type Cry11Aa, Cry11Aa mutants and Bt wild type strains mutants were also visualized using either the Zeiss LEO 1530 scanning electron microscope of the SEM facility at the ESRF or a Thermo Fisher Prisma environmental SEM (ESEM) available for users at the European XFEL (EuXFEL, Hamburg, Germany). For ESEM at the European XFEL, samples

Table 4 .

 4 OCP wild type and mutant samples used in this project.

Extended water-mediated interactions formed by Y449 and D501, connecting two monomers via interface #1

	Supplementary
	Interaction partner 1	Interaction partner 2	Distance (Å)*
	D514 (OD2)	S249 (O)	2.9 / 2.9
		S251 (OG)	2.4
	D514 (OD1)	S251 (N)	3.1
		W253 (NE1)	2.4 / 3.0
		Y272 (OH)	2.7
		R222 (NH1)	2.4 / 3.1
	Y272 (OH)	R222 (NH1)	3.2 / 3.1
	D507 (OD1)	R222 (NH1)	2.5
	D507 (OD2)	R222 (NH2)	3.2
		Y203 (OH)	2.7 / 2.7
		W267 (NE1)	2.9
	Interaction partner monomer 1	Interaction partner monomer 2	Distance (Å)*
	Y449 (OH)	V499 (N)	2.7 / 2.8
		V499 (O)	2.9 / 2.8
		T448 (OG1)	2.9 / 2.6
		D501 (OD1)	2.9 / 2.8
	D501 (OD2)	Q281 (NE2)	2.8 / 2.9 / 2.6 / 3.0
	d, Hydrogen bonds made by Y349
	Interaction partner monomer 1	Interaction partner monomer 2	Distance (Å)
	Y349 (OH)	E295 (OE1)	3.1

* Multiple distances indicate water-mediated hydrogen bonds. c, * Multiple distances indicate water-mediated hydrogen bonds.

Table 1 .

 1 Interactions in the mutated interfaces. Hydrogen bonds up to 3.2 Å are listed.

b,

Extended water-mediated interactions formed by E583 and other residues involved in this network

				Toxicity
	Mutant	Aedes		Anopheles	Culex	References §
		aegypti		stephensi	quinquefasciatus
	Cry11Aa R90E	Not toxic		N.D.*	N.D.	31
	E97A	Not toxic		N.D.	N.D.	31, 33
	Y98E	Not toxic		N.D.	N.D.	31
	V104E	No crystal formation	No crystal formation	No crystal formation	31
	S105E	Not toxic		N.D.	N.D.	31
	V142D	Not toxic		N.D.	N.D.	33
	P261A No change / 3-fold reduced	N.D.	N.D.	48, 49
	V262A No change / 5-fold reduced	N.D.	N.D.	48, 49
	V262E	Not toxic		N.D.	N.D.	48
	E266A No change / 21-fold reduced	N.D.	N.D.	48, 49
	Cry11Ba G257A	Strongly reduced		Reduced	Strongly reduced	53
	I263A	Not toxic		Not toxic	Strongly reduced	53
	S264A	Not toxic		No change	Reduced	53
	K269A	Strongly reduced		No change	Strongly reduced	53
	I306A	Not toxic		No change	Reduced	53
	* N.D.: Not determined; § Reference numbers as used in main text
	Interaction partner 1	Interaction partner 2	Distance (Å)*
	E583 (OE1)		T197 (OG1)	2.8 / 2.9
			L193 (O)		2.8 / 2.8
			V192 (O)		2.6 / 2.7 / 3.0 / 3.1
			A233 (O)		2.6 / 2.7 / 2.8 / 2.9 / 2.6 / 2.9
	W584 (N)		V192 (O)		3.2 / 2.7 / 3.0 / 3.1
			A223 (O)		3.2 / 2.7 / 2.8 / 2.9 / 2.6 / 2.9
	Q511 (NE2)		V192 (O)		3.0 / 2.8 / 3.0 / 3.1
			A223 (O)		3.0 / 2.9 / 2.6 / 2.9
	D510 (O)		V192 (O)		3.0 / 2.9 / 2.8 / 3.0 / 3.1
			A223 (O)		3.0 / 2.6 / 2.9
	D510 (OD2)		R196 (NE)	3.1
			R196 (NH2)	3.3
	* Multiple distances indicate water-mediated hydrogen bonds.
				207

interpreted as a second proteolytic step taking place post-insertion of Cyt1Aa in the membrane. Source data are provided as a Source Data file.

