Cécile Morlot

Thierry Vernet

Laure Roux

Xavier Henry

Florence Gerin

Isabelle Demachy

Jacqueline Ridard

Bernard Levy

Mikolaj Feliks

Martin Field

Sylvie Kieffer-Jaquinod

Von David

Stetten

Christophe Guerin

Jérémie Gaillard

Laurent Guillon

Laurent Blanchoin

Martin Weik

Virginia Guillon

Mudalige S Gunewardene

Sergiy Avilov

Mariam El Khatib

Nicolas Coquelle

Jaques-Phillippe Colletier

Damien Clavel

Maxime Jacq

Christian Frank

Chenxi Duan

Virgile Adam
email: virgile.adam@ibs.fr

Martin Byrdin

Cećile Morlot

Delphine Arcizet

Dominique Bourgeois
email: dominique.bourgeois@ibs.fr

Mariam El Kathib

Rational Design of Enhanced Photoresistance in a Photoswitchable Fluorescent Protein

Keywords: Fluorescent proteins, Photoswitching, Dronpa, RSFPs, Protein dynamics, cis -trans Isomerization, Proton transfer, Super-resolution microscopy

Je n'arrive pas à exprimer mes sentiments complexes maintenant. Le temps passe si vite, au moment de ma soutenance, ça fait déjà quatre ans dans l'équipe PIXEL. En revanche, mon souvenir de la première journée dans cette équipe est plus clair que celle d'hier. Je voudrais bien remercier particulièrement mes directeurs de thèse : Martin BYRDIN et Dominique BOURGEOIS. Vous m'avez appris les connaissances en physique, optique et spectroscopie petit à petit. Etant un biologiste de base, ce n'est pas une tâche facile. Je vous remercie d'avoir une patience importante envers moi surtout quand j'ai fait des bêtises. Je tiens à remercier à Virgile ADAM, chercheur dans l'équipe. Avec lui, j'ai acquis énormément de connaissances sur la cristallographie, les protéines fluorescentes et la microscopie, qui me permettent d'avancer mes projets en douceur. Romain BERARDOZZI, doctorant en 2 ème année. Grace à lui, je me suis familiarisé avec notre nouveau bâtiment IBS puisque j'étais en Allemagne lors qu'on déménageait vers ce nouveau bâtiment. Je te souhaite une bonne continuation et un bon avenir. Un grand merci à Delphine ARCIZET, l'ancienne post-doc d'équipe qui était ma tutrice de stage. Une personne avec pleine de charme et gentillesse. Merci beaucoup de ton

Fluorescent proteins (FPs)

Around the 1970s, wtGFP was firstly studied by a Japanese scientist named Osamu Shimomura [START_REF] Shimomura | Extraction, Purification and Properties of Aequorin, a Bioluminescent Protein from the Luminous Hydromedusan, Aequorea[END_REF]. The protein was purified from a marine Hydrozoa species: Aequorea victoria, a jelly fish found off the West coast of North America. However, wtGFP didn't attract attention of biologists until 1992, when Douglas Prasher cloned and sequenced its gene although at that time few amino acids were missing [START_REF] Prasher | Primary structure of the Aequorea victoria green-fluorescent protein[END_REF].

Two years after, Martin Chalfie expressed the gene in E. coli and C. elegans cells [START_REF] Chalfie | Green fluorescent protein as a marker for gene expression[END_REF] and successfully observed green fluorescence upon illumination with UV light.

However, wtGFP has some drawbacks such as its major excitation peak located in the UV region (395-397nm) [START_REF] Chattoraj | Ultra-fast excited state dynamics in green fluorescent protein: multiple states and proton transfer[END_REF]) (Fig1. A), and poor folding at 37 °C [START_REF] Tsien | The green fluorescent protein[END_REF]. Roger Y. Tsien began to engineer wtGFP by mutagenesis to produce GFP derivatives.

The first important improvement was a single point mutation (GFP-S65T) [START_REF] Heim | Improved green fluorescence[END_REF]. This mutation largely improved the spectral characteristics of GFP by shifting the major excitation peak to 488 nm (Fig1. B), and increased fluorescence brightness and photostability [START_REF] Cubitt | Understanding, improving and using green fluorescent proteins[END_REF]. Although organic dyes are reported to be more stable against photobleaching, FPs are still largely used as fluorescent markers thanks to their advantages such as labeling specificity, non-invasive labeling and one-to-one labeling that facilitates quantitative counting. [START_REF] Tsien | The green fluorescent protein[END_REF]

Fig1. Absorption spectra of GFP (A)

, and EGFP (B ).

Tsien and collaborators continued to make various GFP mutants aiming at designing proteins with better brightness and different colors, such as yellow, cyan, blue and red, which cover almost the whole range of visible colors (Fig2.). Another important mutant of GFP, Enhanced GFP (EGFP) developed by Thastrup and Falkow's lab has two mutations compared to wtGFP: F64L and S65T [START_REF] Cormack | FACS-optimized mutants of the green fluorescent protein (GFP)[END_REF]. The F64L mutation increased largely the folding efficiency at 37 °C, which, combined with the improvements of S65T, makes EGFP more practical to use as compared to wtGFP.

Fig2. FP variants that emit from blue fluorescence to red fluorescence. (Image from internet)

The Remington group firstly published the crystal structure of GFP-S65T [START_REF] Ormö | Crystal structure of the Aequorea victoria green fluorescent protein[END_REF] and subsequently the wtGFP crystal structure was published by the Phillips group [START_REF] Yang | The molecular structure of green fluorescent protein[END_REF]. GFP has a beta barrel structure with a length of 42 Å and a diameter of 24 Å. It is composed of 230 amino acids that form eleven beta strands connected by short polypeptide turns. There is one alpha helix going through the beta barrel with a chromophore 4-(p-hydroxybenzylidene)-5-imidazolinone (p-HBI) formed by the three central amino acids of this helix and situated in the center of the beta barrel. It is believed that this structure, fully conserved within all FPs, largely protects the chromophore from solvent interactions. However, within this thesis, our studies show that this strong protection might be overestimated. In wtGFP, the chromophore is formed by the three amino acids Ser65-Tyr66-Gly67. After protein folding, these three amino acids undergo a complex maturation reaction following a multi-step process including cyclization, oxidation and dehydration [START_REF] Zhang | Reaction progress of chromophore biogenesis in green fluorescent protein[END_REF]) (Fig3.). In the first step, cyclization of Tyr and Gly initializes the maturation of the chromophore, and then with the presence of molecular oxygen a hydrogen peroxide is produced by the oxidation reaction. A hydroxylated cyclic imine is formed in this step. At last the final product, a double bonded imidazolinone ring, is formed and a matured chromophore that is fully conjugated is ready to fluoresce [START_REF] Zhang | Reaction progress of chromophore biogenesis in green fluorescent protein[END_REF]. The maturation of the chromophore is an autocatalytic process and it doesn't need any external factor except molecular oxygen.

Fig3. Maturation of the chromophore. [START_REF] Zhang | Reaction progress of chromophore biogenesis in green fluorescent protein[END_REF] The resolved structures allowed researchers to visualize the chromophore that is responsible for fluorescence as well as its environment and interactions with neighboring residues. With the help of these crystal structures, engineering of new GFP variants by directed mutagenesis was accelerated and rationalized.

We are far from completely understanding all residues' roles in the FPs, rational engineering of FPs is usually very difficult and brings unexpected consequences. Thus, random mutagenesis is a standard method used in developing FP mutants as well.

PhotoTransformable Fluorescent Proteins (PTFPs)

A subfamily of fluorescent proteins called PhotoTransformable FPs (PTFPs) has the unique optical property that their fluorescent state can be quantitatively modified by actinic illumination. Most of the PTFPs were found in Anthozoan corals unlike wtGFP that is found in Hydrozoan Jellyfish. However, engineering of Hydrozoan FPs can also result in phototransformability. Thanks to their subtle manipulation by light, PTFPs are more and more in the focus of research and in fact, they revolutionized the field of fluorescent microscopy.

PTFPs can be classified in three groups: PhotoActivatable Fluorescent Proteins (PAFPs) that can be activated from a non-fluorescent state to a fluorescent state irreversibly by proper light; PhotoConvertible Fluorescent Proteins (PCFPs) that exhibit a nonreversible photoconversion from one fluorescent emission state to another, normally from green to red; and finally, Reversibly Switchable Fluorescent Proteins (RSFPs) that are able to be reversibly switched between nonfluorescent state (off-state) and fluorescent state (on-state) many times.

PhotoActivatable Fluorescent Proteins (PAFPs)

A single site mutation T203H of wtGFP [START_REF] Patterson | A photoactivatable GFP for selective photolabeling of proteins and cells[END_REF] generated PhotoActivatable Green Fluorescent Protein (PA-GFP). Its chromophore is initially found in its protonated, non-fluorescent state. The T203H substitution made the side chain of Glu222 (GFP numbering) rotate away from His203. Thus it occupies a slightly different position than that in wtGFP and stabilizes the protonated chromophore that hence cannot fluoresce under 488-nm illumination (Henderson et al., 2009). By illumination with high energy violet light, the highly conserved Glu222 undergoes an oxidative decarboxylation (Kolbe mechanism) followed by a reorganization of the H-bond network, stabilizing the deprotonated chromophore. The fluorescence brightness under excitation by 488-nm light is thus increased 100 times after photoactivation [START_REF] Patterson | A photoactivatable GFP for selective photolabeling of proteins and cells[END_REF] (Fig5.). Other PAFPs such as PA-mRFP1 [START_REF] Gurskaya | Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light[END_REF], PA-mCherry1 [START_REF] York | Confined activation and subdiffractive localization enables whole-cell PALM with genetically expressed probes[END_REF], PA-TagRFP (Subach et al., 2010a) can also be activated by UV light to emit red fluorescence.

Fig5. Photoactivation of PA-GFP.

PhotoConvertible Fluorescent Proteins (PCFPs)

Green-to-Red photoconvertible fluorescent proteins were discovered by chance. Once, Ando et al left a sample of Kaede protein on the lab bench without any protection from sunlight. The next day, they noticed that the exposed sample emitted red fluorescence instead of green fluorescence [START_REF] Ando | An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein[END_REF]. Further studies revealed that this green to red conversion mechanism is due to the cleavage of the protein backbone, between the amide nitrogen and Cα atoms of His62 (Mizuno et al., 2003) (Fig6.). This cleavage resulted from the absorption of UV light in the protonated state of the chromophore and produced an extension of the electron conjugated system of the chromophore, which induced a red-shifted fluorescence emission. Other proteins such as EosFP [START_REF] Wiedenmann | EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion[END_REF], mEosFP [START_REF] Wiedenmann | EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion[END_REF], mEosFP2 [START_REF] Mckinney | A bright and photostable photoconvertible fluorescent protein[END_REF], Dendra [START_REF] Gurskaya | Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light[END_REF], Dendra2 (Evrogen) all belong to this protein type. PCFPs are mostly used in PALM (PhotoActivated Localization Microscopy) technique thanks to their good brightness in the green form that is observed at the ensemble level and in the red form that is observed at the single molecule level.

Fig6. Photoconversion of Kaede.

Reversibly Switchable Fluorescent Proteins (RSFPs)

The phenomenon of reversible photoswitching was first observed in wtGFP and its yellow variants (Dickson et al., 1997). However, their switching contrast (ratio of emitted fluorescence in the on and off states) was limited. The first well studied RSFP was Dronpa (Mizuno et al., 2008) due to its good photoswitching behavior. Thanks to the unique ability of repetitive switching, RSFPs open up a number of new possibilities including photochromic FRET (Giordano et al., 2002), and super-resolution microscopy such as two-color PALM (Shroff et al., 2007), RESOLFT (REversible Saturable OpticaL Fluorescence Transitions) (Hofmann et al., 2005) and SSIM (Saturated Structured Illumination Microscopy) (Rego et al., 2012) approaches. With years of development, by using site-directed mutagenesis or random mutagenesis, the RSFP family has largely expanded. In particular, Anthozoan RSFPs and their variants are widely used such as mGOS family (Chang et al., 2012), rsTagRFP (Subach et al., 2010b) and the Dronpa family: Dronpa (Ando et al., 2004), Dronpa2 (Ando et al., 2007), Dronpa3 (Ando et al., 2007), bsDronpa (Andresen et al., 2008), rsFastLime (Stiel et al., 2007) and Padron (Andresen et al., 2008). In recent years, by protein engineering of Hydrozoan EGFP, Mut2Q, EYQ1, EYQ2 (Bizzarri et al., 2010), Dreiklang (Brakemann et al., 2011) , modBFP [START_REF] Jablonski | Optically modulatable blue fluorescent proteins[END_REF], rsEGFP (Grotjohann et al., 2011) and rsEGFP2 (Grotjohann et al., 2012) were introduced and reported to have a very good performance in photoswitching.

RSFPs can be subdivided in three types: negative RSFPs, positive RSFPs, and decoupled RSFP.

Crystallographic structures suggested that in negative and positive RSFPs, photoswitching is the consequence of cis-trans isomerization of the chromophore, whereas spectroscopic studies showed that light induced protonation/deprotonation were also involved in the photoswitching.

In negative RSFPs such as Dronpa and its variants, in the native state of the proteins, the chromophores are in their on state. The excitation light makes the proteins fluoresce and in the meantime, on-to-off photoswitching competes with the fluorescence. Generally, the chromophore is changed from the cis conformation to the trans conformation and the protein absorption peak switches from blue to UV, which means off switched proteins are protonated.

Fluorescence emission can decrease to a very low level. However, the fluorescence emission level can be brought back when the protein is illuminated by UV light (Fig7.). In 2011, the Jakobs group reported a RSFP with a different photoswitching mechanism: Dreiklang. Dreiklang is neither a negative nor a positive RSFP. Based on crystallography studies, it is proposed that a water molecule close to the imidazolinone ring can be covalently attached or detached, upon photoswitching. This hydration and dehydration is triggered by light that is not absorbed by the on or off states, hence decoupled from excitation (Fig9.).

Fig9. Photoswitching of Dreiklang.

In recent years, researchers aimed to develop switching speed and contrast of RSFPs to adapt them to advanced imaging technics. For example, rsEGFP2 is a fast switcher with high switching contrast, which allows it to be a good fluorescent marker for RESOLFT.

Thanks to its fast switching speed, it significantly accelerates the scanning speed of RESOLFT.

IrisFP : A combination of RSFP and PCFP

IrisFP is derived from EosFP, a PCFP that is able to be converted from green to red emission by 405 nm light. Mutation of F173S together with a silent mutation F191L generated IrisFP, the first FP reported to combine the properties of RSFPs and PCFPs at the same time. IrisFP is able to photoswitch efficiently in its green state. Upon UV illumination, it can be converted into a red form in which it is also able to switch (Fig10.).

Fig10. Photoswitching and photoconversion of IrisFP. *Red-trans structure is not available

Based on the crystallographic structures of IrisFP-green-on (PDB entry 2VVH), IrisFP-green-off (PDB entry 2VVI) and IrisFP-red-on (PDB entry 2VVJ), we can see that this protein undergoes a cis-trans isomerization of the chromophore during photoswitching in green form. Like in EosFP, absorption of UV light can lead to protein backbone cleavage and convert IrisFP to its red form. Although the structure of IrisFP in its red-off state could not be obtained, it is reasonable to think that in the red state the chromophore isomerizes as well.

The reason why the F173S mutation can turn EosFP into a photoswitchable protein is that Phe173 in EosFP occupies a large space. Upon substitution by the smaller serine residue, a considerable space is freed, which becomes occupied by a labile water molecule w2166 that makes an H-bond to both Ser173 and Thr59. Moreover, Met159 is rotated towards Ser173, which results in another new water molecule w2188 filling the cavity. This water molecule is H-bonded to Ser142, lowering the bond energy of this residue to the chromophore [START_REF] Moeyaert | Structural insight into fluorescent protein properties by rational design and characterization of photoconvertible, photoswitchable and phototoxic fluorescent proteins[END_REF].

When illuminated by 488nm light (absorption peak in green-on form corresponding to deprotonated chromophore) we can see the decrease of both fluorescence at 512 nm and absorption at 488 nm, meanwhile the absorption peak at 390 nm corresponding to the protonated chromophore increases. When the off switched protein is illuminated by UV light (405 nm), the absorption of the protonated chromophore decreases and that of the deprotonated chromophore increases together with fluorescence emission. In the red form, the situation is similar, except that red fluorescence emission is at 580 nm and that 551 nm light is responsible for on-to-off switching and 450 nm light is responsible for off-to-on switching (Fig11.). Monomeric variants exist that combine properties of RSFPs and PCFPs such as

Photobleaching and photofatigue

Photobleaching (Fig12.) is the permanent loss of fluorescence emission capacity, in contrast to photoblinking that is reversible and photophysically more complex. We don't discuss the photoblinking in this thesis.

Fig12. Photobleaching of EGFP. Fluorescence decays upon excitation of 473 nm light (~100W/cm²).

In fluorescence microscopy, photobleaching is always a problematic issue since it can introduce difficulties for data accuracy and analysis. For example, in time-lapse fluorescence microscopy, a series of images are recorded as a function of time. Photobleaching of fluorescent markers makes the time of observation of live cells limited. Most published studies about photobleaching deal with organic dyes [START_REF] Zheng | Ultra-stable organic fluorophores for single-molecule research[END_REF]. Oxygen is generally considered as the main factor that is responsible for photobleaching of these fluorophores [START_REF] Christ | Watching the Photo-Oxidation of a Single Aromatic Hydrocarbon Molecule[END_REF][START_REF] Song | Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy[END_REF][START_REF] Zondervan | Photobleaching of Rhodamine 6G in Poly(vinyl alcohol) at the Ensemble and Single-Molecule Levels[END_REF]. It is reported that fluorophores in their singlet excited state can hardly react with oxygen due to the short lifetime (several ns) of these states [START_REF] Gollnick | Merbromin (mercurochrome) and other xanthene dyes: Quantum yields of triplet sensitizer generation and singlet oxygen formation in alcoholic solutions[END_REF]. Because of the considerably longer lifetime of the triplet state, it is usually considered to be the departure point of the harmful reactions that can induce photobleaching [START_REF] Song | Influence of the triplet excited state on the photobleaching kinetics of fluorescein in microscopy[END_REF]. Typically, electron transfer or energy transfer from a fluorophore in the triplet state to molecular oxygen (whose ground state is triplet state), can return the chromophore to its ground state and generate superoxide radical or singlet oxygen that are very reactive [START_REF] Zheng | Ultra-stable organic fluorophores for single-molecule research[END_REF]. The reactive oxygen species (ROS) can easily attack and destroy the fluorophore by introducing irreversible chemical modifications, typically oxidation. The generated ROS also have the possibility to diffuse and react with closeby molecules, which causes phototoxicity. Thus people often add antifading reagents (acting thanks to their reducing property), or oxygen scavenging enzyme [START_REF] Cordes | On the Mechanism of Trolox as Antiblinking and Antibleaching Reagent[END_REF][START_REF] Penttilä | Active Oxygen Intermediates and Chlorophyllin Bleaching[END_REF], or both of them [START_REF] Rasnik | Nonblinking and long-lasting single-molecule fluorescence imaging[END_REF] to the cell culture medium to reduce photobleaching effects. Some studies showed that the photobleaching rate non-linearly increases as a function of excitation power intensity, indicating multi-photon absorption by the chromophore [START_REF] Hoogenboom | Power-law-distributed dark states are the main pathway for photobleaching of single organic molecules[END_REF].