Quantum Electronics Laboratory, Faculty of Physics, Adam Mickiewicz University in Poznań, Uniwersytetu Poznańskiego 2, Poznan

 Poland Keywords: cyanobacteria -flash photolysis -photosynthetic pigments -structure function relationships -X-ray diffraction

tree between the five OCP variants, and reveals that alike Tolypothrix, the Plankthotrix OCP belongs to a different sub-clade than Synechocystis, Limnospira and Anabaena OCP.

Acknowledgments/Remerciements

Abbreviations

This fall back strategy was crucial to enabling success with the second research axis, focused on OCP and its photoactivation mechanism. The main goals of the second axis consisted in providing a complete study -from in vitro crystallization to structure determination -of the previously structurally and functionally uncharacterized OCP from the cyanobacteria Planktothrix agardhii. My work was devoted to: (i) the establishment of favorable crystallization conditions, subsequent optimization of crystal growth and data collection on these by conventional crystallography at synchrotron facilities; (ii) the development of an optimized experimental protocol enabling the production of gram quantities of holo-OCP for the in vitro growth of microcrystals, suited for TR structural studies and (iii) the expression, crystallization and structural characterization of ~30 point-mutants of OCP Planktothrix which have been designed with the purpose to challenge hypothesis on the photoactivation mechanism and possibly to identify OCP variants with an increase photoactivation yield. In addition, a major part of my PhD work was devoted to the structural investigation of large conformational changes that accompany OCP photoactivation on a longer time-scale (µs-ms), in solution. By combining static and time-resolved small and wide-angle X-ray scattering (TR-S/WAXS), it was possible to shed light on the role played by oligomerization in the OCP photoactivation mechanism and to assign the time-scale on which the first large structural changes takes place in the protein.

The overall structure of my thesis covers four main chapters, including the already presented Introduction section. The Chapter 2 comprises the Materials and Methods section wherein the in vivo recombinant crystallization strategies as well as all unpublished experimental protocols are extensively described. The results of my PhD are present in Chapter 3 (entitled "In vivo crystallization in Bt") and Chapter 4 (entitled "Orange Carotenoid Protein") and are described in 4 published articles. Chapter 5 "Discussion and Conclusions ", develops in greater detail our findings and their implications, as well as evokes results either unpublished and/or not presented in this manuscript. The expertise acquired during my thesis indeed allowed me to participate and contribute to the publication of articles whose subject was not directly pertaining to my thesis project (Appendices section).

Additional publications unrelated to my thesis work:

Libera V., Andreeva E. A., Martel A., Thureau A., Longo M., Petrillo C., Paciaroni A., Schirò G., and Comez L. Porphyrin Binding and Irradiation Promote G-Quadruplex DNA Dimeric Structure. The Journal of Physical Chemistry Letters 2021 12 (33), 8096-8102

Purification of Bt toxins by discontinuous sucrose density gradient

The collected spores-crystals mixture was resuspended in ultrapure water. Sporulated cells and crystal inclusions were washed multiple times in distilled water. Cells were lysed by sonication on ice for 15 min and then centrifuged at 4000 g for 45 min. After sonication the bacterial pellet was resuspended in water and loaded on the top of discontinuous sucrose gradient (67%, 72%, 79%) in 38.5 mL Beckman tubes (Open-Top Thinwall Ultra-Clear Tube 25 x 89 mm, Beckman Coulter).