However, in case of FPs, the photobleaching mechanism is more complicated. Unlike in the organic dyes that are fully exposed to the solvent environment, the chromophore of FPs is buried in the center of the beta barrel and has close interactions with the surrounding residues. The beta barrel limits the access of oxygen molecules. A study demonstrated that EGFP can generate singlet oxygen but with lower efficiency than that of the isolated HBDI chromophore exposed to the solvent, suggesting that the beta barrel provides shielding of the chromophore (Jimenez-Banzo et al., 2008). Therefore, less oxygen could produce less phototoxicity. However, photobleaching in FPs is facilitated due to the complex interaction of the chromophore with its surrounding residues. The generated ROS are not only able to attack the chromophore itself but also other residues inside the FP molecule. Another study showed that, for example, in RFP, photobleaching is caused by chromophore photoreduction [START_REF] Vegh | Chromophore Photoreduction in Red Fluorescent Proteins Is Responsible for Bleaching and Phototoxicity[END_REF]. Photobleaching also depends on the cell environment (Malkani and Schmid, 2011).

Photobleaching can be used at advantage in biological applications as well. The most widely used technics are FRAP (Fluorescence Recovery After Photobleaching) [START_REF] White | Photobleaching GFP reveals protein dynamics inside live cells[END_REF], FLIP (Fluorescence Loss In Photobleaching) [START_REF] Wustner | Quantitative fluorescence loss in photobleaching for analysis of protein transport and aggregation[END_REF]) that allow investigators to measure molecule mobility in vivo. Photobleaching can also be applied in counting protein molecules by stepwise photobleaching, which allows researchers to quantify molecule number in a cluster or determine the protein oligomerization sate [START_REF] Groulx | Single Molecule Fluorescence Study of the Bacillus thuringiensis Toxin Cry1Aa Reveals Tetramerization[END_REF][START_REF] Zijlstra | Molecular Composition of Sub-stoichiometrically Labeled α-Synuclein Oligomers Determined by Single-Molecule Photobleaching[END_REF]. PALM is based on the principle of repeated single molecules activation, localization and photobleaching.

Different FPs display diverse photobleaching kinetics that result from different chromophore photostability and environments. Usually the fluorescence curve doesn't show a mono-exponential decay suggesting that multiple photophysical processes may occur.

Within RSFPs, upon excitation, fluorescence emission competes with other photophysical processes, in particular, on-or off-switching and photobleaching. For example, under illumination, negative RSFPs quickly lose their fluorescence due to a combination of off-switching and photobleaching. In one photoswitching cycle, due to the much lower photobleaching quantum yield (typically 10 -5 ) than photoswitching quantum yield (10 -2 -10 -3 ), most of the chromophores are switched to their off state and are still capable to fluoresce when switched on again. However, upon a large number of photoswitching cycles, the photobleached chromophores accumulate. Overall, at the ensemble level, the amplitude of fluorescence of each cycle decreases and the achievable number of photoswitching cycles is thus limited, a process known as photofatigue (Fig13.).

Fig13. Photofatigue of IrisFP. The maximum of fluorescence intensity at each cycle decreases

because of photobleaching.

Goal of thesis

Due to their unique property of reversible switchability, RSFPs have become a very powerful tool in the field of bio-imaging, reversible data storage, viscosity measurement, optogenetics (Fig14.) Fig14. Applications of RSFPs (Duan et al., 2014). Amongst these technics, super-resolution imaging such as RESOLFT, Photochromic SOFI (pcSOFI) (Dedecker et al., 2012), Nonlinear Structured Illumination Microscopy (NSIM) (Gustafsson, 2005) essentially depend on a large number of switching cycles of RSFP molecules. Thus a high resistance against photofatigue is required. For instance, in RESOLFT, a confocal system is employed. At each position of the scanner, a donut shaped dump light is applied to switch off all the molecules in the peripheral area leaving only molecules in the beam center active. A readout beam is then applied to detect the active labeled molecules and their signals are recorded. At the end, a reset beam is employed to restore molecules back to their on state, then the detector head moves to the next point. To obtain a high spatial resolution, the scanning step is required to be very small. Thus, a given molecule should be able to undergo several dozens of switching cycles (typically ~100 to achieve a spatial resolution of 1/10 of the PSF width in both X and Y dimensions).

As mentioned before, the photobleaching mechanism of FPs is not clearly understood.

And this also applies to the photobleaching mechanisms within RSFPs. Random mutagenesis and large scale screening are usually used to generate better RSFPs mutants in terms of brightness, fluorescence quantum yield, photoswitching speed, photoswitching contrast. However, screening of mutants with enhanced photoresistance would be more delicate because it is notably time consuming. Thus, it would be strongly desirable to design better performing mutants in a rational manner. Although our understanding of brightness or fluorescence quantum yield is increasing, we still have very limited knowledge about photobleaching. Therefore, the understanding of the photobleaching mechanisms of RSFPs is very important. However, there was no previous study of photobleaching of RSFPs before this thesis. Thanks to our experience in combined kinetic X-ray crystallography and optical spectroscopy (absorption, fluorescence and Raman spectroscopy) and good knowledge of PTFPs, and in collaborating with researchers specialized in QM/MM and molecular dynamics modeling, we have acquired the tools to explore the photobleaching mechanisms of RSFPs.

We have chosen IrisFP as our model FP to investigate the photobleaching mechanism in Anthozoan RSFPs since its photophysical properties have been extensively studied in our team, including its crystallographic structures in green-cis, green-trans, red-cis (Adam et al., 2008) and blinked states (Adam et al., 2009). Based on our understanding of the photobleaching mechanism of IrisFP from these studies, we rationally designed an IrisFP mutant with increased photostability that could be more suitable to the above-mentioned techniques.

Abstract of article 1

The first photobleached structure of IrisFP that we observed was from a crystal irradiated by X-rays at cryotemperature (100K). It displayed a decarboxylation of Glu212 and a distorted chromophore. However, this condition is far from the condition that is found in a microscopy experiment. We were interested to know if these structural changes also happen under real experimental conditions. Thus, we tried to carry out experiments at room temperature using the PALM microscope that was set up in our lab. Using the PALM setup, we were able to photobleach protein crystals under PALM conditions. X-ray crystallography then showed that an oxygen-independent photobleaching occurred where dramatic structural changes happened (characterized by light-induced decarboxylation of the strictly conserved Glutamate 212 coupled with chromophore conformational changes and H-bond network rearrangement, which destroy the π conjugated system of the chromophore). To try to figure out the chemical nature of the photobleached chromophore, molecular dynamics simulations were performed by our collaborators at Physical Chemistry Laboratory at University of Paris 11. Suggested by the simulation, under high-intensity illumination, photobleaching is a redoxbased process, where the methylene bridge of the chromophore was reduced resulting in the destruction of the π conjugated system. Due to the high laser intensity, the crystals were often damaged. In order to preserve crystal quality, we largely decreased the laser intensity. To our surprise, although crystals survived photobleaching, they had even worse diffraction quality.

We found that, in contrast to the high-intensity illumination, under low-intensity illumination, an oxygen-dependent photobleaching occurred, which introduces only few structural changes.

The photobleached structure shows an intact chromophore and surrounding H-bond network.

The decarboxylation of Glu212 doesn't take place. Crystallographic data showed positive electron densities close to Met159 and Cys171. To interpret this data, we collaborated with Sylvie Kieffer-Jaquinod at Laboratoire de Biologie à Grande Echelle at CEA. Confirmed by mass spectrometry, Met159 and Cys171 are found sulfoxided. The neighboring Met159 is supposed to be responsible for the photobleaching, because when sulfoxided, it forms a strong H-bond with the chromophore, trapping it in a protonated state that is non-fluorescent.

Structural Evidence for a Two-Regime Photobleaching Mechanism in a Reversibly Switchable Fluorescent Protein

■ INTRODUCTION

Recent progresses in fluorescence bioimaging techniques have benefited from the rapid development of a large palette of fluorescent proteins (FPs). 1 However, the limited photostability of FPs has remained a major impediment to their successful use in many approaches including single-molecule, FRET, time-lapse or super-resolution microscopies. FPs typically can only emit ∼10 5 photons before their chromophores fall victim to irreversible photodestruction. 2 The detailed photophysical mechanisms leading to photobleaching in FPs remain largely unknown, although some structural insight has been obtained in the case of KillerRed, a highly phototoxic FP. 3-5 Successful efforts to obtain more photostable variants have mainly relied on directed evolution approaches. 2,6-8 In reversibly switchable fluorescent proteins (RSFPs), 9 photobleaching manifests itself in a process referred to as "photofatigue". RSFPs can be repeatedly photoswitched between a fluorescent (on) and a nonfluorescent (off) state by illumination with visible light of appropriate wavelengths. Photoswitching capabilities are central to a growing number of advanced techniques including a variety of super-resolution modalities, 10-14 photochromic FRET, 15 optical lock-in detection, 16 frequency-domain imaging, 17 optogenetic manipula-tion 18 and bio data-storage. 19,20 However, because photobleaching competes with photoswitching, a progressive decrease in fluorescence intensity at each on-off cycle (that is, photofatigue) is observed in ensemble experiments, and at the single molecule level, the number of achievable on-off cycles is limited. Thus, photofatigue fundamentally limits the achievable resolution in nanoscopy approaches such as optically linear fluorescence transition microscopy (RESOLFT) 10 and nonlinear structured illumination microscopy (NSIM), 14 or the contrast enhancement capability in frequency-domain based microscopy schemes. 16,17 Efforts to develop fatigue-resistant RSFPs have been made recently, again essentially based on directed-evolution approaches. 20-22 Several parameters exert an influence on the photostability of fluorescent proteins. Although the compact FP β-barrel partially shields the chromophore against molecular oxygen, a number of experimental, 23-25 and theoretical 26,27 investigations concluded that O 2 plays an important role in photobleaching of FPs. However, the exact mechanism by which oxygendependent chromophore photodestruction occurs has not been directly visualized. Light-induced redox chemistry has also been described to cause a variety of chromophore phototransformations 28 and the photostability of GFP and some derivatives has been shown to depend on the presence of redox-active components in the environment. 29,30 Decarboxylation of the strictly conserved Glu212 (IrisFP numbering, corresponding to Glu222 in GFP) has been described in several cases and is notably involved in photoactivation 31-33 or the formation of super-red species. 34 Such decarboxylation was also observed in experimental conditions not routinely used in fluorescence microscopy, such as at cryogenic temperature 5,35 or under X-ray irradiation. 36,37 The possible involvement of Glu212 decarboxylation in photobleaching under commonly used fluorescence microscopy schemes, nevertheless, has not been documented. In general, it has been repeatedly reported that the photostability of fluorescent proteins may strongly depend on illumination conditions. 2,38-41 Here, we set out to study the photofatigue mechanism of the biphotochromic RSFP named IrisFP, 42 under experimental conditions typical of wide-field diffraction-limited and superresolution microscopy. Our previous investigations had revealed the structural and spectroscopic signature of IrisFP in its green and red on-states (chromophore in cis conformation), in the corresponding off-states (chromophore in trans conformation), and in a reversible dark state (displaying a transiently distorted chromophore geometry consistent with a radical species protonated at atom Cα of the methylene bridge 37,43 ). In the present work, employing a combination of kinetic X-ray crystallography, in crystallo UV-vis absorbance, fluorescence and Raman spectroscopies, mass spectrometry, and molecular dynamics simulations, we discovered two distinct photobleaching mechanisms in IrisFP. The first mechanism is oxygen-independent, whereas the second is oxygen-dependent. A switch from the first to the second mechanism was observed as the illumination power density was decreased from levels typical of localization-based nanoscopy to those of standard wide-field microscopy.

■ EXPERIMENTAL PROCEDURES

Experimental procedures are described in details in the Supporting Information.

■ RESULTS

Photofatigue at High Illumination Intensity. In green IrisFP, illumination by 488-nm (cyan) light isomerizes the fluorescent cis anionic chromophore to a trans nonfluorescent neutral configuration (quantum yield = 3.2 × 10 -3 ). Backswitching to the fluorescent cis state is efficiently achieved with 405-nm (violet) light (quantum yield =0.15). 42 We thought of studying the photofatigue of green IrisFP in crystallo under experimental conditions typical of super-resolution PALM microscopy, that is, under relatively high excitation laser power density (∼0.1 kW/cm 2 at 488 nm, see the Supporting Information, Figure S1A). After 10 min of alternating illumination at 488 and 405 nm, corresponding to about 260 switching cycles, the fluorescence intensity was largely reduced (Figure 1). The decay of the fluorescence envelope appears biphasic and can be fitted with a biexponential model in which ∼75% of the molecules undergo rapid bleaching and ∼25% undergo ∼10 times slower bleaching (see discussion in the Supporting Information). Absorbance by the anionic chromophores largely decreased without any increase of the neutral species, suggesting that most molecules were irreversibly bleached (Figure 2A). Similar behavior was observed in solution (Figure S2, Supporting Information).

Experimental difference electron density maps between fatigued and nonfatigued parts of a single crystal (Figure 2C and Table S1, Supporting Information) revealed a complex set of structural modifications confined in the chromophore pocket (Figure S5A, Supporting Information). Strong negative electron density at Glu212 suggested decarboxylation of this residue, as unambiguously confirmed by mass spectrometry analysis (Table S2, Supporting Information). The hydrogen-bond network around the chromophore was largely perturbed, with three water molecules being dislocated and Arg66 and His194 adopting a conformation similar to that found in the trans state of the chromophore. The phenolate group of the chromophore appeared largely disordered, together with the hydroxyl group of Ser142 normally H-bonded to the chromophore phenolate. The imidazolinone ring, however, remained planar. These crystallographic data suggest that the chromophore is no longer properly held in place by the protein matrix and may have lost its electron conjugation.

To gain further insight into the chemical modifications of the chromophore, we performed in crystallo Raman spectroscopy of IrisFP crystals illuminated in the same manner. Upon photobleaching, the spectra (Figure 2B) showed considerable modifications (see discussion in the Supporting Information), notably a complete disappearance of the bands at 1503 cm -1 and 1545 cm -1 (anionic chromophore) and 1564 cm -1 and 1604 cm -1 (neutral chromophore). The strongly resonanceenhanced band at 1545 cm -1 has been assigned to a mode that combines stretching of the CαC5 exocyclic double bond and deformation of the imidazolinone moiety of the chromophore 44 (for chromophore atoms nomenclature, see Scheme S1 in the Supporting Information). In the neutral state of the chromophore, this band is shifted to 1564 cm -1 , consistent with a more localized electron density in this state. The loss of these bands, together with the vanished UV-vis absorbance, strongly suggests that the Cα carbon atom of the chromophore methylene bridge converts from an sp 2 -hybridized to an sp 3hybridized configuration, possibly as a result of intramolecular electron and proton transfer. 43 Based on these findings, as well as on further molecular dynamics simulations (see below) a model of photodamaged IrisFP was refined (Figure 2D).

Overall, these data suggest that under high-intensity illumination IrisFP suffers from a redox-based photofatigue mechanism, which is associated to decarboxylation of Glu212.

Photofatigue at Low Illumination Intensity. Submitting IrisFP crystals to repeated high-intensity laser illumination is a harsh procedure that often compromises their diffraction quality. In an attempt to better preserve the samples, we photofatigued IrisFP crystals at ∼10 times lower power density (∼10 W/cm 2 at 488 nm, see the Supporting Information, Figure S1B). After 100 min of alternating cyan and violet illumination (460 switching cycles), fluorescence emission was again largely reduced (Figure 3). However, the fluorescence photofatigue decay profile (Figure 3, red curve) significantly differed from that under high illumination, although a biexponential model was still required to achieve a satisfactory fit (see discussion in the Supporting Information).

To our surprise, the diffraction quality of the crystals was not improved. Instead, it was generally even more degraded. Furthermore, crystallographic analysis revealed neither any substantial structural changes of Glu212 nor any significant distortion or disorder of the chromophore (Figure 4C and Table S1, Supporting Information). Instead, additional positive electron density close to the sulfur atoms of both Met159 and Cys171 was noticed in the difference electron density maps (Figure 4C and Figure S5B, Supporting Information). Absorption spectra of crystals fatigued in this way showed a decreased anionic band but an increased neutral band (Figure 4A). A titration experiment revealed that the pK a of the photofatigued chromophore increased to ∼12 (Figure S8, Supporting Information) instead of 5.7 under native conditions, 45 suggesting that the chromophore has been trapped in a nonfluorescent protonated state. Raman spectrometry of a partially fatigued crystal confirmed this finding, displaying an increase of the vibrational bands associated with the neutral chromophore (1564 cm -1 , 1604 cm -1 ) at the expense of those associated with the anionic chromophore (1503 cm -1 , 1545 cm -1 ) (Figure 4B), but without signs of a ruptured π-system. Finally, peptide analysis of a photofatigued sample by mass spectrometry revealed a substantial increase in the level of oxidation of a number of fragments containing methionine, cysteine, and tryptophan residues (Figure S9, Supporting Information). In particular, fragments that contained Met159 had ∼320% higher mono-oxidation levels than those of intact IrisFP. Oxidation levels of fragments containing Cys171 were also raised significantly (see Figure S9 caption in the Supporting Information). Conversely, the decarboxylation level of Glu212 remained unaltered as compared to nonbleached IrisFP (Table S2, Supporting Information). These data suggest that, under low-intensity illumination, photofatigue of IrisFP results in sulfoxidation of Met159 and Cys171, followed by oxidation of other residues more remote from the chromophore pocket. Comforted by the mass spectrometry results, we modeled the two positive features near Met159 and Cys171 in the difference electron density map as oxygen atoms covalently bonded to the sulfur atoms of these residues, respectively (Figure 4D). A new water molecule was also modeled next to Met159, above the chromophore hydroxybenzylidene ring. The sulfoxidized Met159 was found to form a tight H-bond (2.8 Å) between the newly added oxygen atom and the presumably protonated chromophore phenol moiety. This tight H-bond is consistent with the extremely high pK a measured for the fatigued chromophore. Interestingly, the level of Met159 and Cys171 sulfoxidation observed in the electron density maps strongly varied between the four IrisFP monomers. Whereas significant electron densities were observed in monomers A, B, and D, no sign of sulfoxidation could be detected in monomer C (Figure S6A-D, Supporting Information). This difference could be explained by a variable diffusion capacity of oxygen molecules into the chromophore pocket via pores in the IrisFP β-barrel, probably due to crystal packing effects. In monomers A, B, and D, inspection of the IrisFP static structure showed a pore between the residues Glu140 and Ile196, in the close vicinity of the chromophore phenolate (Figure S6E-H, Supporting Information) at a location previously identified in other FPs such as His148Asp-YFP 46 or the Arthropoda TurboGFP. 47 This pore is occluded in monomer C.

Overall, the data suggest that under low-intensity illumination IrisFP suffers from an oxidation-based photofatigue mechanism, which is initiated by the production of singletoxygen within the chromophore pocket.