The crystal inclusions were then separated from the spores by centrifugation at 23 000 g for 12h at 4 °C. The crystals were collected from the 67-72% sucrose gradient interface and resuspended in distilled water. Several rounds of centrifugation at 4000 g for 45 min were performed in order to eliminate as much sucrose as possible and obtain highly pure crystals 191,199 . Figure 26. Representative scheme of the construction of sucrose density gradients within a centrifuge tube from top to the bottom. A mixture of spores and crystals (5 mL) is deposited on 11 mL of sucrose 67% (pink), 11 mL of 72 % (pale pink), and 11 mL of 79 % (violet). After ~ 12 hours of centrifugation at 23 000 g a separation of the crystals from the spores occurs. The crystals are situated on the top of the tube and present a milky aspect and the spores are found at the bottom of the tube. An intermediate phase comprising spores and crystals may form in the middle which can be subsequently purified again on sucrose gradient.

Quality assessment of purified protein samples by polyacrylamide gel electrophoresis

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a widely used technique that provides information about protein's size (MW in kDa) and quality. The Bt crystals purified by ultracentrifugation were electrophoresed on a 12% SDS-PAGE gel. The 12% gels were cast in two steps: (i) the first step consisted in preparation of a resolving gel composed of 12% polyacrylamide prepared in 1.5M Tris-HCl at pH 8.8 and 10% SDS. The preparation of the resolving gel was followed by preparation of stacking gel, composed of 5% polyacrylamide, 0.5M Tris-HCl pH 6.8 and 10 % SDS. For the migration, 10 μL of each sample was mixed with Laemmli Heat stability assays were performed for wild type Cyt1Aa crystals and related solubilized mutants.

The stability tests were assayed at different pHs and at increasing temperatures. Crystals of wt Cyt1Aa were solubilized in 0.1 M Na2CO3 buffer at pH 11.8, centrifuged and the supernatant containing soluble protoxin dimers was collected. Suspensions were diluted 50 times in buffers at different pHs. Na2CO3-NaHCO3 buffer solutions at 0.1 M were used for pH 9, 10, and 11, and Na2HPO4-NaH2PO4 buffer solutions 0.1 M, for pH 7, 8, and 9, following guidelines from the Sigma Aldrich Buffer Reference Center. Nearly, complete buffer exchange was achieved by performing two steps of concentration using an AMICON ultrafiltration unit. Briefly, the protoxin suspension at pH 11 was concentrated 50 times using a centricon with a 10 kDa cutoff (Sigma Aldrich, France), and then diluted 50 times in the buffer of interest (pH 7, 8, 9, or 10). This two-step procedure was repeated twice to achieve a nearly complete buffer exchange. Suspensions were then added to the Laemmli buffer 4X devoid of DTT. To test the stability of the disulfide-linked dimer, each sample at each pH was heated for 5 min at 95, 100, 105, 110, 115, 120, 125, or 130 °C prior to loading on an SDS-PAGE 12% gel. Each temperature was tested in triplicate. Gels were stained by overnight incubation in Instant Blue (Sigma Aldrich, France), washed twice in ultrapure water and digitized using a ChemiDoc XRS imaging system controlled by the Image Lab software version 6.0.0 (Bio-Rad, France). The relative intensity of the bands corresponding to Cyt1Aa dimers was estimated using the software ImageJ v1.51k.

Solubilization assays of Cyt1Aa and Cry11Aa toxin crystals

To assess the pH sensitivity of wild type and mutant crystals of Cyt1Aa and Cry11Aa, i.e. to determine the pH at which they dissolve, solubilization tests were performed. To that end, purified crystal suspensions were centrifuged at 11 000 g for 2 min and the pellets were resuspended in 50 μL of Na2CO3-NaHCO3 0.1 M buffers at pHs ranging from 9.2 to 11.8, or in 50 μL of 0.1M Na2HPO4-NaH2PO4 buffers at pH ranging from 7.0 to 9.0. The series of Na2CO3-NaHCO3 buffers were prepared following guidelines from the Sigma Aldrich Buffer Reference Center. Crystal solutions were incubated for 1h at RT at each pH, and then centrifuged at 11 000 g for 2 min. The supernatants were collected and the concentration of solubilized toxins was quantified using a Nanodrop 2000 (Thermo Fisher Scientist) by measuring the OD at 280 nm. The corresponding values of molar extinction coefficient (𝜀) and molecular weight (in kDa) were calculated with the ProtParam tool of ExPASy (https://www.expasy.org) using the Cyt1Aa and Cry11Aa proteins sequences available under UniProt accession numbers "Q7AL78" and "P21256", respectively. To increase the reproducibility, the tests were performed in duplicates. Solubility of the wild type Part 2: Orange Carotenoid Protein project