Further Insight into IrisFP Photobleaching under High-Intensity Illumination. The novelty of the decarboxylation-based photofatigue mechanism of IrisFP, its complex structural signature and its potential relevance for highresolution microscopy prompted us to investigate this mechanism in more details. Notably, we asked the four following questions: (i) Which wavelength (488 or 405 nm) is primarily responsible for photodestruction? (ii) What is the temporal order of the structural events leading to the observed photofatigued structure? (iii) What is the chemical nature of the photobleached chromophore? (iv) Is a two-consecutivephoton absorption process involved? Question i can be readily answered in the case of lowintensity photobleaching, as the chromophore clearly adopts a cis configuration in this bleached state, strongly suggesting that photobleaching results from the absorption of a 488-nm photon. The question is more delicate to answer in the case of high-intensity photobleaching, as two arguments could favor the hypothesis that photodestruction originates from violet light illumination. First, it has been reported that decarboxylation reactions occurring via a Photo-Kolbe mechanism are favored in the UV range. 48 Second, the conformational switch of the His194-Arg66 pair observed in our structure (Figure 2C and 2D) seems at first glance consistent with the chromophore being photobleached in its trans configuration by violet light. However, close inspection of the difference electron density map of Figure 2C shows that residue Ile157 maintains a conformation typical of the cis chromophoric state. 42 This suggests that the His194-Arg66 switch could be a consequence of photobleaching by cyan light in the cis configuration of the chromophore. To test this hypothesis, crystals of green IrisFP were illuminated at 100 K at 488 nm for a prolonged time. At this temperature, chromophore isomerization is prevented, likely due to the lack of sufficient conformational flexibility of the IrisFP chromophore pocket, and thus no switching by cistrans isomerization can occur. Despite a deterioration of the crystalline order resulting from this harsh sample treatment, difference electron density maps clearly showed that 488 nm light is able to induce extensive Glu212 decarboxylation, similarly to 405 nm light (Figure S4, Supporting Information). The conformational switch of the His194-Arg66 pair subsequent to Glu212 decarboxylation in the cis chromophoric state was then confirmed by molecular dynamics simulations (see below). These arguments, together with the fact that the sample was exposed to ∼600 times more cyan than violet photons, favor the hypothesis that photodestruction upon repeated switching in IrisFP predominantly results from light absorption at 488 nm by the cis chromophore.

We next attempted to disentangle the order of the structural events leading to chromophore destruction (question ii) by collecting a high-quality structure of IrisFP en-route to photobleaching. Knowing that X-rays efficiently induce IrisFP photobleaching through Glu212 decarboxylation, 37 we reasoned that collecting a pair of high-resolution crystallographic structures at 100K at different X-ray doses might uncover structural differences representative of early events along the photobleaching pathway. The results, presented in Figure S7 (Supporting Information) (see also Tables S1 andS3, Supporting Information), reveal decarboxylation of Glu212 as well as the disappearance of several water molecules participating in the hydrogen bond network surrounding the chromophore. However, instead of the disorder of the phenolate moiety observed in the photofatigued structure, the chromophore exhibits a clear distortion with an upward tilt of the chromophore phenolate and a downward bend of the methylene bridge. This distortion is also consistent with sp 3hybridization of the C α carbon atom (Figure 2D). Furthermore, His194 and Arg66 are not significantly displaced as compared to their native conformation. If we admit that photobleaching pathways induced by high-intensity visible and X-ray light are both initiated by redox processes leading to Glu212 decarboxylation, it is reasonable to associate these structural changes with an early intermediate state along the highintensity photofatigue pathway.

To further investigate whether the X-ray bleached cryostructure is a plausible intermediate state along the high- intensity photofatigue pathway, we conducted molecular dynamics simulations using software specifically tailored for fluorescent proteins simulations. 49 These simulations also allowed us to examine the chemical nature of the fatigued chromophore (question iii). Starting from the putative intermediate state with the chromophore adopting various chemical structures, we analyzed whether or not the IrisFP conformation evolved toward the experimentally observed fatigued structure. In order to ensure the sp 3 -character of the Cα atom revealed by Raman spectroscopy while excluding an unstable radical state of the fatigued chromophore, the latter must be considered as formally reduced by two hydrogen atoms. Among all available choices tested (Figure S10, Table S4, Supporting Information), the reduced structure protonated at positions Cα and N4 (referred to as CαN) was found to be the only one compatible with our experimental data (Figure 5A). Protonation at other positions either altered the planar character of the imidazolinone ring or weakened the hydrogen bond between that ring and Arg91 (see further discussion in the Supporting Information). Using structure CαN in our MD simulations, we noticed that the conformational switch of His194 and Arg66 to their positions observed in the fatigued structure occurred within a nanosecond (Figure 5B and Figure S14, Supporting Information). Furthermore, large fluctuations of the chromophore torsions around the Cα atom were observed afterward (the 80% confidence interval of the dihedral τ angle ranged from -5°to 55°), reflecting the interplay between the intramolecular energy landscape of the reduced chromophore CαN (Figure S11, Supporting Information) and the decreased constraints due to the protein environment. These fluctuations are consistent with the phenolate disorder observed experimentally.

Finally, in order to investigate whether the high-intensity bleaching mechanism involves a two-consecutive-photon absorption process (question iv), we measured the initial photofatigue rate as a function of the illumination power density in the absence of oxygen (Figure S3, Supporting Information). The results confirm that O 2 is indeed not required in this mechanism. The best linear fit to a doublelogarithmic plot of the data showed a slope of 1.8 ± 0.3 (Figure S3, Supporting Information), indicating a mechanism mainly involving two photons with a possible weak residual from a one-photon contribution.

■ DISCUSSION

Our study of the photofatigue mechanism of the reversibly switchable protein IrisFP reveals a two-regime photodestruction pathway. At illumination intensities of ∼100 W/cm 2 , photobleaching of IrisFP involves decarboxylation of Glu212 probably via electron transfer to the chromophore in a photo-Kolbe reaction. 31,48 We propose that this leads to a prebleached dark state, represented by the cryo-trapped structure of Figure S7 (Supporting Information), in which the chromophore is already reduced and protonated at the Cα atom and at the N4 nitrogen atom. In this state, denoted pBl red , the chromophore has lost its electron conjugation and adopts a tilted geometry. (Note that pBl red differs from the blinked radical state DH • described earlier by us. 43 ) Subsequently, while the chemical structure of the chromophore does not evolve further, the hydrogen bond network surrounding it collapses, and the His194-Arg66 pair flips to a conformation resembling that of IrisFP in its switched-off state. The phenolate moiety of the chromophore loses its anchoring to the protein matrix, resulting in large fluctuations of the τ and φ dihedral angles. A complete loss of absorbance and fluorescence is thus observed in this bleached state, referred to as Bl red , and the presence of oxygen is not required.

At illumination intensities ten times lower (∼10 W/cm 2 ), a completely different scenario takes place. The chromophore reacts in its triplet state T 1 with molecular oxygen to produce singlet oxygen 1 O 2 . We propose that singlet oxygen then rapidly reacts with the nearby Met159 and a water molecule to produce sulfoxidized-Met159 and hydrogen peroxide H 2 O 2 . 50 H 2 O 2 in turn attacks Cys171 to give sulfoxidized Cys171 and a hydroxyl molecule. 50 Because the S-O bond in a sulfoxidized methionine is highly polarized, with partial negative charge on the oxygen atom, the chromophore is trapped in a protonated state due to the establishment of a strong H-bond interaction. Loss of fluorescence is observed, but partial absorption by the neutral chromophore is maintained. Alternatively to oxidizing Met159 and Cys171, singlet oxygen may also diffuse further throughout the IrisFP matrix and attack more remote amino acids, as revealed by mass spectrometry. We refer to either of these oxygen dependent bleached states as Bl oxy . Singlet oxygen may also undergo a different fate: it can escape into the solvent and generate cytotoxicity. This is consistent with the estimated 1 O 2 lifetime and diffusion length in cells of 250 ns and 45 nm, respectively. 51 Diffusion out of the IrisFP β-barrel could occur via the pore identified in this work, or through other pores opening transiently that will necessitate future investigations by molecular dynamics simulations. 26,27 Sulfur-containing residues are major targets of reactive oxygen species (ROS) and are most susceptible to oxidation. 52 Notably, methionine oxidation by singlet oxygen has been described in the context of chromophore assisted light inactivation (CALI), typically resulting in enzyme inactivation. 53 Here, low-intensity photobleaching of IrisFP can be viewed as a special case of intramolecular CALI. We note that it is unlikely that methionine sulfoxide reductases, which are enzymes known to revert methionine sulfoxidation 54 would salvage IrisFP fluorescence, because Met159 is buried into the β-barrel. Further oxidation of remote sensitive residues could also be associated with a loss of fluorescence possibly linked to partial unfolding of the IrisFP β-barrel, in turn corroborating the loss of diffraction observed in crystals treated with prolonged low-intensity light. This observation is reminiscent of the loss of enzymatic activity observed in enzymes submitted to a high-level of oxidation by ROS, 50,55 and it is consistent with the loss of fluorescence observed in other FPs submitted to ROS by pulse radiolysis. 56 The photophysical scheme of Figure 6 recapitulates the proposed photobleaching pathways. Upon excitation, the ground-state singlet chromophore S 0 reaches the first electronically excited state S 1 , from which it can either fluoresce, deactivate nonradiatively (through isomerization, giving rise to off-switching), or undergo intersystem crossing to the longlived triplet state T 1 . From T 1 , a number of possible pathways can be envisaged. T 1 can relax to S 0 either spontaneously or, more efficiently, by triplet-triplet quenching if molecular oxygen is present near the chromophore. This results in the production of 1 O 2 which may, as described above, attack the fluorescent protein or escape into the medium. The photofatigued state Bl oxy is produced. Alternatively, the high oxidation potential of the excited chromophore may drive electron transfer from Glu212 without the need for oxygen, leading to the nonfluorescent prebleached state pBl red from which the photofatigued state Bl red occurs.

It is interesting to determine which factors control the switch from one photobleaching regime to the other. Two hypotheses can be proposed. First, the entry rate of molecular oxygen into the IrisFP β-barrel could play a role. A slow O 2 entry rate relative to the rate of photon absorption would disfavor the reaction of the excited IrisFP chromophore with oxygen, leaving redox chemistry as the dominant route to photobleaching. Second, as strongly suggested by our data (Figure S3, Supporting Information), the onset of redox photobleaching at high intensity could result from a mechanism relying on the consecutive absorption of two photons. The increase of the T 1 lifetime while oxygen is absent from the chromophore pocket would further boost such mechanism. In order to test these hypotheses, the complete kinetic model illustrated in Figure S15 (Supporting Information) was challenged by simulations predicting the relative fraction of the two photofatigued species Bl red and Bl oxy as a function of the applied laser intensity, assuming realistic rates for entry/exit of oxygen in/from the chromophore pocket (see discussion in the Supporting Information and Figure S16). The model consistently predicts dominant oxygen-dependent and oxygen-independent photobleaching pathways under the experimental low-and high-intensity illumination conditions, respectively. We propose that the photobleached state Bl red can be reached via one-or two-consecutive-photon excitation. In the first case, the radical state DH • could be involved, as suggested by our previous simulations using QM/MM approaches. 43 In the second case, absorption of the second photon could take place in T 1 or another state of the chromophore with micro-to millisecond lifetime, thus accelerating formation of Bl red at high excitation rates. The existence of a two-consecutive-photon absorption route leading to Glu222 decarboxylation has been noticed in GFP 57 and is consistent with the high laser intensities observed to build-up the super-red species in DsRed. 34 Furthermore, by analogy to the results found in GFP, 57 it may be speculated that conventional two-photon-excitation schemes, in view of the high laser power-densities used, could favor the oxygenindependent bleaching pathway.

In the crystalline state, assuming a typical concentration of oxygen of 200 μM, there is a large excess of IrisFP monomers (>100). Assuming a bulk diffusion coefficient of 2 × 10 -5 cm 2 / s, a single molecule of O 2 is able to explore only ∼20 unit cells in a microsecond. Thus, it is likely that the rate at which oxygen molecules penetrate into a given crystalline IrisFP monomer is lower than the excitation rate of the chromophore, at least under our high-intensity illumination conditions (5 × 10 4 s -1 ). In solution samples or in biological cells this situation might differ, with more molecular oxygen available per fluorophore. This may possibly shift upward the intensity threshold between the two photobleaching pathways. More generally, we expect this threshold to depend not only on the fluorescent protein and molecular oxygen concentrations but also on the redox environment, as well as on other parameters such as pH, viscosity of the medium, or even the nature of fusion constructs.

Photo-Kolbe reactions in fluorescent proteins have been described several times. 31-33,48,58 The invoked mechanism, however, always leaves the chromophore intact. Indeed, it is generally proposed that following electron transfer from Glu212 to the chromophore, an electron (and proton) is transferred back from the chromophore to the CH 2

• radical of the decarboxylated Glu212. By contrast, the chromophore of IrisFP is chemically altered, with the methylene bridge ending up in an sp 3 -hybridized state as demonstrated by Raman spectroscopy. The photobleached chromophore is thus likely to be photochemically reduced, which implies the net transfer of two electrons and two protons. Our MD simulations favor such a process, suggesting the formation of a zwitterionic imidazolinone ring in which the oxygen atom is negatively charged and stabilized by two H-bonds to the guanidinium groups of Arg66 and Arg91 while the N4 nitrogen atom is protonated and positively charged. Whereas the proton ending up on the Cα carbon could originate from Arg66, 43 we speculate that the second electron and proton could be provided by a reducing molecule in the surrounding medium. 29 Further studies will be needed, however, to precisely unravel the redox mechanism allowing these transfers.

Studying the photobleaching mechanisms of RSFPs is important because these markers play an increasing role in several advanced fluorescence microscopy applications. It could be argued, however, that the photoswitching capabilities of RSFPs complicate the investigation of irreversible photodestruction processes. On the contrary, and consistently with previous work, 41 we propose that the investigation of photobleaching pathways through repeated photoswitching provides the advantage that uncontrolled accumulation of reversible dark states ("shelving") 2 is largely reduced: the overall slow photofatigue of RSFPs facilitates the recovery of thermally unstable dark states, and repeated illumination by 405 nm light often promotes fast recovery of light-sensitive dark states, as is the case for the main trans-isomeric off state. Overall, studying photobleaching of RSFPs facilitates the decoupling of irreversible and reversible photophysical processes.

The photobleaching mechanisms described here for IrisFP might be conserved in other photoconvertible and photoswitchable fluorescent proteins derived from Anthozoan species such as Dronpa, mTFP0.7, Kaede, EosFP, Dendra2, and their variants. 39 Indeed, all these proteins display very similar structural organizations of their chromophore pocket, with nearly identical hydrogen bond networks involving Glu212, His194, Arg66, and Glu144. Their sequence identity and global structural similarity is also high, suggesting the possibility of a common pathway for oxygen diffusion toward the chromophore and/or common redox mechanisms. Moreover, all these proteins, except mTFP0.7, contain a methionine at position 159. The observation of a trapped neutral chromophore upon Met159 sulfoxidation might therefore be a general feature of this family of proteins. We note that, in the context of the present study, the presence of this residue at this position can be considered as a fortunate feature that allowed us to provide the first structural evidence of singlet oxygen production within a FP chromophore pocket. Interestingly, during the directed evolution of mCherry from mRFP1, it was reported that the mutation Met163Gln was entirely responsible for the 10-fold enhanced photostability of mCherry. 2 This finding could relate to our observations in IrisFP, although recent molecular dynamics simulations proposed that this mutation essentially served to block the entry of molecular oxygen within the barrel. 27 In general, the photobleaching mechanisms observed in IrisFP are unlikely to apply to Hydrozoan RSFPs such as rsEGFP. 20,21 While oxygen diffusion pathways probably differ, Glu212 decarboxylation in Hydrozoan FPs typically induces photoactivation rather than photobleaching. Nevertheless, competing photobleaching routes probably also exist in Hydrozoan RSFPs and would deserve investigations similar to those described in this work. Likewise, non-RSFPs of Anthozoan origin are likely to exhibit yet different photobleaching mechanisms. In the red fluorescent protein KillerRed, for which structures of photobleached states under low intensity illumination have been published, 3,4 the chromophore was found either disordered 3 or distorted, 4 possibly consistent with sp 3 -hybridization of the methylene bridge, and no substantial modifications of the chromophore environment was noticed. These differences with our low-intensity IrisFP bleached structure might arise from a type I photosensitization mechanism in KillerRed 3 versus a type II mechanism in IrisFP and be a consequence of the different chromophore structures and environments in the two proteins.

Our results provide some of the molecular basis required for the rational design of more photoresistant Anthozoan RSFPs. For example, in IrisFP, mutations Met159Ala 45 or Glu212Gln 59 could be carried out and their effect on photobleaching investigated, with the potential caveat that mutations enhancing photoresistance may also modify important properties such as photoswitching yields or maturation efficiency. Further directed-evolution based engineering of electron transfer pathways and oxygen entry routes will probably be required to obtain optimal photoresistant variants.

■ CONCLUSION

Despite considerable advances in fluorescent proteins research in the recent past, the high susceptibility of FPs to irreversible photobleaching still remains a major bottleneck to their use in advanced microscopy applications. Here, we have explored the photobleaching mechanisms in IrisFP, a representative of the important class of reversibly switchable fluorescent proteins. Under low-intensity illumination (∼0.01 kW/cm 2 ) typical of standard wide-field fluorescence microscopy, an oxygendependent mechanism dominates, that damages the IrisFP chromophore environment mainly through sulfoxidation reactions and releases singlet oxygen in the medium. Under high-intensity illumination (∼0.1 kW/cm 2 ) approaching laserlight levels used in localization-based super-resolution microscopy, an oxygen-independent mechanism takes over, that damages the chromophore itself via decarboxylation of the strictly conserved Glu212.

As a consequence of this work we speculate that highintensity illumination of Anthozoan phototransformable FPs might generate less cytotoxicity than low-intensity illumination at constant integrated dose. This prediction is consistent with previous findings that light-induced damage of cellular samples labeled with fluorescent proteins mostly originates from ROS released by the fluorescent markers under typical widefield illumination regimes. 60,61 However, at high-intensity, additional effects due to, e.g., singlet oxygen release by endogenous chromophores (such as flavins) and rapid depletion of ROS scavenging cellular components (such as glutathione) may play an adverse role. Overall, our results might be related to the observation that prolonged PALM illumination of Anthozoan FPs generally induces tolerable phototoxicity in live cells, 62-65 but call for further quantitative investigations of the lightintensity dependence of cytotoxic effects in biological samples. The two-regime photobleaching mechanism characterized in this work may also play a role in the frequently observed discrepancy between bleaching kinetics measured under a variety of conditions, typically low versus high intensity widefield or wide-field versus confocal. In the latter case, however, the short illumination dwell-times and typically much higher instantaneous power densities (>10 kW/cm 2 ) probably call for yet other photobleaching mechanisms to be explored in the future.

■ ASSOCIATED CONTENT

* S Supporting Information Materials and methods for crystal growth, X-ray data collection and structure determination, photobleaching procedures, optical spectroscopy and mass spectrometry, quantum mechanics and molecular dynamics simulations. Discussions of laser beam heterogeneity, QM/MD simulations, modeling of kinetic scheme, Raman spectra from photobleached crystals. This material is available free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author dominique.bourgeois@ibs.fr

Notes

The authors declare no competing financial interest

■ ACKNOWLEDGMENTS

The ESRF and SLS synchrotron facilities are acknowledged for providing beamtime. We thank Martin Fuchs, Guillaume Pompidor, and Florian Dworkowski for help in X-ray data collection at the SLS, Aline Faro for scientific discussions, and David von Stetten for support in using the Cryobench laboratory of the ESRF. We acknowledge technical help by Laure Roux, Jeŕeḿie Gaillard and Christophe Guerin. We thank Gabriella Jonasson and Bernard Levy for fruitful discussions and invaluable help. D.B. acknowledges financial support by the ANR (ANR-2011-BSV5-012-01 NOBLEACH). This work used the platforms of the Grenoble Instruct center (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) with support from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). This work was granted access to the HPC resources of CINES under the allocation 2012-c2012086318 made by GENCI (Grand Equipement National de Calcul Intensif).

S1

Supporting Information

Structural evidence for a two-regime photobleaching mechanism in a reversibly switchable fluorescent protein.

Chenxi Duan, Virgile Adam, Martin Byrdin, Jacqueline Ridard, Sylvie Kieffer-Jaquinod, Cécile Morlot, Delphine Arcizet, Isabelle Demachy and Dominique Bourgeois

Materials and Methods

Expression and purification

IrisFP was expressed in Escherichia coli BL21 (DE3), using a pQE32 plasmid encoding the full-length IrisFP as described previously. 1 The bacterial culture was grown at 37°C to OD 600 nm = 0.6. Overexpression was induced by adding 0.1 mM IPTG and the culture was incubated 7 days at 4°C. After centrifugation, the cell pellet was resuspended in a solution containing 50 mM HEPES pH 7.5 and 150 mM NaCl, and then lysed by sonication. The Histagged recombinant protein was purified in two steps, using a pre-packed Talon metal affinity column (Clontech Laboratories, California, USA) and then a Hiload 16/60 Superdex 75 gelfiltration column (GE healthcare, Pennsylvania, USA). Fractions suitable for spectroscopic characterization and crystallization trials were concentrated to 20 mg/ml and dialyzed against a 50 mM sodium phosphate buffer (pH 7) containing 300 mM NaCl.

Crystallization

Crystals were grown at 293 K using the hanging drop method, using 2.1 M (NH 4 ) 2 SO 4 and 100 mM Bicine pH 8.0 as crystallization buffer. Rod-shaped yellowish crystals appeared within 24 h and reached their final size (typically 50 × 100 × 1000 µm 3 ) within a few days.

Cryoprotection

For X-ray diffraction and UV-visible microspectrophotometry, photofatigued crystals were rapidly transferred to a cryoprotectant solution (10% glycerol/ 2.1 M (NH 4 ) 2 SO 4 /100 mM Bicine pH 8.0) before being flash-frozen in gaseous nitrogen at 100 K. Glycerol was not used for Raman spectroscopy to avoid spectral contamination by this molecule.

Photobleaching procedures

Photobleaching at room temperature

To photofatigue IrisFP crystals under high-intensity conditions, we used our super-resolution PALM microscope. Crystallization drops were deposited on a glass slide, and were surrounded by several drops of crystallization medium to prevent drying. The drops were covered with a coverslip and sealed with grease. The samples were then placed on the stage of an inverted microscope (IX-81, Olympus) equipped with a ×20 objective. 488-nm and 405nm lasers (Gaussian beam FWHMs: ~100 and ~200 µm) were then focused onto the part of the crystal to be treated (Figure S1A). Continuous illumination at 488 nm (73 W/cm²) was applied at room temperature for 10 minutes while the 405-nm laser (1 W/cm²) was turned on for 0.3 s every 2 s to restore the cis conformation of the chromophore. Only few crystals withstood this harsh treatment, which often resulted in significant unit-cell expansion in the bleached region, preventing the computation of accurate difference electron density maps.

Higher laser power-densities resulted in crystal cracking and could not be used. To monitor photofatigue decays, the EMCCD camera of the microscope was replaced by a CCD-based spectrometer (AvaSpec-ULS2048, Avantes, Eerbeek, The Netherlands).

Solution samples were treated similarly, except that the crystallization drops were replaced by films of polyvinyl alcohol (PVA) containing purified IrisFP at a concentration of 0.74 mM (Figure S2 andS3).

To photofatigue IrisFP crystals under the low-intensity conditions, the same invertedgeometry setup turned out to be inappropriate, as the much longer experiment time required to achieve extensive photofatigue typically resulted in crystal drying. Thus, we designed an alternative setup based on an upright microscope which allowed photobleaching crystals directly in their crystallization trays (Figure S1B). Laser light was brought to the samples with a 200 µm diameter optical fiber, the tip of which was positioned above the crystal of interest with the aid of the microscope eyepieces. The measured FWHMs of the 488-nm and 405-nm laser beams obtained in these conditions at the sample position were ~220 µm (a smaller value than anticipated, possibly due to a lens effect of the crystallization drops). Crystals were continuously illuminated by the 488-nm laser (9 W/cm²) for 2 hours while the 405-nm laser (10 mW/cm²) was turned on for 4 s every 12 s to restore the cis conformation of the chromophore. The same optical fiber was used to detect fluorescence emission, which was redirected with a dichroic mirror to a CCD-based spectrometer.

In all cases, the switching cycles were terminated by a final exposure to 405-nm light to avoid the presence of residual non-fluorescing IrisFP molecules in their switched-off state.

Photobleaching at cryogenic temperature Photofatigue of crystalline IrisFP at cryogenic temperature (Figure S4) was performed using a microspectrophotometer on which samples were mounted with standard cryoloops.

Crystals were flash-cooled to 100 K by a nitrogen gas cryo-stream (Oxford cryostream, Series 600, Oxford, UK). For photobleaching of the cis-state, crystals were mounted and illuminated by laser light (51 W/cm²) for 1 hour. For photobleaching of the trans-state, crystals were first illuminated in their crystallization drops by unfocused 488-nm light in order to isomerize the chromophore to the trans conformation. They were then mounted onto the spectrometer and illuminated by 405-nm laser light (40 W/cm²) for 1 hour. X-Ray data collection X-ray diffraction data sets were collected at 100 K at the European Synchrotron Radiation Facility (ESRF) on beamlines ID14-2 and ID29, with an ADSC Q210 and a PILATUS 6M detector, respectively, and at the Swiss Light Source (SLS) on beamline PXII, with a PILATUS 6M detector. Diffraction experiments on ID14-2 and PXII were combined with online microspectrophotometry in order to properly locate photobleached areas on the crystals, based on absorption spectra.

The structure of photofatigued IrisFP under high-intensity illumination conditions (Figures 2C,2D) was obtained at beamline ID14-2 of the ESRF. Two data sets were collected on the same crystal, one centered on the photobleached zone and the other on an intact zone.

The X-ray beam size was 100 × 100 µm², somewhat larger than the photobleached zone.

Many crystals showed a reduced diffraction quality in the photobleached area, usually coupled to a substantial increase in the unit-cell dimensions.

The structure of photofatigued IrisFP under low-intensity illumination conditions (Figures 4C,4D) was obtained at beamline PXII of the SLS. Two data sets were collected on the same crystal, one centered on the photobleached zone and the other on an intact zone. The X-ray beam size was 50 × 50 µm². Many crystals showed poor diffraction quality in the photobleached area. Only a few moderately photofatigued crystals conserved acceptable diffraction quality, and the unit-cell dimensions always increased in the fatigued zone.

The structure of X-ray bleached IrisFP (Figure S7) was collected at beamline ID29 of the ESRF. Two data sets were helically collected along the rod-shaped crystal in order to minimize the spread of X-ray damage within each data set.

X-Ray data processing

All crystallographic data sets were reduced with the XDS package. 2 Data collection statistics are compiled in Table S1. Experimental difference electron density maps (F obs, bleached -F obs, green ) were calculated with CCP4 3 after Bayesian q-weighting of the difference structure factor amplitudes 4 performed with IDL (ITT, Boulder, Colorado, USA) and using phases from the green structure of IrisFP (PDB entry 2VVH). Such maps are able to reveal subtle conformational changes by a small fraction of the molecules in the crystal, but only in the absence of significant changes in the unit-cell dimensions. Under high-intensity illumination conditions, one crystal showed no such change and provided the data presented in Figure 2C.

Due to the larger X-ray beam size relative to the bleached crystal volume, the fraction of bleached molecules observed crystallographically was estimated to be ≤ 20%. Under lowintensity illumination conditions, unit-cell expansion in the bleached area was always too large to compute reliable experimental difference electron density maps. Instead, standard difference Fourier maps (F obs, bleached -F calc, green ) were generated (Figure 4C). NCS-averaged maps were calculated with Coot. 5 Figures presenting crystallographic data were prepared with Pymol (Schrödinger, LLC., New York, USA).

Model refinement

In order to obtain refined models of pBl red and Bl red , we employed the difference refinement method 6 after a molecular replacement step using the program MOLREP 7 with PDB entry 2VVH as a search model. We used extrapolated structure factors:

) ( 1 Green Bleached Green Bred F F m m q q F F - × × × + = α
where α is a scaling factor corresponding to the fraction of photobleached chromophores, q is the weight calculated from the ''q-weighting'' procedure 4 and m is Read's figure of merit. 8 Stereo-chemical restraints for the chromophore were taken as published in Roy et al. 9 in order to properly describe the methylene bridge C α carbon in a sp 3 -configuration and a planar imidazolinone ring. The structures were refined with PHENIX 10 along with manual inspection and corrections in Coot. 5 In order to obtain a refined model of Bl oxy , a standard refinement procedure using PHENIX 10 and Coot 5 was employed.

Refinement statistics can be found in Table S1. The relatively poor R cryst and R free factors obtained for the structure of photobleached IrisFP under high-intensity illumination conditions is explained by the low occupancy of the photobleached state in the crystal volume probed by X-rays (α ≈ 0.2), resulting in rather high sigma values for the extrapolated structure factor amplitudes. However, the refined structure was found to correctly match the experimental difference electron density features of Figure 2C, providing credit to its significance.

Atomic coordinates and structure factors amplitudes of IrisFP in states Bl red, pBl red and Bl oxy have been deposited in the Protein Data Bank (www.pdb.org) under the PDB accession codes 4LJB, 4LJC and 4LJD, respectively.

Optical microspectrophotometry

Absorption spectra from IrisFP crystals (Figures 2 and4) or PVA-embedded samples (Figure S2) were recorded at 100 K and at room temperature, respectively, using a microspectrophotometer setup described previously 11 . Solution spectra (Figure S2) were measured at RT using a 3-window quartz-cell (50 µL, optical path 3 mm). The cell was connected to the lamp and spectrometer of our microspectrophotometer setup through 100-µm optical fibers. Actinic light was delivered at right angle via a third fiber of 200-µm diameter, so that the laser beams covered completely the third window. For anaerobic measurements, the quartz-cell was nitrogen-bubbled beforehand and sealed (Figure S2 andS8). Spectroscopic data were processed using homemade routines based on the MATLAB software (The MathWorks Inc., Natick, Massachusetts, USA). Absorption spectra were corrected for background using a polynomial baseline subtraction. Model fitting of the photofatigue decays was performed with Origin (OriginLab, Northampton, Massachusetts, USA). Fluorescence emission spectra were corrected for background using an offset baseline subtraction at 450 nm.

Raman spectroscopy

In crystallo Raman spectroscopy was performed at the Cryobench laboratory (ID29-1, ESRF). Off-resonance Raman spectra were recorded at 100 K from IrisFP crystals mounted in standard cryoloops, using a 785-nm laser. Samples were photofatigued using the same protocols as described above, except that, for the low-intensity illumination condition, crystals were prepared using the inverted PALM microscope, which allowed achieving only partial photobleaching before crystal drying occurred. As for X-ray data, Raman spectra were recorded on both bleached and non-bleached parts of the same crystal. Spectra were recorded in 10 min over the spectral window (200 -2000 cm -1 ). Baseline subtraction was carried out with an automated baseline-flattening routine. 12 Spectra were scaled with MATLAB (The MathWorks Inc., Natick, Massachusetts, USA), after normalization at the amide I band (1665 cm -1 ).

Mass spectrometry

Mass spectrometry was used to confirm Glu212 decarboxylation and amino acids oxidation levels under high-and low-intensity illumination conditions, respectively. Crystals were prepared following the same protocol as described above, except that the entire crystal volume was treated. The crystals were then dissolved in 20 µl Tris buffer (50 mM, pH 8.0); Solution samples were directly prepared either in PVA films (to prevent molecular diffusion, high-intensity conditions, 1 µl, 740 µM) or in a quartz cuvette (low-intensity conditions, 30 µl, 10 µM). Samples in 50 mM Tris were diluted to have a final concentration of 10% of acetonitrile; heated at 90°C during 5 min then cooled with an ice bath to unfold the FP structure. A 1/20 ratio of trypsin was added to the mixture and the digestion was extended overnight. ~ 100 ng of each trypsin digested sample was analyzed in triplicate by nano-liquid chromatography coupled to tandem mass spectrometry (Ultimate3000, Dionex and LTQ-Orbitrap XL, Thermo Fischer, Waltham, Massachussets, USA). Fragments were identified using the Mascot software (version 2.4, Matrix Science) against a custom database containing the target protein. 13 We specified Met/Trp/His/Tyr oxidations, Cys di-and tri-oxidation and Glu decarboxylation as possible variable modifications (Figure S9, Table S2). ESI-TRAP was selected as the analytical instrument, semi-Trypsin as the enzyme and 3 missed cleavage were allowed. Precursor and fragment mass error tolerances were set respectively at 10 ppm and 0.6 Da. For the evaluations of the ratio between oxidized and non-oxidized forms, the 6 raw files were deconvoluted and aligned using the Decon-2ls and MultiAlign software suite.

Quantum mechanical and molecular dynamics simulations

To assess the chemical structure of the chromophore in both the pBl red (X-ray bleached) and Bl red photobleached structures (a prerequisite to perform the molecular dynamics simulations), quantum mechanical (QM) calculations at the B3LYP/6-31G* level using Gaussian 14 were performed. In the absence of experimental evidence concerning the charge state of the chromophore in these bleached structures, the anionic form, with a deprotonated phenolate ring, was assumed. The only simple way to account for the observed chromophore tilted geometry is that an H atom is added on the bridge carbon atom Cα, as previously described. 9 In such conditions, however, the chromophore becomes a free radical and something else necessarily happens. We examined if a second H atom may be accommodated by the imidazolinone ring. The requirement of the observed planar geometry of that ring excludes all sites except the oxygen atom O and the nitrogen atom N4 (see Figure S10). Three chemical structures were thus explored : two structures with an hydrogen atom on O, that differ from each other by the orientation of the hydroxyl group, either towards the phenolate ring (structure referred to as CαOI ) or in the opposite direction (CαOII), and the one with an hydrogen atom on N4 (CαN) (Figure S10).

The QM calculations showed that the three structures are stable with respect to the addition of the second hydrogen atom, by an energy amount of 40-60 kcal/mol depending of the structure.

Molecular dynamics (MD) simulations were performed in order to examine whether the X-ray bleached structure (Figure S7 and Table S3) might spontaneously transform into the Bl red photobleached structure at room temperature. The chromophore force field, apart from the dihedral potential, was obtained as described previously. 15 The missing carboxylate of Glu212 was replaced by a hydrogen atom. For all standard amino-acids the AMBER force field "ff99" was used. 16 MD simulations were run with the parallel module PMEMD of the AMBER suite 16 modified by Jonasson et al. 15 that allows using coupled dihedral potentials. The MD protocol consists of minimization and equilibration phases, where constraints on the protein are progressively removed, followed by 4-ns free runs at constant temperature (300 K) and pressure (1 atm). In the case of the CαN chemical structure, four free runs were carried out, starting with different snapshots of the last equilibrium phase. For each of the two structures CαOI and CαOII three free runs were carried out in the same way. A run with the chromophore in its fluorescent cis form (see Jonasson et al. 15 for the force field) was also performed in order to get some information about the influence of Glu212 decarboxylation and of chromophore denaturation.

An essential point in the molecular dynamics simulations is the torsional behavior of the chromophore around Cα. It is expected to significantly differ from a free rotation profile, due to the two rings. We used coupled dihedral potentials V(τ,φ) (see Figure S10 for the definition of τ and φ) determined as in Jonasson et al. 15 For each of the three reduced chemical structures, a specific potential was obtained by fitting an ab initio two dimensional potential energy surface (PES) calculated at the B3LYP/6-31G* level using Gaussian. 14 The three PES are shown in Figure S11. The iso-energy curves around the minima are ellipses with axes largely tilted by respect to the τ and φ axes, showing that the usual additive form V(τ)+V(φ) would have been inappropriate to fit the dihedral profile.

Laser beam heterogeneity

In all cases, photofatigue experiments were carried out using laser beams adopting a near-Gaussian two-dimensional profile rather than a top-hat profile. Therefore, the beam powerdensities were not homogeneous throughout the samples. Notably, under our "high-intensity" illumination conditions, a fraction of the IrisFP molecules at the periphery of the illuminated profile rather experienced "low-intensity" illumination conditions. For a monophasic photobleaching process, it has been shown that the observed fluorescence emission decay of a sample bathed in a Gaussian-shaped laser beam can be expressed as: 17 ( ) ( )

0 0 2 / / exp 1 τ τ t t ka t F - - =
where a is the radius of the laser beam and τ 0 is the inverse of the bleaching rate at the beam center. Attempts to fit our photofatigue decays (Figures 1 and3) using such a model did not provide satisfactory results, suggesting that in both cases the photobleaching processes are multiphasic. The most satisfactory fit with a biphasic model under both low-and highintensity illumination conditions is likely to incorporate both the effects of the beam heterogeneity and the various bleaching mechanisms that may be present. The different decay profiles observed in the two illumination conditions, however, strongly suggest that a distinct distribution of bleaching mechanisms are involved in the two cases. The more pronounced biphasic shape of the photofatigue decay under high-intensity illumination is consistent with a large fraction of the molecules experiencing oxygen-independent bleaching while a smaller fraction, remote from the beam center, experiences slower, oxygen-dependent bleaching.

It should be noted that the various techniques used in this work to probe photobleaching mechanisms typically interrogate volumes of the treated crystals that differ in size: mass spectrometry used the entire crystal volume (photofatigued on multiple spots), crystallography typically employed a 100 µm diameter X-ray beam of approximately the same size as the photofatigued region (but possibly slightly offset), fluorescence spectroscopy used exactly the same size (with no offset), optical absorption microspectrophotometry used a 50-µm whitelight beam slightly smaller (with little offset), and Raman spectroscopy used a ~10-µm near-IR-laser beam much smaller than the photofatigued region (with little offset).

We also note that, on the PALM microscope, the size of the 405 nm beam was larger than that of the 488 nm beam. As a result, for samples treated on this microscope, volume peripheral to the photofatigued region underwent green-to-red photoconversion (Figure S12A). Residual difference electron density map corresponding to this photoconversion is visible in figure 2C around the Phe61-His62 backbone linkage. A calculated difference density map between the models of green and red photoconverted IrisFP (Figure S12B) confirmed that no other density features observed in Figure 2C may originate from unwanted photoconversion.