OCP gene constructions and samples

Expression of WT, single and double mutants of OCP from Synechocystis (listed in Table 4) was carried out in the lab of Dr. Diana Kirilovsky at I2BC institute (Gif-sur-Yvette, France). Purified proteins were sent in our laboratory for additional purification steps, crystallization trials and biophysical characterization. Expression of wild type OCP from Planktothrix agardhii was also performed in Dr. Diana Kirilovksy's lab and the protein was sent for crystallization trials in our laboratory. All the constructions were achieved following transformation and purification procedures published previously [START_REF] Wilson | A photoactive carotenoid protein acting as light intensity sensor[END_REF][START_REF] Gwizdala | In vitro reconstitution of the cyanobacterial photoprotective mechanism mediated by the Orange Carotenoid Protein in Synechocystis PCC 6803[END_REF]189 . However, some of the constructions were sent from Dr. Diana Kirilovksy's lab and expression vectors were amplified using the transformation protocol described below. Point mutations of OCP from Planktothrix agardhii were designed in our laboratory and were subsequently constructed by Elisabeth Hartmann (laboratory of Prof. Ilme Schlichting at Max Planck Institute (MPI) Heidelberg, Germany). The co-transformed OCP expressing BL21 DE3 E. coli cells were sent back to our laboratory for further expression and purification steps. holo-OCP, using prepackaged phenyl-sepharose columns, was done in collaboration with Elisabeth Hartmann (MPI, Heidelberg). "Salting out" or salt induced precipitation is a widely used method in biochemistry that enables the strengthening of the hydrophobic van der Waals forces between ligands and protein. Salt precipitation is often used when triggering protein precipitation or fractionating of two or more protein forms characterized by differences in their intrinsic hydrophobicity. The precipitation is triggered by adding high salt concentrations into the protein solution. The two most studied salt agents for this type of procedure are ammonium sulfate (NH4)2SO4) and sodium chloride (NaCl).

Indeed, these two behave differently, with ammonium sulfate being classified as a good salting Guillaume Tetreau [START_REF] Hohmann-Marriott | Evolution of photosynthesis[END_REF]12 , Anne-Sophie Banneville [START_REF] Hohmann-Marriott | Evolution of photosynthesis[END_REF]12 , Elena A. Andreeva [START_REF] Hohmann-Marriott | Evolution of photosynthesis[END_REF]12 , Aaron S. Brewster 2,12 , Mark S. Hunter 3 , Raymond G. Sierra 3 , Jean-Marie Teulon 1 , Iris D. Young 2 , Niamh Burke 1 , Tilman A. Gruenewald 4 , Joël Beaudouin 1 , Irina Snigireva 4 , Maria Teresa Fernandez-Luna 5 , Alister Burt 1 , Hyun-Woo Park 5,6 , Luca Signor 1 , Jayesh A. Bafna 7 , Rabia Sadir 1 , Daphna Fenel 1 , Elisabetta Boeri-Erba 1 , Maria Bacia 1 , Ninon Zala

Supplementary Information Supplementary Note 1 A difference-density based mutation strategy

The Fo 'DTT' -Fo 'pH7' map indicates that soaking with DTT results in rupture of the disulfide bridge (strong negative peak on the C7-Sγ-Sγ-C7 bond) and, consequently, in a relaxation of the Nterminal propeptide resulting in an expansion at crystal packing interface #3 where the N-terminal propeptide (notably D11), the C-terminal end of β6, and the N-terminal ends of β5 (notably Q168) and β7 interact (Fig. 2 and Supplementary Fig. 3 and Supplementary Table 1). Marked by a positive peak between D11 and Q168, this expansion seemingly impacts the hydrophobic core of the protein, with negative peaks observed on the aromatic side chains of F147, Y171 and Y173.