Detailed results on QM and MD simulations

The three torsional landscapes of the chromophore corresponding to the three putative chemical structures of the bleached pBl red and Bl red states largely differ from each other (Figure S11). In the case of the CαN structure it presents a deep energy minimum around the geometry τ=0°, φ=±90°. In the case of CαOI a deep minimum is also observed, centered on τ=180°, φ=±90°. Concerning CαOII, the energy profile is very flat compared to the preceding ones, allowing very large torsional movements away from the minimum energy point centered at τ=120°, φ=-75°.

The main results of the MD simulations are summarized in Table S4.

CαOI structure: No switch of Arg66 and His194 is observed in any of the three runs.

The internal forces of the chromophore drive it to conformations with τ and φ average values equal to respectively 169° and -74°, near the equilibrium geometry of the isolated chromophore. Inspection of the chromophore environment in the MD trajectories shows that in these conformations the phenolate ring hinders the movement of the Arg66 sidechain that would drive this residue to its conformation in Bl red (Figure S13A). CαN structure: With this chemical structure, the mean values of the dihedral angles τ and φ are 25° and -85°. In such geometries the phenolate ring lets the Arg66 sidechain free to move (Figure S13B), and the switch is observed in all four runs. This results in the loss of the H-bond between His194 and Glu144, and in the formation of two new H-bonds: a strong one between His194 and Arg66, and a non-permanent one between His194 and Ser142. Figure S14 shows that these H-bond modifications are simultaneous. In addition a first movement of Arg66 was observed in the last part of the equibration phase, leading to the formation of a Hbond with Glu144.

The mean value of τ (25°) is in good agreement with the experimental values in the Bl red photobleached structure. The large fluctuations observed (standard deviation of 16°, much larger than the 6° deviation observed for the fluorescent chromophore) are consistent with the disorder of the phenolate in the crystallographic structure. The mean value of φ is -85°, far from the crystallographic values. However this comparison is not pertinent due to the observed disorder of the phenolate.

It is worth noting that the rearrangement of the switch of Arg66 and His194 is observed with the fluorescent planar cis chromophore as well, as long as Glu212 is decarboxylated. Thus, Glu212 decarboxylation, resulting in an H-bond missing for His194, appears to be sufficient to induce this rearrangement, provided that the phenolate does not hinder the displacement of Arg66.

These results show that IrisFP structural dynamics at 300 K is indeed able to drive His194 and Arg66 from their conformations in the cryo-X-ray-bleached structure towards those in the Bl red photobleached one, adding value to the notion that the former structure may be viewed as an intermediate along the Bl red bleaching pathway. More generally, they strongly suggest that the decarboxylation of Glu212 is sufficient to induce the large displacement of His194 and Arg66. The results also provide an answer to question (iii) of the main text, concerning the chemical structure of the chromophore : the most probable structure is the zwitterionic CαN.

Modeling of kinetic scheme

In order to simulate the observed two-regime bleaching behavior, we consider the kinetic scheme S15.

The chromophore in its singlet ground state S 0 absorbs photons with rate k ex , exciting it to the first singlet excited state S 1 from where it can return to S 0 by fluorescence or nonradiative decay (both processes described by rate k F ). Alternatively, the singlet excited chromophore can undergo intersystem crossing to the triplet excited state T 1 with rate k IS . T 1 may then engage into a number of pathways.

First, it can spontaneously convert to the ground state (rate constant k T1,1 ), or be quenched by triplet oxygen (rate constant k T1,2 ), creating highly reactive singlet oxygen. 1 O 2 may in turn face three different fates : intersystem crossing to restore triplet oxygen (k 1 ), exit from the protein to the solvent (k 2 ) or reactive attack on the FP itself, thus leading to permanent oxygenic bleaching of the chromophore fluorescence (state Bl oxy formed with k 3 ).

Second, T 1 may experience electron/proton transfer with rate constant k 4 , leading to a doublet radical state DH

• . This « blinked » dark state may either recombine with rate constant k 5 to the intact singlet ground state or further react with rate constant k 6 to a permanently (« redox ») bleached state Bl red . 9 Third, T 1 may undergo a second excitation to a higher excited triplet state T n that may rapidly return to the ground state or go through electron/proton transfer to the permanently bleached state Bl red (we assume that this bleached state is close or identical to that reached S12 through DH

•

). The overall rate for this latter process is unknown but is likely to be limited by the preceding second excitation; therefore the whole process is described by the rate constant k T1,ex .

Within the framework of this simplified kinetic scheme, there exist two possible mechanisms that can introduce a dependence of the prevailing bleaching pathway on the excitation rate k ex .

A) The T 1 state is populated by k ex via k IS and depopulated essentially by k T1,2 , fuelling the k 3 oxygen dependent bleaching pathway. If, in the high-energy case, oxygen replenishment by k O2,in and k 1 is too slow to compete with k ex , the lack of available oxygen within the FP barrel will be limiting the Bl oxy pathway. The entry and exit rates of oxygen in and out of the barrel are poorly known and therefore, we introduce k O2,in as well as k O2,out as adjustable parameters.

B) The weight of the T n pathway will increase with k ex , as the chance of the absorption of the second photon necessary for this pathway is directly proportional to the excitation power density, ie k T1,ex .α k ex . However, currently the absorption cross section of T 1 is not known, and we use k T1,ex as an adjustable parameter for the probability of this pathway.

Fixing the rate constants to the values given in the legend of Figure S16 and varying the excitation power density from 10 to 100 W.cm -2 , we can actually observe the gradual change from prevailing oxidative damage to more and more redox and second photon induced decarboxylation with increasing energy (Figure S16).

Raman spectra from photobleached crystals

Modifications of the Raman spectra of IrisFP crystals induced by photobleaching at high illumination intensity are drastic throughout the whole investigated wavenumber range (Figure 2B), indicative of the chemical transformation of the chromophore. In addition to the complete disappearance of the bands at 1503 cm -1 , 1545 cm -1 , 1564 cm -1 and 1604 cm -1 described in the main manuscript, many bands in the range 1000 to 1500 cm -1 are also strongly decreased, while a few others appear or are increased. A precise assignment of these vibrational bands has not been performed, but some indications can be given based on previous work on the HBDI chromophore. 18 The decreased band at 1037 cm -1 can be assigned to a coupled imidazolinone C-C stretching mode and C=O mode. The decreased bands at 1093 cm -1 and 1111 cm -1 , and the increased band at 1069 cm -1 are unassigned. The strongly increased band at 1137 cm -1 is possibly assigned to an imidazolinone mode, while the vanished bands at 1154 and 1172 cm -1 could be assigned to a phenol C-H deformation band.

Decreased bands at 1217 and 1260 cm -1 are unassigned. The increased band at 1304 cm -1 , and the decreased bands at 1321 and 1338 cm -1 are possibly assigned to phenol modes. The decreased band at 1375 cm -1 and the increased band at 1395 cm -1 are unassigned. The slightly decreased strong band at 1448 cm -1 could possibly be assigned to a phenol C-H deformation mode, and the slightly increased band at 1484 cm -1 remains unassigned. The disappearance of the ill-defined band around 1665 cm -1 could correspond to an imidazolinone C=O stretching mode. Although the exact interpretation of all these changes remains out of the scope of this paper, they strongly suggest that the chemical structure of the chromophore is not only altered at the methylene bridge, but also at the phenol and imidazolinone cyclic moieties, consistent with our proposal of a reduced chromophore.

Modifications of the Raman spectra induced by photobleaching at low illumination intensity appear much less pronounced throughout the entire wavenumber range (Figure 4B), although in this case an incompletely bleached crystal was interrogated, which limits the accuracy with which spectral modifications can be detected. The reason for partial bleaching was to probe the Raman signature of the Met159 sulfoxidation reaction, while avoiding the overall barrel destabilization due to oxidation of residues more remote from the chromophore that was found to occur upon long illumination. Apart from the protonation of the chromophore, as described in the main manuscript, these Raman spectra suggest that the chemical structure of the chromophore remains chemically unaltered under these conditions. Due to sample pre-treatment (heating and digestion) before injection into the mass spectrometer, some background oxidation of sensitive residues is generally observed on the intact, non-bleached IrisFP, as is commonly the case. 19 The high scatter of oxidation state of the very sensitive Met159 from the reference sample is explained by the time dependent oxidation of the sample during the analytical process. The only way to evaluate Cys171 oxidation was through a low abundance miss-cleaved peptide. The non-oxidized form was not observed in any condition, but various oxidation states appeared in the illuminated sample. It was therefore impossible to derive a ratio for the corresponding peptide. For Cys195, the evaluation of the oxidation corresponded to the ratio of two oxidation states. Overall, these data clearly suggest that, upon low-intensity illumination, photo-oxidation occurs not only on residues close to the chromophore but also on more distant ones. and Bl oxy (red squares) are calculated as a function of excitation power density upon illumination for a time chosen so that 15% of molecules are bleached. Rate constants described in Figure S15 were chosen as: Table S3. Distances in the crystallographic structures (chain A).

k F =0.33×10 9 s -1 , k ex =5-50×10 3 s -1 , k T1,ex =0.15×k ex , k IS =0.3×10 6 s -1 , k 1 =4.0×10 3 s -1 , k 2 =3.9×10 6 s -1 , k 3 =1.2×10 5 s -1 , k T1,1 =5.0×10 3 s -1 , k T1,2 =1.2×10 6 Mol -1 s -1 , k 4 =3.0×10 4 s -1 , k 5 =1.0×10 4 s -1 , k 6 =1.0×10 1 s - 1 , k O2,in =5.0×10 7 Mol -1 s -1 , k O2,out =1.0×10 5 s -1 with [O 2 ] medium =200 µM.

X-ray bleached structure

High-intensity photobleached structure Glu144(OE1)-His194(NE2) 2.9

5.8 Glu144(OE1)-Arg66(NE) 3. Reversibly Switchable Fluorescent Proteins (RSFPs) [4] offer the widest panel of applications. RSFPs can be switched back and forth between a fluorescent and a nonfluorescent state, which has recently led to the development of a number of smart methods including reversible data biostorage [5,6],

photochromic FRET [7] and a variety of nanoscopy approaches. Amongst the latter, photochromic Stochastic Optical Fluctuation Imaging (pcSOFI) [8], Nonlinear Structured Illumination Microscopy (NSIM) [9] and REversible Saturable Optical Linear Fluorescence Transitions (RESOLFT) [10] critically rely on the ability of the RSFPs to perform a high number of switching cycles. For example, in RESOLFT nanoscopy, a confocal setup is used and at each scanner position, a donut-shaped beam with a central zero intensity is applied to switch off the RSFP labels at the periphery of the Point Spread Function (PSF). A probe beam is then applied to readout the fluorescence from the labels that are still in their on-state at the center of the PSF, followed by a reset beam to restore the on-state over the entire PSF. To achieve a high spatial resolution, small laser-scanning steps are chosen and a given RSFP is thus submitted to many photoswitching cycles (typically ~100 to achieve a spatial resolution of 1/10 of the PSF width in both X and Y dimensions), requiring a strong resistance to photofatigue in addition to other key parameters such as a high fluorescence brightness and a high switching contrast.

Continued engineering efforts have been dedicated to the development of RSFPs with improved photophysical properties [1,3,4]. Structural views of the chromophore isomerization based switching mechanism have facilitated the rational design of RSFPs switching at various rates [11,12,13]. In contrast, the lack of a precise understanding of photobleaching mechanisms has only allowed the engineering of photoresistant variants by directed-evolution approaches [13,14].

Nonetheless, the development of hydrozoan RSFPs based on EGFP and EYFP has resulted in fatigueresistant switchers such as rsEGFP [6] and Dreiklang [15] and recently rsEGFP2 [16] Here, we replaced Met159 by Alanine in IrisFP in order to increase photofatigue resistance at least under low intensity illumination condition. A structural view of IrisFP-M159A is presented and photofatigue experiments in solution, in polyvinyl-alcohol (PVA) gels and in bacterial cells were performed at low and high illumination intensities to compare the achieved performance of the designed variant with that of its parent protein.

Materials and Methods

Expression and purification

The M159A mutation in IrisFP/pQE32 was introduced by site-directed mutagenesis with oligonucleotide primers purchased from Invitrogen (Life Technologies, Saint Aubin, France). E.coli BL21(DE3) bacteria were transformed by plasmidic DNA for expression of both parent and mutant His-tagged proteins. Bacterial cultures were grown at 37°C and the overexpression was induced by adding 0.1 mM IPTG once the optical density at 600 nm reached 0.6. Cultures were then incubated at 4°C for 7 days. Cells were harvested by centrifugation and resuspended pellets (150 mM NaCl/50 mM HEPES pH 7.5) were lysed by sonication. Supernatants containing crude protein extracts were purified using a pre-packed Talon metal affinity column (Clontech Laboratories, California, USA) followed by a HiLoad 16/60 Superdex 75 gel-filtration column (GE Healthcare, Pennsylvania, USA). Fractions were pooled and concentrated to 20 mg/ml.

Crystallization, X-ray data collection and data processing

Crystals of IrisFP-M159A were grown at 293 K by the hanging drop method, using a 1:1 ratio of concentrated protein and 2.1 M ammonium sulfate / 0.1 M Bicine (pH 8.1). The rod-shaped yellowish colored crystals dimensioned 50 × 100 × 500 µm 3 . X-ray data were collected at 100 K at the European Synchrotron Radiation Facility (ESRF, Grenoble) on the beamline ID14-4 (X-ray wavelength λ = 0.939 Å) equipped with an ADSC Q315r detector. Data sets were integrated and scaled at 2.0 Å resolution with XDS [19]. The crystal structure of IrisFP-M159A (PDB ID: 4R6B) was solved by molecular replacement with Molrep [20], using the coordinates of one monomer of IrisFP (PDB ID: 2VVH) as a search model. Refinement was made with Phenix [21]. Data collection and model refinement statistics are compiled in Table 2. Figure 2 was prepared with PyMOL [22]. The structure of the protein in its switched-off state could not be obtained due to compromised diffraction after laser illumination of crystals.

Photophysical characterization in solution

Purified proteins were diluted in Tris buffer (pH 8.5) to ensure that chromophores were completely in the anionic state and concentrations were adjusted to an optical density OD = 0.1 at 488 nm. Samples were placed in a 10 mm optical path quartz cell with blackened walls and two opposite 3

x 5 mm 2 clear windows (Starna, Pfungstadt, Germany). The cell was filled to the upper limit of the window, closed with a plastic stopper and placed between optical fibers, coupled to excitation lasers on one side and, via a 500 nm cutoff filter (EdgeBasic, Semrock), to a CCD-based spectrometer (AvaSpec-ULS2048L, Avantes, Eerbeek, the Netherlands) on the other side. The switching-off laser (488 nm, CrystaLaser, Reno, USA) also served for readout and thus was on permanently, whereas the activation laser (405 nm, CrystaLaser, Reno, USA) was only on during periods of on-switching. Highresolution photoswitching cycles were recorded by placing a microlens array (Thorlabs) between the fiber output and the quartz cuvette entry window so as to produce a fully homogenized excitation beam. Data were recorded for ~1 h with a cycle time of 10 min and an on-switching duration of 3 min.

(405 nm: 1.3 mW/cm², 488 nm 8 mW/cm²). For photofatigue experiments, sufficient laser power densities could not be obtained with the microlens array, which thus was replaced with a collimating lens despite the loss of homogenized beam profiles. Photofatigue was studied for >18 h with a cycle time of 20 s and an on-switching duration of 3 s. (405 nm: 0.03 W/cm², 488 nm 0.04 W/cm²). Laser powers were measured behind the exit window of the water-filled cell.

Microscopy setup

Photobleaching experiments were performed on an inverted microscope (IX-81, Olympus) with a 100× oil immersion objective equipped with a NPS nosepiece (Olympus). Samples were illuminated by circularly polarized 488-nm (Spectra-Physics, Santa Clara, USA) and 405-nm

Photofatigue in PVA gel

Purified proteins were diluted to micromolar concentration in a solution of 1% PVA (Mowiol 4-88, Sigma-Aldrich) in Tris buffer at pH 8.5. Glass slides were coated with a solution of PVA containing the protein of interest to form a thin film, which was let to dry for 1-4 hours. The dried matrix ensured an efficient embedding of the proteins, preventing their diffusion.

Photofatigue in cellulo

E.coli BL21(DE3) bacteria were transformed by plasmidic DNA coding for each of the two proteins and bacterial cultures were grown at 37°C until reaching an optical density at 600 nm between 0.4 and 0.8. For fixed-cell experiments, three consecutive centrifugations were performed. Cells were first resuspended in PBS buffer and then in paraformaldehyde (%4 w/v) for 30 min for fixation.

Finally, paraformaldehyde was washed by the last centrifugation and cells were resuspended in PBS.

The fixed cells were immobilized by incubating the bacterial suspension during 1h at room temperature on chitosan-coated glass slides. For live-cell experiments, after a centrifugation at 150 rpm, cells were resuspended in 80 µL LB medium. 10 µL of the cell suspension were spread on a freshly prepared 1-mm thick agarose pad (2 % w/v agarose / PBS pH 7.5) on a slide and incubated for 10 mn at room temperature. A cover slide was then placed over the pad and sealed before the measurements.

Fluorescence quantum yield and pKa measurements

Excitation and emission spectra were recorded with a Synergy H4 Hybrid microplate reader (Bio-Tek, Winooski, USA). The fluorescence quantum yield of IrisFP-M159A was calculated by the method described by Williams et al. [23]

Extraction of photoswitching parameters

Image processing was achieved with ImageJ (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997-2014). Background subtraction was achieved in the following manner: In PVA the measured EMCCD-offset of 500 counts was subtracted; for in-cellulo experiments, non-transformed cells were mixed with transformed cells so that a correct background corresponding to the EMCCD offset plus the autofluorescence signals from the bacteria and the LB medium (for live cells) could be substracted. To obtain photofatigue profiles, the average fluorescence signal inside a region of interest contouring a chosen transformed cell was computed and the average fluorescence signal inside a region of interest contouring a nearby nontransformed cell was subtracted, using ImageJ.

On-and off-switching quantum yields were obtained by fitting individual switching cycles with a simple kinetic model involving one-to-one reversible conversion between the on and off state.

However, in the case of PVA and fixed cells, as molecules may not be excited at the same rate in a rigid medium, the model allowed for a variable anisotropic distribution of the chromophore dipoles.

Also, the fitting model was designed to take into account the limited time-resolution afforded by the used integrating detectors. Model fitting and further processing of the photofatigue decay profiles was done in Matlab (see Supporting Information).

Results

Photophysical and structural characterization of IrisFP-M159A

To test the hypothesis according to which photobleaching of IrisFP under moderate illumination intensity mainly results from sulfoxidation of Met159, we produced the single mutant Met159Ala (IrisFP-M159A) with the expectation that resistance to photofatigue should be enhanced.

Mutation of Met159 in several RSFPs has previously been shown to affect various photophysical properties, such as the off-and on-switching rates in Dronpa-2 (Met159Thr) and Dronpa-3 (Met159Ala) [12,24], blue shifting of excitation/emission spectra in bsDronpa (Met159Cys) [25], or reversal of switching mode in Padron (Met159Tyr) [25]. Thus, to evaluate such possible effects in addition to fatigue resistance, we carried out an extensive biophysical characterization of IrisFP-M159A and compared the results with IrisFP. IrisFP-M159A has its excitation and emission maxima at 484 nm and 513 nm (Figure 1), slightly blue shifted by 4 nm and 3 nm as compared to IrisFP [17],

respectively, and giving rise to a 29 nm Stokes shift (Table 1). IrisFP-M159A shows a 12 % higher extinction coefficient than its parent at physiological pH (pH 7.3), but a significantly reduced fluorescence quantum yield, resulting in a decreased brightness by a factor of ~2.4. At physiological pH, no neutral absorbance peak around 400 nm is observed, suggesting a low chromophore pKa.