Refinement of the 'DTT' structure confirms that DTT has no further effect on the protoxin structure at pH 7 (Fig. 2b and Supplementary Figs. 2 and4).

Features in the Fo 'pH10' -Fo 'pH7' map differ, with strong positive and negative peaks at the DS interface and no peaks on disulfide bridge atoms (Fig. 2b and Supplementary Fig. 4). A positive peak is again seen between D11 and Q168 at crystal packing interface #3, reminiscent of the observation made in the Fo 'DTT' -Fo 'pH7' map. Refinement of the 'pH10' structure confirms changes in interactions at the DS interface (Fig. 2 and Supplementary Figs. 2 and4), with loss of the bifurcated intermolecular H-bond between the side chains of E32 (β1-β2 loop) and E45 (β2-αA loop) from facing monomers, and of the intramolecular H-bond tethering the αC/αD region (Q138) to the tip of β2 (E45). As a result, in each monomer, strands β1, β2 and the C-terminal propeptide draw away from strand β3, at the opposite end of the β-sheet, but also from the αC, αD and αE helices, which together cover the hydrophobic face of the β-sheet (Supplementary Fig. 2). To the contrary, the αA/αB face of the β-sheet is not affected by pH elevation, in line with Fo 'pH10' -Fo 'pH7' map observations. We challenged by mutagenesis the role of residues pinpointed by the strongest peaks in the Fo 'DTT' -Fo 'pH7' and Fo 'pH10' -Fo 'pH7' maps, hypothesizing that they would be central to crystal formation and dissolution, and possibly function (Fig. 3). We first probed the role of disulfide-bridge chaining by expressing a C7S mutant. The C7S crystals are spherical and significantly smaller than the WT crystals, but they are produced at a similar yield and diffract to a similar resolution (2.0 vs. 1.85 Å, respectively, for similarly sized datasets), revealing a structure nearly indiscernible from the 'DTT' structure (Table 1, Figs. 2b and 3 and Supplementary Figs. 2 and4). Thus, cysteine chaining of WT Cyt1Aa monomers is nonessential for the production of diffraction-grade crystals and could Supplementary Fig. 2. Structural differences between the Cyt1Aa protoxin and toxin structure. a, From left to right, intramolecular difference distance matrices highlight changes in Cα-Cα distances with respect to the protoxin structure (' pH7' structure) upon proteolytic activation (3ron), soaking with DTT (' DTT' structure), pH elevation (' pH10' structure) or C7S mutation (' C7S' structure), respectively. b, Porcupine plot highlighting the displacement of Cα atoms between the protoxin (' pH7' structure) and toxin structures (3ron). Each arrow shows the direction along which a Cα atom moves upon proteolytic activation, with the length of the arrow scaling to the amplitude of the motion; here, arrows are 10 times larger than the distance travelled by Cα atoms. No data is shown for residues 234-238, which concentrate the largest conformational changes and whose Cα-arrows would dominate the plot, rendering visualisation of other motions difficult. c, The H-bond between E138 and E45, which tethers the αC/αD hairpin to the β-sheet (left panel; same colouring as in b), is affected by proteolytic activation, being either preserved in a different conformation (chain A of the asymmetric unit; middle panel) or absent (no density for E45 in chain B of the asymmetric unit; right panel) in the activated toxin structure (3ron). Red cycles highlight the side chain conformational changes of interest. d, Porcupine plot highlighting the displacement of Cα atoms between the protoxin structure at pH7 (' pH7' structure) and at pH 10 (' pH10 structure' ). Each arrow shows the direction along which a Cα atom moves upon pH elevation, with the length of the arrow scaling to the amplitude of the motion; here, arrows are 10 times larger than the distance travelled by Cα atoms. e, From left to right, intermolecular difference distance matrices highlight changes in Cα-Cα distances between monomers in the domain-swapped dimer upon soaking with DTT, pH elevation or C7S mutation, respectively. Supplementary Fig. 3. The natural Cyt1Aa crystals are highly packed. a, Natural Cyt1Aa crystals display a solvent content of ~33% and feature chains of domain-swapped (DS) dimers associated by disulfide bridges. For more details on crystal packing interfaces, please see Supplementary Table 3.