Indeed, a pH titration of the absorption bands of IrisFP-M159A revealed a pKa of 4.7, significantly lower than IrisFP (pKa=5.7) (Figure 1). The overall crystallographic structure of the IrisFP-M159A mutant, obtained at 2.0 Å resolution, is nearly identical to that of the parent protein, with an rms deviation over all C α atoms of only 0.174 Å. The interface between monomers is entirely preserved so that IrisFP-M159A is, as

IrisFP, an obligate tetramer. Inspection of the chromophore and its surrounding residues (Figure 2)

shows only minimal rearrangements. Because alanine has a side chain much shorter than that of Despite the newly established hydrogen bond network, the absence of Met159 likely results in a reduced rigidity of the chromophore environment promoting non-radiative de-excitation pathways and consistent with the observation of a lower fluorescence quantum yield in the mutant. Moreover, in the absence of Met159, the electrostatic environment of the chromophore hydroxybenzylidene moiety is altered, which likely accounts for the reduced pKa and slightly blue-shifted excitation spectrum.

Switching behavior and photofatigue studies in solution

A first comparative investigation of the switching behavior of IrisFP-M159A and IrisFP was performed in solution. Purified protein solutions were placed in a quartz cuvette, with concentrations adjusted to provide identical optical density at 488 nm (OD=0.1). First, a very low laser power density (6 mW/cm² at 488 nm) was applied in order to record high resolution switching kinetics over a few cycles (300 points per cycle). As its parent, IrisFP-M159A displays negative photoswitching, that is, off-switching results from illumination at the peak excitation wavelength. The extracted quantum yields for off-and on-switching, the off-and on-switching brightness (product of switching quantum yield by extinction coefficient at the switching wavelength) and the switching contrast (ratio of emitted fluorescence in the on and off states) are shown in Table 3. It is seen that the off-switching quantum yield of IrisFP-M159A is about 4 times that of IrisFP, consistent with the idea that the larger flexibility of the mutant protein should facilitate cis-trans isomerization of the chromophore. The onswitching quantum yield of IrisFP-M159A, however, is about 30% lower than that of IrisFP, despite the apparent higher rate of on-switching (Figure 3). This is due to the fact that, as the 488 nm laser is kept on during on-switching to allow proper monitoring of the emitted fluorescence, the apparent rate is the sum of the actual on-and off-switching rates. For the two proteins, the switching kinetics follow an almost ideal monophasic behavior, suggesting a homogeneous population of molecules and a oneto-one switching behavior between the on-state and the off-state under these experimental conditions. 3C and3D. IrisFP showed a much faster photofatigue decay than the M159A mutant: its fluorescence was halved after ~80 cycles whereas it took about 3500 cycles to achieve the same decrease with the mutant. The photofatigue decay of IrisFP is clearly biphasic, which suggests the possible buildup of a long-lived dark state retarding photofatigue during the slow phase of the experiment through a shelving effect. It is important to note that the difference in the number of achievable cycles by a factor of ~43 is not representative of the true difference in photostability between the two proteins, due to the fact that IrisFP-M159A switches more rapidly. Indeed, it can be estimated by integrating the emitted fluorescence signal along the photofatigue decay curve and correcting for the difference in extinction coefficient that IrisFP-M159A is about 5.2 times more photostable than its parent protein under these experimental conditions. Furthermore, since IrisFP-M159A also has a reduced fluorescence quantum yield, its total photon budget is only ~1.8 times that of IrisFP.

Comparative absorbance spectra recorded before and after photofatigue decay of the two proteins show that, whereas a substantial increase in the neutral band peaking at around 390 nm is observed for IrisFP after photofatigue, this is not the case for the mutant, which in contrast displays a completely flat spectrum (Figure 3C, 3D, inset). We previously have shown that the increase in absorption at 390 nm by IrisFP upon photofatigue at low illumination intensity follows from sulfoxidation of Met159, trapping the chromophore in a nonfluorescent protonated state. The present data thus confirms that no such trapping occurs in IrisFP-M159A, which most likely accounts for the observed superior photofatigue resistance of this mutant.

Switching behavior and photofatigue studies in vitro and in cellulo

It is now recognized that the switching behavior of RSFPs strongly depends on both illumination and nanoenvironmental conditions [16,26]. Therefore, we next carried out photofatigue decay experiments in vitro and in cellulo at two different illumination intensities (~40 W/cm² and ~400 W/cm²), aiming at comparing the behavior of the two proteins. All results are summarized in Figure 4 and Table 4. At high illumination intensity, our EMCCD camera based setup did not provide 4 were extracted.

A first set of experiments were performed in polyvinyl-alcohol (PVA) gels. Interestingly, the switching speed of both proteins appeared to be strongly reduced as compared to solution studies (Table 4). The photofatigue half decay of IrisFP was reduced by a factor of ~3 as compared to solution studies and, surprisingly, that of IrisFP-M159A was decreased by a factor of nearly 80. This finding is corroborated by the observation that the switching kinetics of the mutant was dramatically slowed down, to the point that in PVA the global behavior of the M159A mutant became relatively similar to that of its parent. Nevertheless, a slight photoresistance enhancement by a factor of ~1.9 was still obtained with the mutant protein. Under high-intensity illumination, the two proteins also behaved similarly, with IrisFP even showing a slightly slower photofatigue decay. Of interest is the complex shape of the overall photofatigue decay profiles of the two proteins recorded under low illumination conditions. These profiles exhibit a clear biphasic decay of the fluorescence maxima and an intriguing evolution of the fluorescence minima at early time points, pointing at complex photophysical processes taking place. Overall, these data suggest that PVA strongly modifies the switching behavior of IrisFP and its mutant possibly due to reduced oxygen access, redox effect that are known to be promoted by this polymer [27,28,29], or motional restriction. In fact, although macromolecules are expected to freely tumble in the aqueous cavities within the PVA gel, we observed that individual switching cycles were best fitted with a kinetic model that assumes fixed dipole orientations of the fluorescent proteins. a High corresponds to 400 W/cm 2 , low corresponds to 40 W/cm 2 b Calculated as the product of switching quantum yield and absorption coefficient taken from Table 1 c Calculated over the first halftime of the photofatigue decay d Calculated from the ratio of the integrated photofatigue decays × extinction coefficients taken from Table 1 e Values between parentheses are normalized to IrisFP in solution (Table 3) f Determination of switching brightness limited by time resolution ND: Not determined Next, we recorded photofatigue decay profiles in fixed E. coli BL21(DE3) cells. Again, the switching speed of both proteins appeared to be strongly reduced as compared to solution studies.

However IrisFP-M159A displayed a well behaved photofatigue decay profile under low illumination conditions and was found to exhibit a much greater number of switching cycles than its parent protein under both illumination conditions. The photoresistance enhancement of IrisFP-M159A in fixed cells Finally, photofatigue data were recorded in live E. coli cells. In this case, it appeared that the switching kinetics of both proteins were too rapid to allow reliable fitting of individual cycles, suggesting that in such environment, the fluorescent protein markers adopt a behavior close to that in solution. Inspection of the switching cycles also suggests that molecules freely tumble when expressed in the cytoplasm of live E. coli cells. Consistent with faster switching, the achievable number of photoswitching cycles by both proteins was found higher than in PVA or fixed cells, and the photo resistance enhancement achieved by IrisFP-M159A was estimated to be ~2.0.

It is worth noting that a switching contrast much lower than in solution was observed in all tested conditions, and particularly in live cells. Such lower contrast may result from the higher laser power densities used in widefield illumination conditions, but this issue remains to be explored.

Discussion and Conclusion

In this work, we have shown that rational engineering of an RSFP by a single mutation leads to significantly enhanced photoresistance. As expected from our previous structural investigation [18], the M159A mutation in IrisFP increased fatigue resistance at low laser illumination intensities (< ~40 W/cm²). This is explained by the suppression of a sulfoxidation reaction that in the IrisFP parent protein results in trapping the chromophore irreversibly in a nonfluorescent protonated state. The mechanism that leads to sulfoxidation of Met159 likely involves quenching of the chromophore triplet state by oxygen, which releases highly reactive singlet oxygen within the chromophore pocket. Singlet oxygen is prone to attack any neighboring sulfur group, and thus rapidly reacts with the nearby Met159. The observed enhancement in fatigue resistance by a factor > 5 in solution reaches beyond expectations, considering that other sources of photobleaching certainly exist (e.g. destabilization of the protein scaffold by oxidation reactions involving singlet oxygen and other residues such as cysteins, tryptophans or tyrosines). Thus, the Met159 sulfoxidation pathway appears to be the major source of photobleaching in IrisFP in solution and at low illumination intensities, and this conclusion may also hold for popular phototransformable fluorescent proteins such as mEos2, Dronpa or Dendra2, which all have a methionine at position 159 located next to the chromophore. However, in PVA and in E. coli cells, the increase in photoresistance afforded by the M159A mutation was variable (Table 4) suggesting that the complex nanoenvironment in these samples may promote other photobleaching pathways.

The M159A mutation in IrisFP also resulted in about a 4-fold faster off-switching speed, which is similar to the effect obtained in e.g. Dronpa variants [12,24]. The combination of more rapid switching and enhanced photoresistance yielded a considerable increase in the number of achievable on-off switching cycles, reaching a factor > 40 in solution. In PVA and in E. coli cells, the situation was again different: a strong decrease of the switching speed of the two proteins was noticed and the enhancement in the number of achievable switching cycles by the mutant was not so high (factors of ~2, ~7.5 and ~1.8 in PVA, fixed cells and live cells, respectively).

Under high illumination intensities (~0.4 kW/cm²), the improvement in photostability achieved in the M159A-IrisFP mutant could not be assessed due to the limited time-resolution of our experimental setup, which did not allow to accurately monitor individual switching cycles.

Nevertheless, the gain in the number of on-off switching cycles achieved by IrisFP-M159A was on average reduced as compared to low light-level conditions (with the intriguing exception of live cells, Table 4). This is consistent with the hypothesis that other photobleaching pathways predominate at high illumination intensities. We indeed observed that at such intensities photobleaching in IrisFP essentially proceeds through a consecutive two-photon absorption process (CTPA) that leads to disruption of chromophore π-conjugation through decarboxylation of the fully conserved Glu212 residue, without involving Met159 nor the presence of oxygen [18]. Yet, the improvement in photofatigue decay achieved by IrisFP-M159A at high intensities suggests that Met159 could indirectly participate in this redox based photobleaching mechanism.

Overall, our data suggest that IrisFP-M159A, or preferably monomeric variants less prone to impair fused protein function such as NijiFP-M159A, Dendra2-M159A [11], or pcDronpa2-M159A [30], could facilitate the use of advanced techniques based on photochromism such as pcFRET [7],

OLID microscopy [31] or pcSOFI [8].

Our widefield-based setup did not allow testing IrisFP and its mutant under the experimental conditions of RESOLFT nanoscopy. Based on our results and on the published switching properties of rsEGFP and rsEGFP2, which exhibit ~1100 and ~2100 cycles, respectively, before half decay in polyacrylamide [16], we speculate that the latter RSFPs remain superior for RESOLFT nanoscopy.

However, the successful use of Dronpa-M159T with this technique [32] suggests that IrisFP-like M159A variants could still perform well under RESOLFT conditions. The use of mIris-M159A or NijiFP-M159A could thus open the door to the development of two-color pulse-chase RESOLFT nanoscopy [33].

The putative superior photofatigue resistance of EGFP-like RSFPs could be explained by two factors. First, rsEGFP and its derivatives do not have sulfur containing residues in their chromophore pocket, de facto achieving the same effect as the one we rationally engineered in the case of IrisFP-M159A. Second, Glu222 decarboxylation in these proteins may not necessarily result in photobleaching, as suggested by the observation that such decarboxylation rather activates proteins of the GFP family [34], the most notable example being photoactivatable PA-GFP [35]. Nevertheless, a precise understanding of the strong photoresistance of rsEGFP and its variants calls for a structural characterization of these proteins in their on and off states as well as in their photofatigued states.

In conclusion, we have shown that Met159 plays a critical role in controlling the photostability of IrisFP and possibly other RSFPs of anthozoan origin. Importantly, the photoswitching behavior of RSFPs appears to be strongly influenced by the local nanoenvironment, consistent with the growing evidence that, despite embedding by the β-barrel matrix, the photophysics of fluorescent protein's chromophores is largely controlled by the external environment. In particular we observed that PVA gels or the use of paraformaldehyde for cell fixation drastically changed the photoswitching properties of IrisFP and its mutant. The rationale behind such observation remains to be investigated, but could involve redox effects or reduced conformational freedom. Finally, the complex shape of photofatigue decay profiles observed in this work suggests the existence of subtle photophysical processes that could, in particular, involve the formation of long-lived dark states yet to be characterized.

Acknowledgment

We thank Stefan Jakobs and Tim Grotjohann for insightful discussions. D.B. acknowledges financial support by the ANR (ANR-2011-BSV5-012-01 NOBLEACH).

(assumed to be uniform throughout the illuminated sample), N A is the Avogadro number, h is the Planck constant and c is the speed of light.

The model then simply writes:

[ ]( ) [ ]( ) [ ]( ) t OFF k t ON k dt t ON d ON OFF φ φ 1 1 - + - = [ ]( ) [ ]( ) 0 C t OFF t ON = +
where C 0 is the total concentration of the fluorescent protein.

During switching-off periods (488 nm laser on and 405 nm laser off), we have:

488 1 1 488 1 1 , - -= = k k k k Note that 488 1 -
k corresponds to residual on-switching due to absorption at 488 nm of the OFF state. This residual on-switching may not be negligible, and will essentially determine the observed switching contrast. 488 1 -k depends on the extinction coefficient 488 OFF ε ie the absorption of the OFF state at 488 nm, which is unknown but can be fitted from the data.

During switching-on periods (488 nm laser on and 405 nm laser on), we have:

405 1 488 1 1 405 1 488 1 1 , - - - + = + = k k k k k k
As 488 1 k is very large as compared to 405 1 k because 488 ON ε , the absorption of the ON state at 488 nm, is much larger than 405 ON ε (which is also unknown), and the illumination power density at 488 nm is typically much higher than that at 405 nm, 405 1 k is neglected.

The emitted fluorescence is then:

( ) [ ]( ) t ON t F ∝
To take into account the limited time resolution of the EMCCD detector, the detected signal at every frame is then integrated over the frametime:

( ) ( ) ∫ ∝ frametime EMCCD dt t F frame S

Case of fixed molecules:

For a fixed oriented molecule with spherical coordinates θ and φ, the excitation rate of the absorbance dipole is angle dependent. For a circularly polarized laser beam whose electromagnetic field is assumed to be parallel to the objective focal plane, the excitation rate is given by: ( )

θ θ 2 cos 2 3 k k =
, where θ is measured from the focal plane. The model then writes:

( ) [ ]( ) ( ) ( ) [ ]( ) ( ) ( ) [ ]( ) t OFF k t ON k dt t ON d ON OFF θ φ θ θ φ θ θ 1 1 - + - = ( ) [ ]( ) ( ) [ ]( ) ( ) θ θ θ 0 C t OFF t ON = + ( ) ( ) ( ) 0 0 cos cos C d C θ θ θ θ ∫ =
For each θ the calculations proceeds in the same way, however, the fluorescence is not emitted isotropically, so that based on Fourkas et al (Opt Lett 26: 211-213, 2001), the detected signal is:

( ) ( ) [ ]( ) ( ) ∫ ∝ θ θ θ d E t ON t F
with the detection efficiency ( ) θ E taking the form:

( ) ( ) θ θ 2 sin b a E + =

Case of variable tumbling propensity:

In real situations, molecules might be partially tumbling. We modeled this case as the fixed molecule case except that the excitation rates were then obtained as weighted averages of those rates for fully tumbling and fixed molecules.

( ) ( )k w k w k - + = 1 cos 2 3 2 θ θ
where w is a tumbling propensity.

RESOLFT Reversible saturable optical linear fl uorescence transitions SSIM Saturated structured illumination microscopy (pc)SOFI (Photochromic) stochastic optical fl uctuation imaging IR Infra-red HSQC Heteronuclear single quantum coherence XFEL X-Ray-free electron laser

Introduction

Fluorescent proteins (FPs) have become indispensable tools to investigate the interrelations between cell structure, function, and dynamics [ 1 ]. Their three-dimensional structure is quite simple: the peptide chain forms an 11-stranded β-barrel resembling a "soda can," which wraps around a three-residues-based endogenous chromophore (Fig. 1 ). This 4-( p -hydroxybenzylidene)-5imidazolinone ( p -HBI) chromophore only requires oxygen as an external cofactor to become mature. A stunning palette of fl uorescent proteins displaying a wide range of emission colors (450-650 nm) has been engineered, typically by rational or random mutagenesis of the chromophore itself or of its close environment [ 2 ]. The discovery of FPs from Anthozoan species (e.g., corals or anemones), which exhibit a high structural similarity but only a small sequence identity (<30 %) with the more classical Hydrozoan FPs (e.g., jellyfi shes), launched the development of red fl uorescent proteins [ 3 ], and considerably boosted FP research in recent years.

In addition to suitable colors, useful FPs should exhibit a high expression level, fast maturation, and high fl uorescence brightness so that they can be imaged with suffi cient signal-to-noise ratio soon after cell transfection. Furthermore, they should be monomeric and should not induce cytotoxicity, to avoid cellular dysfunction. Importantly, they should be photostable and possible modifi cations of their fl uorescence properties as a function of light excitation or environmental parameters (e.g., pH, redox potential, oxygen level) should be understood.

The protonation state of the p -hydroxybenzylidene moiety of the chromophore plays a key role in the fl uorescence properties of FPs. Two protonation states can generally be adopted, the proportion of which is determined by the interactions with neighboring residues in the chromophore pocket and may (or not) vary with pH. The protonated (neutral) chromophore absorbs in the socalled "A-band," whereas the deprotonated (anionic) chromophore absorbs in the "B-band" (Fig. 1 ). For a green-emitting FP, those bands usually peak at ~400 nm and ~490 nm, respectively. In their anionic state, FPs are typically highly fl uorescent, whereas in their neutral state they are dimly or not fl uorescent. However, upon excitation of the neutral state, the FP chromophore becomes a strong acid with a p K a near zero. Ultrafast conversion to a deprotonated excited state may thus occur, resulting in high yield and strongly Stokes-shifted fl uorescence.

The high fl uorescence quantum yield of FPs (generally > ~0.5) is commonly attributed to the idea that the chromophore is held rigidly within the protein matrix through a set of tight non-covalent interactions. This is a too simplistic view. In fact, FPs exhibit a highly dynamic behavior, in line with their observed complex photophysics. Upon light absorption, and especially when intersystem crossing to the long-lived triplet state takes place, the FP structural plasticity may allow a number of photophysical or photochemical transformations to occur. For example, like any fl uorophore, FPs undergo transient stochastic switching events to nonfl uorescent dark states ("blinking"), and eventual conversion to a permanent off state ("bleaching").