Supplementary Table 1 | Summary of crystal packing interactions.

Interface number

Description

Overall

The asymmetric unit monomer is shown as a ribbon colored in slate. In the natural crystals, it establishes contact with nine symmetry-related mates, shown as ribbons of different colors, with each molecule having the same colour coding in all panels. In the following panels, atoms involved in the discussed interface(s) are shown as spheres, with carbon atoms colored as the ribbon, and nitrogen, oxygen and sulfur atoms colored in blue, red and yellow, respectively. Below, the reported H-bonds are those found by PISA, further filtered on the basis of a minimal distance of 3.2 Å between non-hydrogen atoms. We also used PISA to calculate the per-monomer buried surface area. For the sake of concision, only H-bonds expected to be pH-sensitive are explicitly discussed below. These include H-bonds wherein an acidic residue is involved, with specific focus on those wherein the acidic oxygen is an obligate donor, because such H-bonds are bound to break upon deprotonation due to elevation of pH.

7

Disulfide-bridged interface.

Residues 6-8 (N-terminal propeptide) from both chains are involved, with a disulfide bridge at position C7.

There is no H-bond or salt-bridge at this interface.

Per monomer buried surface area: 71 Å 2 8Residues 72 (tip of the αA/αB hairpin) and 167 (β4-β5 loop) from both chains are involved.

There is no H-bond or salt-bridge at this interface, which nonetheless participates in reducing access to Q168.

Per monomer buried surface area: 37 Å 2

Supplementary Information

Supplementary Fig. 1. Cry11Aa and Cry11Ba exhibit high similarity but limited sequence identity. Cry11Aa and Cry11Ba were aligned using strap [START_REF] Doniach | Small and wide angle X-ray scattering from biological macromolecules and their complexes in solution[END_REF] . This shows that 54.0% (392) of Cry11Aa and Cry11Ba residues are identical. Domain I is shown in blue; domain II is shown in orange except for the αhβh-handle and βpin which are shown in purple and red, respectively; domain III is shown in pink. Relative distribution of distances between residues at the bottlenecks of channels #1

Supplementary tables.

Supplementary

Contribution to this work

In the present work, I prepared OCP protein samples for static and TR-X-ray scattering experiments. I also performed all static X-ray scattering experiments and afterwards I analyzed all data. Additionally, I performed OCP thermal recovery experiments and TR-SAXS experiments.

Together with my supervisor, we wrote the paper with input from other coauthors.

Elena A. Andreeva At each concentration, the molecular envelopes determined for light-adapted OCP R R27L-Ntag and OCP R wt-Ctag are similar, and consistent with the presence of an OCP R dimer, at 0.7 and 3.5 mg/ml, and a higher-order OCP R oligomers at 10.5 mg/ml, e.g., a compact tetramer or a more extended trimer, wherein one monomer associated by the NTD interface would be more opened than those associated via the CTD.

Supplementary Information

Supplementary Tables:

Table S1: Data collection and processing parameters for static SAXS measurements recorded at the BioSAXS SWING beamline (Synchrotron SOLEIL, France)

Chapter

-Conclusions and Discussion

The goal of my thesis work was to attain a better knowledge on the photoactivation mechanism of the Orange Carotenoid Protein. So far, various experimental approaches have been used aiming to elucidate the precise sequence of events leading to the protein photoactivation. Mainly due to the lack of structural information regarding the events occurring on the ultra-fast time-scales(fsps), as well as the lack of structural data occurring on longer time-scales (µs-s) over which largescale structural changes take place, the complete understanding of OCP photoactivation remains incomplete.

In order to address these issues, we intended to characterize the structural changes that accompany formation of the active OCP red state (OCP R ), as well as the time-scale on which they occur. In this context, we used a multipronged study combining X-ray solution scattering and Xray crystallography. Both structural approaches were conducted in their steady-state and timeresolved fashion. While static and time-resolved X-ray solution scattering experiments are routinely performed at synchrotrons, the advent of X-ray free electron lasers (XFELs) has revolutionized structural biology, as it enabled to perform time-resolved (TR) experiments on ultrafast time-scale (fs-ps) and to solve macromolecular structures from nanocrystals. Indeed, smaller crystals are preferable to use for TR crystallography experiments because of the reduced light penetration in large crystals of optically dense proteins like OCP. The production of calibrated micro-to nanometer sized crystals in-vitro usually remains challenging. To that end, we used the Gram-positive crystalliferous spore-forming bacterium Bacillus thuringiensis (Bt), characterized with the ability to produce nanocrystals naturally. Specifically, we intended to recombinantly crystallize OCP as fusions with naturally crystalline Bt toxins, acting as crystallization chaperones.

However, an extensive structure-function study of these toxins was first required in order to elucidate the mechanism by which these form crystals in vivo. In this context, an important part of my work was to expand the current knowledge on the in vivo crystallization of two Bt toxins, Cyt1Aa and Cry11Aa.

In the present Chapter, I discuss the main findings of my thesis projects and their implications.

The following discussion is divided into two topics, i.e. "In vivo crystallization in Bacillus thuringiensis" and "Structural and functional insights into the photoactivation and recovery of the Orange Carotenoid Protein", in order to distinguish the two projects and to facilitate the reader.

5.2.B. Functional comparison between OCP Planktothrix and Synechocystis

Generally, members of OCP1 clade are characterized by a slow OCP R to OCP O thermal recovery (8 °C) that is accelerated by the addition of FRP (ref). However, it has been proposed that OCPrelated NPQ mechanism is more efficient in Planktothrix compared to as Synechocystis [START_REF] Djediat | Light stress in green and red Planktothrix strains: The orange carotenoid protein and its related photoprotective mechanism[END_REF] . In this context, we performed a comparative structure-function study between these with the aim to assess (i) their photoactivation and recovery kinetics; (ii) the impact of His-tagging, present either on the N-terminal or C-terminal, on the OCP photocycle and the proteins interaction with the PBS;

(iii) the influence of the functionalizing carotenoid, i.e. ECN vs. CAN, on OCP excited and intermediate states dynamics.

We found that OCP Planktothrix photoactivates and recovers faster than OCP Synechocystis.

Intriguingly, such a fast recovery is unusual for the members of the OCP1 clade, and has been observed only for members of the OCP2 and OCPX clades. In presence of FRP, the thermal recovery of OCP Planktothrix was accelerated, which furthermore confirmed its belonging to the OCP1 clade. The introduction of a histag also influences the OCP recovery kinetics. This In the biological context the extent to which canthaxanthin can be used as a functionalizing carotenoid for OCP remain unclear. Indeed, when expressed in their parent strains, members of OCP1 clade bind mostly hECN. To date, only OCP Tolypothrix was found to bind canthaxanthin (CAN) when overexpressed in Tolypothrix cells. However, when overexpressed and isolated from CAN-producing E. coli cells, all OCP1 members bind CAN. Recently it was reported that the relative populations of the carotenoid S1 and ICT excited-states depend on the OCP-bound carotenoid. Formation of ICT state was lacking when OCPs were functionalized with CAN, reassociation of OCP O monomers into OCP O dimers represents the limiting step in the recovery process, as it is characterized by the longest time (min). The OCP O dimer formation can be slowed down in presence of an his-tag at the N-termini of the protein, which is not surprising given that the NTE is involved in the dimerization interface of both OCP O and OCP R .