In the subfamily of fl uorescent proteins termed Phototransformable FPs (PTFPs), specifi c phototransformations can be quantitatively induced by light, which is at the basis of a number of revolutionary developments in advanced fl uorescence microscopy. These PTFPs, mostly found in Anthozoan stony corals, but also engineered from Hydrozoan FPs have become the focus of intense research since a few years [ 4 -6 ]. Three types of phototransformations may be distinguished in PTFPs (Fig. 2 ): nonreversible activation from a nonfl uorescent to a fl uorescent state (referred to as "photoactivation," fl uorescent proteins of this group are called Photoactivatable FPs (PAFPs)), nonreversible conversion between two fl uorescent states with different emission colors ("photoconversion," fl uorescent proteins of this group are called Photoconvertible FPs (PCFPs)), and reversible switching between a fl uorescent on-state and a nonfl uorescent off-state ("photoswitching," fl uorescent proteins of this group are called Reversibly Switchable FPs (RSFPs)). In this chapter we give a structural perspective on the fascinating photophysical properties of RSFPs.

Discovery and Classifi cation of RSFPs

A list of the currently developed RSFPs with their main photophysical properties is provided in Table 1 .

The fi rst observation of reversible photoswitching of a fl uorescent protein at room temperature was made with yellow derivatives of Aequora victoria GFP at the single molecule [ 7 ] and at the ensemble levels [ 8 -10 ]. However, photoswitching of these Hydrozoan FPs appeared limited, probably involving only minor Wavelengths required for reversible transitions. Background colors represent the emission colors of the proteins (left) and the colors of lights used to reversibly switch proteins (right). M, monomer; T, tetramer; NA, not applicable; ND, not determined subpopulations of molecules. Effi cient photoswitching was initially reported of the weakly fl uorescent and tetrameric asFP595 from the Anthozoan sea anemone Anemonia sulcata . The phenomena was described as reversible "kindling," because the fl uorescence emission from asFP595 was enhanced by the fl uorescence excitation light (568 nm) and quenched by blue light (450 nm) [ 11 ].

The fi rst RSFP of suffi cient quality to successfully conduct biological experiments was obtained upon engineering a Pectiniidae coral FP, yielding the well-known Dronpa [ 12 ]. In contrast to asFP595, fl uorescence emission in Dronpa is quenched by the excitation light (490 nm) and recovered by illumination with violet light (405 nm). A large number of mechanistic investigations were then performed on these two proteins [ 13 -28 ].

In view of the growing interest of RSFPs for advanced fl uorescence microscopy applications, substantial efforts were made in the last years to develop variants with improved properties such as higher fl uorescence brightness (the product of fl uorescence quantum yield and molar extinction coeffi cient), enhanced switching contrast (ratio of fl uorescence emission of the on and off states), tunable switching quantum yields, increased photoresistance, or red-shifted emission (Table 1 ). Several new Anthozoan RSFPs were thus introduced such as a number of Dronpa variants [ 20 , 29 , 30 ], mTFP0.7 from Clavuliara sp. [ 31 ], rsTagRFP from E. quadricolor [ 32 , 33 ], mGeos from L. hemprichii [ 34 ], and others (Table 1 ). An interesting case was IrisFP (and later NijiFP), which combines onoff photoswitching with green to red photo-conversion properties [ 35 , 36 ], introducing the possibility to achieve dual color photoswitching with a single protein. Recently, Hydrozoan GFP-based RSFPs were also developed, showing that high-performance photoswitching is not restricted to Anthozoan FPs. Single-mutation of the strictly conserved Glu222 (GFP numbering) into Gln conferred high-contrast switching properties to variants of YFP [ 37 ], while a combined rational and random mutagenesis approach yielded the photoresistant rsEGFP [ 38 ] and rsEGFP2 [ 39 ].

All these RSFPs can be classifi ed as "negative" or "positive." In negative RSFPs such as Dronpa, off-switching results from illumination at wavelengths absorbed by the protein in its on-state. In positive RSFPs such as asFP595, on-switching results from illumination at wavelengths absorbed by the protein in its on-state (Table 1 ). In all cases, switching is thought to primarily result from a light-induced cis -trans isomerization of the chromophore accompanied by a change of the p -hydroxybenzylidene protonation state. A different acid-base environment of the chromophore in the two isomeric states modulates its p K a and is key to the photoswitching function. In the fl uorescent on-state, the chromophore is typically in an anionic cis confi guration, whereas in the nonfl uorescent off state it is found in a trans confi guration, either neutral or anionic. In negative RSFPs, the cis confi guration of the chromophore is the thermodynamically stable form, so these proteins are typically highly fl uorescent in their native state. In positive RSFPs, the nonfl uorescent trans confi gurations tends to be more thermodynamically stable, although both states are sometimes observed at equilibrium, so these proteins are normally nonfl uorescent or weakly fl uorescent in their native state.

In 2011, Brakemann et al . [ 40 ] engineered YFP-derivatives previously shown to be partially switchable [ 10 , 41 ] to elaborate Dreiklang, an RSFP that exhibits an entirely new mechanism which does not rely on cis -trans isomerization of the chromophore, but rather involves reversible chemical modifi cations of the imidazolinone moiety. In Dreiklang, photoswitching is neither positive nor negative but is "decoupled," meaning that fl uorescence excitation exerts no infl uence on photoswitching.

Methodology for the Investigation of Photoswitching Mechanisms

Boosted by this rapidly evolving research fi eld, increased attention has been devoted to mechanistic investigations of RSFPs with a dual goal: gaining fundamental insight into this intriguing phenomenon and rationally designing variants with enhanced photophysical properties. Several methods can be used that are briefl y described below.

Confocal or wide-fi eld fl uorescence microscopy approaches based on single-molecule detection allow monitoring the stochastic nature of photoswitching. By recording many singlemolecule fl uorescence traces, histograms of on-times and off-times can be extracted from which key photophysical parameters such as switching quantum yields can be derived. Singlemolecule investigations provide the essential advantage that subpopulations of molecules behaving differently from the average can be identifi ed. The same illumination conditions as those used in cell microscopy experiments can be used and the infl uence of environmental conditions such as redox potential, pH, or oxygen level can be monitored. However, to study single molecule behavior, the RSFPs are typically attached to a glass coverslip or immobilized in a polymer substrate, which might induce deviations relative to the in cellulo behavior. Ultimately, despite complications due to a lower signal-to-noise ratio and a higher molecular density, these methods will be applied directly in the biological sample so as to evaluate RSFPs switching in genuine experimental conditions.

To get a high-resolution structural view of photoswitching, ensemble level techniques must be used, such as X-ray crystallography, NMR, UV-vis optical spectroscopy, or vibrational spectroscopy. The most direct view of the structural signature of photoswitching is provided by crystallography. However, the nonstandard concepts of "kinetic" protein crystallography need to be used [ 42 ], whereby photoswitching is induced in crystallo . Indeed, the photoswitched chromophore is usually not stable for more than at most a few hours, a time much too short to achieve crystal growth. Thus, to generate the switched state, the crystalline sample is submitted to laser illumination, followed by fl ash cooling to prevent back switching and to minimize radiation damage effects during diffraction data collection. Fortunately, FPs are wellsuited samples for kinetic protein crystallography, as conformational changes induced by light illumination are small enough to be compatible with crystal packing interactions, so that samples are usually not seriously deteriorated upon illumination. However, it should always be kept in mind that the crystalline state may exert an infl uence on the observed switching scenario, for example by selecting out a subset of conformational states not strictly representative of the in-solution behavior. This point has for example been a matter of debate concerning the structure of the off state of Dronpa [ 16 ]. 2D-NMR investigations (typically based on 1 H- 15 N or 1 H- 13 C HSQC experiments) have the advantage that no crystal constraints are present, so the observed conformational changes may be more genuine. However, the usually longer measuring times at room temperature make it trickier to maintain the photoswitched state in the sample. For example, prolonged illumination might be required and result in unwanted photobleaching. In all cases, X-ray or NMR structural views should be complemented by steady-state optical spectroscopy measurements so as to quantify the extent of photoswitching in the investigated samples. Optical microspectrophotometry can be applied in crystallo , in the absorbance, fl uorescence [ 43 ], or even Raman mode [ 44 ]. It should be noted that careful spectroscopic investigations using relatively basic instruments can provide a wealth of insightful mechanistic information complementary to structural approaches [ 45 , 46 ].

To gain further mechanistic insight, putative intermediate states along the photoswitching reaction pathway should be characterized in addition to the switching endpoints. Since photoswitching is typically a very rapid (subnanosecond) process, ultrafast pump-probe UV-vis or IR spectroscopy are the tools of choice to be used. However, a major diffi culty is that photoswitching in RSFPs is a low yield process (10 -4 -10 -1 ) so that the fraction of molecules that can simultaneously follow the desired photoswitching pathway is small (although a larger fraction may engage into the pathway but quickly return to the starting state or deviate to other photophysical states). This explains why almost all ultrafast spectroscopic investigations of RSFPs photoswitching so far concentrated on the back-photoswitching reaction of Dronpa which has a particularly high yield (>0.1).

Yet, another approach to track intermediates consists in performing temperature-controlled experiments. At suffi ciently low temperature, the switching reaction may not be able to be completed if thermal energy barriers are present along the pathway. In such a case, activated molecules may possibly get trapped and accumulate in an intermediate state that can then be probed structurally or spectroscopically. Such an approach was for example used to study the protein Padron [ 47 ]. Working at low temperature, however, always carries the danger that the protein conformational landscape may be altered.

Important insight into photoswitching mechanisms can also be obtained from theoretical investigations such as molecular dynamics simulations or reaction-path-fi nding techniques. In such studies, the RSFP is modeled with quantum mechanics/molecular mechanics (QM/MM) hybrid approaches: the chromophore and its nearby environment are described at the quantum level in the ground or excited state, whereas the rest of the protein is treated with classical force fi elds. Such investigations have provided a number of interesting details about fundamental properties of photoswitching [ 21 , 27 ]. However, they strongly rely on available high-resolution crystallographic input structures, and it should be kept in mind that they remain restricted by necessary simplifi cations and assumptions, due to limited computing power. For example, modeling proton transfer reactions involving the RSFPs chromophore is diffi cult, as the number of atoms in the QM region can typically not be changed along simulations.

Ultrafast time-resolved crystallography experiments on RSFPs based on synchrotron Laue diffraction have so far remained unsuccessful. However, new XFEL sources offer exciting prospects to catch photoswitching in action. In the long term, the dream would be to watch single RSFPs in real time at atomic resolution, e.g., by combining femtosecond stimulated Raman scattering [ 48 ] with XFEL diffraction [ 49 ].

Photoswitching Mechanisms

In this section, we describe in more detail the photoswitching mechanisms as observed in negative, positive, and decoupled RSFPs. Three important remarks should be made beforehand.

First, the p -HBI chromophore in solution has been shown to undergo facile photoinduced cis -trans isomerization caused by twisting around the methylene bridge that links the two cyclic moieties (explaining the lack of fl uorescence of the isolated p -HBI chromophore in solution) [ 50 ]. Thus, chromophore isomerization in RSFPs is primarily a manifestation of intrinsic excited state chromophore dynamics. This process is in general hindered by the protein scaffold in FPs, but it can still occur with low yield in RSFPs. Chromophore isomerization in RSFPs is a single-photon excitation process in both directions [ 13 , 47 ]. However, back-switching is also thermally driven and it should be kept in mind that some FPs are able to change the isomeric state of their chromophore in the ground state following pH changes [ 51 , 52 ].

Second, the isomeric state of the chromophore in general does not dictate per se the occurrence or the lack of fl uorescence. There exist FPs exhibiting strong fl uorescence in the trans state [ 52 , 53 ]. However, in positive or negative RSFPs, the nonfl uorescent state has always been found to correspond to the trans confi guration of the chromophore.

Third, the proton affi nity of the chromophore hydroxybenzylidene group strongly depends on the local protein environment and therefore it is isomer dependent, both in the ground and in the excited states. Widely different chromophore p K a are therefore expected-and observed-in the trans or cis confi gurations. As chromophore protonation has a strong infl uence on the ability to fl uoresce, this isomer-dependent p K a generally plays a key role in fl uorescence switching.

To describe the photoswitching mechanism in negative RSFPs, we take the example of IrisFP, which has been extensively characterized by us [ 35 ]. The X-ray structures and absorption/fl uorescence spectra of the protein in its on and off states are presented in Fig. 3 .

In both states, it can be seen that the chromophore is maintained by a complex set of H-bonding interactions involving Arg66, Ser142, Glu144, Ser173, Tyr177, His194, and Glu212, as well as water molecules. These residues are essentially conserved in all negative RSFPs from Anthozoa species. cis -trans Isomerization is accompanied by a substantial structural change of the chromophore pocket. The tightly H-bonded triad Glu144-His194-Glu212 in the cis confi guration is replaced by the Glu144-Arg66-Glu212 triad in the trans confi guration, with either His194 or Arg66 stabilizing the chromophore by π-stacking and π-cation interactions with the hydroxybenzylidene moiety, respectively. In the cis confi guration, the chromophore phenolate interacts with Ser142 whose hydroxy group is protonated, thus favoring a deprotonated state. This anionic state is further favored by the interaction of Arg66 with the carbonyl group of the imidazolinone moiety, which retains electron density on that ring. Hence, the p K a of the cis chromophore in IrisFP ensures a predominantly deprotonated chromophore at physiological pH (p K a = 5.7). The chromophore is also found in a highly planar confi guration. Thus, the conditions favoring a high fl uorescence yield are fulfi lled. In the trans confi guration, a very different scenario is found, where the phenolate group is interacting with the deprotonated Glu144, and the interaction of Arg66 with the imidazolinone being disengaged. Consequently, there is a substantial change in the chromophore electrostatic environment, and the latter is found completely protonated at physiological pH, with a p K a > 10.

Negative Anthozoan RSFPs: On and Off States

No ESPT (excited state proton transfer) is observed and the chromophore is found to adopt a quite distorted geometry. Thus, the conditions favoring a high fl uorescence yield are not fulfi lled. The trans -conformation is thermodynamically quite stable, taking a few hours to return to the cis -conformation. This stems from the fact that the protein scaffold is able to accommodate the hydroxybenzylidene group of the trans -chromophore with overall little deformation, and by optimally rearranging the H-bond networks within the pocket. Some subtleties contribute to enhancing the stability of the trans -state: for example, Ser142, which maintains a strong Fig. 3 Crystallographic structures of the chromophore, its microenvironment, and the corresponding spectra for IrisFP. ( a ) In the more stable fl uorescent state (PDB ID 2VVH), the chromophore adopts a cis conformation and is deprotonated as observable by the major anionic absorption band peaking at 487 nm ( black spectrum ) that provides fl uorescence peaking at 516 nm ( green dotted spectrum ). ( b ) In the dark state (PDB ID 2VVI), the 3D structure shows that the chromophore clearly adopts a trans conformation and is protonated as observable by the major neutral band peaking at 390 nm ( black spectrum ). The remaining fl uorescence signal ( green dotted spectrum ) is due to a minor fraction of chromophores remaining in the cis conformation. Water molecules are represented as red balls and hydrogen bonds are represented as black dotted lines H-bond with the hydroxybenzylidene moiety in the cis -state, engages with another H-bonding partner once the chromophore has been isomerized, as a compensation. Also, Glu212 directly interacts with the imidazolinone nitrogen in the trans -state, providing further stabilization.

Similar schemes have been described for Dronpa [ 15 ] and mTFP0.7 [ 31 ], with some relatively minor variations. An important peculiarity of Dronpa is that upon off-photoswitching, the seventh β-strand near the chromophore becomes disordered, as shown by NMR experiments [ 16 ]. As this strand forms part of the cross-dimer interface in the tetrameric parent of Dronpa, Dronpa photoswitching modulates its propensity to multimerize at high concentration. This property was ingenuously used to develop a new optogenetic approach [ 54 ]. Likewise, researchers took advantage of the fl exibility-mediated photoswitching of Dronpa to develop a viscosity measurement assay [ 55 ].

Padron is a positive RSFP engineered from Dronpa [ 30 , 56 ]. The two essential Met159Tyr and Val157Gly mutations were suffi cient to completely reverse the switching properties of Dronpa (hence the name "Padron"). The X-ray structures and absorption/fl uorescence spectra of Padron in its on and off states are presented in Fig. 4 . In the on state, the confi gurations of the chromophore and of its immediate environment do not differ much from Dronpa or IrisFP. However, Ser142 adopts a different conformation than in Dronpa, which might be responsible for the substantial increase in the proton affi nity of the chromophore hydroxybenzylidene moiety. Indeed, the chromophore exhibits a p K a of 6.0 (5.3 for Dronpa), meaning that at physiological pH a signifi cant fraction of the switched-on Padron molecules are protonated and nonfl uorescent. In the trans state, structural differences with negative RSFPs are striking: Tyr159 now establishes an H-bonding interaction with the chromophore phenolate, which maintains the latter in an anionic state, with a p K a of 4.5. His193, Arg66, and Glu211 (equivalent to His194, Arg66, and Glu212 in IrisFP) do not change their conformation relative to the cis state, possibly because of the shifted location of Ser142, and this may in turn result in the severe torsion of the trans chromophore. Thus, the trans chromophore is nonfl uorescent but exhibits an absorption spectrum similar to that of the fl uorescent on state (although somewhat broader presumably due to a larger conformational freedom).

Photoswitching kinetics and pathways in RSFPs are controlled by the potential energy surfaces that connect the on and off states of the chromophore. The chromophore isomer conformation and protonation, in the ground and excited state, as well as the protein environment, are all involved. Therefore, modifying the chromophore composition, or its environment, allows tuning properties

Positive Anthozoan RSFPs: On and Off States

Positive and Negative Anthozoan

RSFPs: Reaction Pathways such as photoswitching rates, photon outputs, photostability, or pH-sensitivity. For example, Chang et al. [ 34 ] engineered the mGeos negative RSFPs by modifying the fi rst position of the chromophore tripeptide and found that mGeos-M (with a methionine at this position) had the highest number of photons emitted per switching cycle amongst all green RSFPs. Likewise, Adam et al. [ 36 ] modifi ed the protein environment of the photo-convertible EosFP and Dendra2 PCFPs to engineer bi-photochromic variants with differing properties. Fig. 4 Crystallographic structures of the chromophore, its microenvironment, and the corresponding spectra for Padron. ( a ) In the more stable dark state (PDB ID 3ZUF) the 3D structure shows that the chromophore adopts a trans conformation and is deprotonated as indicated by the major anionic absorption band peaking at 503 nm ( black spectrum ). ( b ) In the fl uorescent state (PDB ID 3ZUJ) the chromophore clearly adopts a cis conformation. Absorption spectra reveal two bands, one peaking at 390 nm and corresponding to the neutral fraction and one peaking at 503 nm and corresponding to the anionic fl uorescent fraction peaking at 518 nm. Water molecules are represented as red balls and hydrogen bonds are represented as black dotted lines

In both positive and negative RSFPs, the exact order of events and the presence of intermediate states along the reaction pathway have been investigated. Because of its high quantum yield (~0.5), the back switching reaction in Dronpa could be experimentally interrogated by ultrafast spectroscopy. ESPT to an intermediate state I was proposed to occur, consistent with the observation of a signifi cant kinetic isotope effect (KIE) using deuterated samples [ 18 ]. In line with this view, theoretical calculations proposed that isomerization and deprotonation events during Dronpa off-on switching are concerted [ 21 ]. This view is also consistent with the elegant theoretical model of Olsen et al. [ 57 ], which suggests that to promote effi cient photoswitching, the protein environment should restrict torsion around the methylene phenoxy bond (P-bond) and promote torsion around the imidazolinone bond (I-bond) through suitable acid-base chemistry. However, these fi ndings were recently questioned by a Fourier Transform infrared study (FTIR) in which a cis protonated ground state intermediate was evidenced, strongly suggesting that deprotonation of the chromophore rather occurs as a subsequent step to isomerization [ 23 ]. These contradictory observations show that the interpretation of ultrafast spectroscopic data is a delicate issue and corroborates the fact that many interrelated factors control photoswitching in RSFPs, the exact roles of which are diffi cult to disentangle. Moreover, hidden processes may take place and further complicate the matter: as an example, the recent study of Gayda et al. [ 45 ] on a mutant of IrisFP (called mIrisGFP) revealed that the neutral cis chromophore of mIrisGFP can isomerize to the neutral trans state much more effi ciently than the anionic cis chromophore (consistent with a barrierless mechanism predicted by the Olsen model [ 57 ]). This phenomenon however typically remains unnoticed, as back switching by the same illumination wavelength (405 nm) is even more efficient, bringing back the neutral trans chromophore "immediately" to the cis confi guration. Several different switching processes may thus occur in parallel, even if at the ensemble level the spectroscopic footprint remains steady. Overall, the precise photoswitching mechanism of negative RSFPs still remains to be elucidated completely.