5.2.E. OCP light-induced structural changes probed by TR-X-ray scattering

The current knowledge regarding the exact photoactivation mechanism of OCP remains incomplete. Until recently, this was partially due to the absence of a high-resolution structure of OCP R and the events occurring on the ultra-fast timescales, as well as because information regarding long timescales over which large-scale structural changes take place were lacking.

In this PhD work we used both TR-SAXS/WAXS and transient absorption spectroscopy with the aim to elucidate the structural (on the fate of the protein scaffold) and spectroscopic (on the fate of the carotenoid) changes associated with the OCP photocycle. The TR-SAXS/WAXS method monitors structural changes, however the low-resolution of this approach does not allow to obtain information regarding carotenoid position and formation on non-predominant species. Both experimental methods required to work at comparatively high concentration of OCP, i.e. with ~ 50 % of the protein in the dimeric state at the lowest tested concentration, and more than 98 % dimers at the highest concentration. We probed the formation of OCP R by pulsed photoexcitation, however we estimated that the probability to activate the two monomers in a dimer by the same pulse is extremely low ≈ 1e -5 , meaning that our TR-SAXS experiment cannot yield the formation of the OCP R .

First, our TR-SAXS/WAXS experiment has provided one of the most unexpected and surprising findings, i.e. the existence of a non-photoprotective OCP intermediate, coined 'numbed' state (OCP NS ) which forms upon non-productive laser excitation and decay to the ground-state up to the µs time scale. This furthermore suggested that the OCP O ground state structure, formed after excitation, remains non-photoactivatable for at least 0.5 µs. By using a multi-pulse approach (reexcitation was triggered every 1 ms for up to 30 pulses) we enabled estimation of the lifetime of OCP NS , the latter lying between 450 ns and 1 ms. Second, in this study we also demonstrate that the first large scale conformational changes associated with OCP photoactivation take place on the tens of µs time scale (15 µs), and that the reversion of these occurs in the tens to hundreds ms (10 ms-200 ms) times-scale. Intriguingly, the reversion is also influenced by the localization of the his-tag in the protein. Indeed, we worked with two OCP Synechocystis variants that differed in possessing a His-tag at either N-or C-terminus of the protein. The Orange Carotenoid Protein (OCP), involved in energy-quenching of the cyanobacterial lightharvesting antennae, is a two-domain protein functionalized by a non-covalently bound ketocarotenoid. To elicit its photoprotection function, the protein must be photoactivated by a bluegreen photon, triggering transition from an orange inactive "dark" state (OCP O ) to a red photoactive "light" state (OCP R ). The photoactivation mechanism, which spans 13 decades in time, involves structural rearrangements at the level of both the photoexcited carotenoid and the protein scaffold.

During my thesis, I investigated the OCP photoactivation mechanism using variety of biochemistry and structural biology methods.

Firstly, through combination of spectroscopy and steady-state and time-resolved (TR) X-ray scattering, we examined the large-scale motions involved in the photoactivation and recovery of OCP. We demonstrated that oligomerization occurs at both the OCP O and OCP R level, and that it partakes in the regulation of the protein photoactivation and thermal recovery. Secondly, by using conventional X-ray crystallography, we determined the previously-uncharacterized crystal structure of Planktothrix agardhii OCP. Structural analysis pointed to the influence of protein flexibility on the photoactivation and recovery rates, and on the energy-quenching activity of this variant. In an effort to characterize early stages of OCP photoactivation by means of TR crystallography, we investigated in vitro and in vivo crystallization methods to produce sub-micronsized OCP crystals. Specifically, we attempted in vivo crystallization in the crystalliferous bacterium Bacillus thuringiensis (Bt), endeavoring to obtain crystals of fusions of OCP with Cyt1Aa and Cry11Aa Bt toxins. While unsuccessful in terms of production of OCP crystals, this work enabled to shed light on the structure and bioactivation cascade of the two toxins, and to investigate the molecular mechanisms at the basis of their crystallization.