In positive RSFPs, work has been done on Padron and asFP595. A study of the asFP595-Ala143Ser mutant (displaying an enhanced fl uorescence quantum yield) by molecular dynamics suggested that on-switching was linked to chromophore trans -cis isomerization via a Hula-Twist mechanism, in which both I and P bonds change in a concerted manner to minimize the volume swept by the chromophore during switching [ 14 ]. This view is however diffi cult to reconcile with the Olsen theory of isomerization through I-bond fl ip. Ab initio calculations and QC/MM molecular dynamics simulations in asFP595 suggested that trans -cis isomerization occurs in the neutral state of the chromophore, followed by a dark state equilibration to a zwitterionic fl uorescent cis state [ 27 ]. In Padron, a different scenario was observed: two fl uorescent intermediates along the on-switching pathway could be cryo-trapped [ 47 ]. The combined spectroscopic and crystallographic data suggested that trans -cis isomerization of the chromophore occurs entirely in the anionic state and precedes protonation. These experimental fi ndings are in line with the latest results by FTIR in Dronpa [ 23 ]. They are also interesting in terms of structural protein dynamics. First, they reveal that full trans -cis isomerization of the Padron chromophore is possible at 100 K, a temperature at which protein dynamical breathing is essentially stalled. As heat dissipation upon photon absorption occurs on the picosecond timescale, a transiently "hot" chromophore could account for this observation. Second, it was observed that protonation of the chromophore following isomerization only took place above the glass transition temperature (~200 K), suggesting that protonation involves exchange with the surrounding solvent. The fact that Padron can be effi ciently photoswitched at cryo-temperature opens interesting potential applications such as cryo-nanoscopy [ 47 ].

Overall, as for negative RSFPs, the mechanisms governing fl uorescence switching in positive RSFPs are still not entirely understood. The extent by which these mechanisms differ between members of each family also remains to be evaluated. Surprisingly little structural information is available on RSFPs evolved from GFP derivatives. Amongst several pieces of indirect evidence, studies by vibrational spectroscopy [ 58 ] suggest that a chromophore cis -trans isomerization process similar to that found in Anthozoan RSFPs also takes place in these proteins. However structural evidence for a trans chromophore in Hydrozoan RSFPs is still lacking.

Positive and negative RSFPs are not optimal in that laser-light used for fl uorescence excitation also induces switching. The protein Dreiklang, evolved from the GFP variant Citrine, nicely overcomes this problem [ 40 ]. In Dreiklang, excitation at 514 nm does not induce signifi cant off-switching, whereas near UV-light at 365 and 405 nm result in on and off switching, respectively. The existence of such decoupling had already been hinted at in previous experiments with EYFP [ 10 , 41 ]. In Dreiklang, this behavior was deliberately exacerbated by random mutagenesis, resulting in an unprecedented switching mechanism. Combined crystallographic and mass spectroscopy analyzes provided evidence that lightinduced hydration-dehydration of the chromophore caused switching by disrupting the π-conjugated electron system (Fig. 5 ). Hydration of the C 65 atom of the imidazolinone moiety appeared to be facilitated by the proper positioning of a water molecule (Wat A ) held in place by hydrogen bonding between Tyr203 and Glu222 and putatively replaced in the switched-off state by another water molecule (Wat B ) garnered from the environment. However, the exact roles of the 4 mutations (Val61Leu, Phe64Ile, Tyr145His,

Hydrozoan RSFPs

Decoupled Switching in Hydrozoan RSFPs: Dreiklang

and Asn146Asp) relative to Citrine remain to be established. Also, the use of UV-light to induce on-and off-switching is prone to generate cytotoxicity. It is also not clear yet how the hydration/ dehydration processes can be light-activated in the protonated state of the chromophore.

Using RSFPs in Advanced Fluorescence Applications

Many exciting fi elds of science are nowadays explored by using photoswitchable fl uorescent proteins, including super-resolution fl uorescence microscopy ( see Chapter 16 of this volume), Fig. 5 Crystallographic structures of the chromophore, its microenvironment, and the corresponding spectra for Dreiklang. ( a ) In the more stable fl uorescent state (PDB ID 3ST4), the chromophore adopts a cis conformation and is deprotonated as observable by the major anionic absorption band peaking at 515 nm ( black spectrum ) that provides fl uorescence at 529 nm ( green dotted spectrum ). ( b ) In the dark state (PDB ID 3ST3) the water molecule Wat A is found to hydrate the carbon C 65 , distorting the geometry of the imidazolinone ring. Absorption spectra reveal that both the neutral and the anionic forms have been converted to a blue-shifted absorption band peaking at 340 nm. Water molecules are represented as red balls and hydrogen bonds are represented as black dotted lines optogenetics, optical lock-in detection, frequency-domain imaging, sensor developments and biotechnological applications (Fig. 6 ). Some examples are described below.

Single molecule localization microscopy (SMLM) was developed in 2006 with techniques such as photoactivated localization microscopy (PALM) [ 59 , 60 ] and stochastic optical reconstruction microscopy (STORM) [ 61 ]. These super-resolution techniques are conceptually identical but use either phototransformable FPs (PALM) or blinking organic dyes (STORM) as highlighters. SMLM is nowadays very popular in that it does not require a complex instrumental setup and provides the best spatial resolutions currently achievable. Irreversible photoconvertible FPs are generally preferred for PALM applications over RSFPs because they typically emit more photons per localized spot and are less prone to complications due to multiple localizations of a single molecule. Multicolor PALM, however, is diffi cult to achieve with PCFPs only, as currently available members do not display a large spectral separation. A solution to this problem has been found by achieving dual-labeling with a green-to-red PCFP such as EosFP and an RSFP such as Dronpa. The method relies on a sequential acquisition protocol: all EosFP molecules are fi rst photoconverted to

Super-Resolution Fluorescence Microscopy by Single Molecule Localization

Fig. 6 Panoply of possible applications using RSFPs in super-resolution imaging and biotechnology. OLID optical lock-in detection, FDI frequency domain imaging their red-emitting state by 405-nm irradiation and detected by 561-nm illumination with minimal effects on the Dronpa molecules that are visualized subsequently with alternating 488 and 405-nm illumination. This protocol succeeded in colocalizing a variety of proteins of interest [ 62 , 63 ].

Interestingly, the rich photophysics of RSFPs also allow acquiring dual-labeling "false-multicolor" PALM data [ 30 ]. This can be accomplished by distinguishing RSFPs variants of similar colors based on properties such as positive vs. negative switching, or different photoswitching rates.

The capacity of RSFPs to produce intense fl uctuations of their emission signal recently permitted their application into a variant of SMLM called stochastic optical fl uctuation imaging (SOFI) ( see Chapter 17 of this volume). In its standard version, SOFI takes advantage of natural fl uorescence fl ickering to enhance image resolution [ 64 ]. Photochromic SOFI (pcSOFI) [ 65 ] demonstrates improved signal-to-noise contrast and a two-to threefold enhancement of the spatial resolution compared to diffraction-limited images, thanks to the possibility of precisely controlling the "fl ickering" (switching) of RSFPs.

RSFPs can be advantageously used in nonlinear imaging because the saturation of their switching transitions leads to distinct minima/maxima of fl uorescence depending on the illumination pattern, even with low light intensities.

In confocal illumination mode, the concept of REversible Saturable Optical Linear Fluorescence Transitions (RESOLFT) [START_REF] Hell | Concepts for nanoscale resolution in fl uorescence microscopy[END_REF] has been proposed as an alternative to the well-known STimulated Emission Depletion (STED) microscopy. In STED, a donut-shaped laser spot applied immediately after excitation by a diffraction-limited laser spot depletes emission of a standard fl uorophore except for the central zero-intensity region of the donut beam. The diameter of the resulting effective point spread function decreases with increasing intensities of the donut-shaped beam, thus providing enhanced resolution. However, an enormous power density is required for this beam (typically MW/cm 2 ). Instead, when RESOLFT is used in combination with RSFPs [ 67 , 68 ], the donut-shaped beam serves to switch off the label, a process which is not limited by the fl uorescence lifetime. Thus, albeit at the expense of a reduced time-resolution, this allows a huge decrease (up to six orders of magnitude) of the light intensity needed to break the diffraction barrier as compared to STED microscopy. This considerably reduces potential cytotoxic effects.

Increasing spatial resolution in RESOLFT implies achieving a higher number of switching cycles of the RSFP, while improving time resolution requires higher switching yields. The engineering of the well-known EGFP into rsEGFP [ 38 ] created an RSFP with much faster photoswitching rates than Dronpa and with a much

Nonlinear Microscopy Applications

enhanced photoresistance, comparable to that of its parent EGFP. Recent modifi cations of this variant resulted in an even faster RSFP, named rsEGFP2, in which, as compared to rsEGFP, the mutations Val150Ala and Ser205Asn have been back-mutated and the fi rst amino acid of the chromophore was modifi ed (Thr65Ala) [ 39 ]. RsEGFP2 allows the application of RESOLFT microscopy with no more light than in conventional confocal microscopy and at unprecedented speed.

Similarly, very weak illumination intensities can be used when RSFPs are employed in combination with Saturated Structured Illumination Microscopy (SSIM), a nonlinear version of SIM [START_REF] Gustafsson | Nonlinear structuredillumination microscopy: wide-fi eld fl uorescence imaging with theoretically unlimited resolution[END_REF]. In this wide-fi eld imaging technique, the sample is illuminated with a sine-shaped wavefront (structured illumination) that is modulated in phase and angle. From the interferences between the patterned illumination and the spatial response of the fl uorescent sample (Moiré fringes) a resolution increase by a factor of 2 can be achieved. To push the resolution further with standard fl uorophores, very intense illumination is required to saturate the singlet excited state S 1 , producing sharp fl uorescent fringes. The use of RSFPs such as Dronpa relies on saturating the long-lived off state instead of the short-lived S 1 , alleviating the need for high intensities [START_REF] Rego | Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution[END_REF].

Biphotochromic FPs combine the photophysical properties of both PCFPs and RSFPs into single PTFPs. IrisFP [ 35 ] was the fi rst member of this family: it can be irreversibly photoconverted from green to red and reversibly switched off in either the green or the red state. The monomeric variant mIrisFP was successfully used as a tool for two-color super-resolved pulse-chase imaging experiments [START_REF] Fuchs | A photoactivatable marker protein for pulse-chase imaging with superresolution[END_REF]. In this technique, a fraction of the molecules are fi rst photoconverted to their red-emitting state and let to diffuse within the cell. Using photochromic-based PALM, both the unphotoconverted (green) and photoconverted (red) molecules are then tracked at nanometric precision. Improved biphotochromic variants have been recently engineered [ 36 ]. In particular, NijiFP is a variant of the monomeric PCFP Dendra2 and demonstrated promising properties: low tendency to oligomerize, good spectral contrast between green and red states, and excellent photoswitching capability of the two forms.

PTFPs have been used to demonstrate the concept of biological data-storage, possibly in 3D [START_REF] Adam | Data storage based on photochromic and photoconvertible fl uorescent proteins[END_REF]. Notably, rsEGFP has been shown to provide a remarkable support for rewritable ultrahigh density data storage [ 38 ]. Biphotochromic FPs open the route to quaternary data storage applications. Photoconverting to the red state half of a biphotochromic FP substrate spread on a surface, it becomes possible to achieve four optical combinations by switching off or on the green and red molecules. A base-4 encoding system is obtained: dark + dark = 0, dark + green = 1, dark + red = 2, red + green = 3, equivalent in binary code to 00, 01, 10, and 11, respectively.

Advanced Microscopy and Biotechnology with Bi-photochromic FPs

The use of RSFPs in optogenetics was recently introduced as an exquisite tool to control specifi c cellular activities through reversible photoswitching [ 54 ]. Dronpa is a monomeric RSFP, but it was derived from a tetrameric parent. A single mutant of Dronpa (Lys145Asn), called PDM1-4 [START_REF] Mizuno | Higher resolution in localization microscopy by slower switching of a photochromic protein[END_REF], was shown to exhibit a propensity to tetramerize in the switched-on state, whereas it remains monomeric in the off state. The crystallographic structure of PDM1-4 revealed a rigidifi ed tetramer interface, consistent with this fi nding [START_REF] Nguyen Bich | Structural basis for the infl uence of a single mutation K145N on the oligomerization and photoswitching rate of Dronpa[END_REF]. A mixture of PDM1-4 and Dronpa was then shown to promote light-induced dimer-to-monomer conversion rather than tetramer-to-monomer conversion with PDM1-4 alone. The idea, then, consists in fusing a protein of interest (POI) at one end with PDM1-4 and at the other end with Dronpa. In the onstate, the two RSFP domains interact, "caging" the POI, while in their off state they split, restoring the POI activity. The method was applied to intersectin, a guanine nucleotide exchange factor that activates Cdc42. Upon 490-nm illumination, activation of Cdc42 by uncaged intersectin was monitored through the growth of fi lopodia, an obvious morphological change that was observed concomitantly with the disappearance of Dronpa fl uorescence due to switching. Using the same concept, it was possible to design a light inducible NS3-4A protease in the hepatitis C virus (HCV).

The interrelation between Dronpa photoswitching and β-barrel plasticity allowed yet another surprising application of this RSFP, this time as a sensor for solvent viscosity. Hydrodynamic changes exerted on Dronpa by an increasing viscosity were found to reduce the photoswitching rate of its chromophore [ 55 ]. Moreover, the authors demonstrated that fast-switching variants and more specifi cally Dronpa[Val157Ile/Met159Ala], named Dronpa-3 [ 29 ], are especially sensitive to viscosity changes: the photoswitching rate of Dronpa-3 slowed down by a factor of 4 when the glycerol concentration was increased from 0 to 90 %. This makes Dronpa-3 an effi cient genetically-encoded reporter for microviscosity in vivo as demonstrated in the case of chromatin: a slightly more viscous environment was repeatedly measured when cells were in mitosis.

The synthesis of photochromic organic fl uorescent dyes allowed, a decade ago, the development of a novel approach for quantitative Förster resonance energy transfer (FRET) [ 75 , 76 ]. In this photochromic FRET (pcFRET) approach, modulations of fl uorescence emission by a donor molecule can be induced by reversible switching of a photochromic acceptor, allowing quantitative and repeatable determination of the FRET effi ciency between two molecules without the need to apply corrections based on reference images.

The recent engineering of the red-shifted RSFPs rsCherryRev [START_REF] Stiel | Structural basis of X-ray-induced transient photobleaching in a photoactivatable green fluorescent protein[END_REF] and rsTagRFP [ 78 ] allowed the conception of similar pcFRET

Optogenetics

Viscosity Sensing

Photochromic FRET (pcFRET)

experiments using solely genetically encoded reporters. Coupled to EYFP as a donor, rsTagRFP revealed to be an excellent photochromic acceptor [ 78 ] and allowed a precise quantifi cation of protein-protein interactions within living cells.

Conclusion: Future Prospects for RSFPs

Reversibly switchable FPs are astonishing macromolecules that nowadays play a key role in state-of-the-art techniques based on light-induced protein manipulation.

In contrast to photoconvertible FPs that are found in nature, all RSFPs known so far are man-made, developed on the basis of FPs from both the Anthozoan and the Hydrozoan classes. Thus, there is probably little functional advantage to such switching capabilities in marine organisms. On the contrary, an incredibly wide palette of applications has already been found by researchers, in the fi elds of advanced fl uorescence microscopy and biotechnology.

The mechanism underlying switching in RSFPs is generally based on cis -trans photoinduced isomerization of the chromophore coupled with a protonation change. However, there are subtle differences between members, and despite signifi cant progress in the last years, reaction pathways and intermediate states possibly involved are still under debate.

An important aspect of future research in RSFPs photophysics will be to understand how blinking and bleaching, these phenomena that are so characteristic in all FPs, interfere with photoswitching. Although hints have been provided already [ 79 , 80 ], these stochastic events are diffi cult to capture and characterize. Yet, they are fundamental in the quest for future optimization of RSFPs, together with other properties, such as red-shifted fl uorescence or decoupled switching.

Our knowledge of fl uorescent proteins is constantly growing. However, experience has shown that the complex architecture of the β-barrel and its links to the chromophore is intricate to a degree that success in developing improved variants on the basis of rational design alone has been so far the exception rather than the rule. It will be interesting to see if multi-residue rational design of a PTFP will eventually be successful, knowing that directed evolution approaches are currently bound to miss a large fraction of possible synergistic combinations of mutations.

Whatever the engineering approaches, surprising new properties will continue to emerge and, based on these, the palette of new RSFPs-based applications will continue to grow, so it is worth to stay tuned for amazing new developments. and Dendra derivatives, this two-regime photobleaching mechanism could be a common phenomenon within these protein families. From our results we make a somewhat counterintuitive prediction that at constant integrated illumination dose, perhaps less cytotoxicity could be produced because under high-intensity illumination, less reactive oxygen species are likely to be generated and released. Based on our previous results on the IrisFP photobleaching mechanism, we rationally designed the IrisFP-M159A mutant in order to increase photoresistance against photofatigue under low-intensity illumination conditions. We obtained its native crystallographic structure and photophysically characterized IrisFP-M159A. We also investigated its photoswitching behavior and observed enhanced photoresistance in solution, in polyvinyl alcohol (PVA) gel, in fixed E.coli cells and in live E.coli cells compared to the parent protein IrisFP.

By characterizing the photophysical properties of the mutant, we found that compared to the parent, it has a lower pKa (4.7 vs. 5.7), as well as slightly blue-shifted excitation and emission maxima at 484 nm and 513 nm, respectively. However it has a lower fluorescence quantum yield and brightness.

As expected, our IrisFP-M159A mutant has a photoresistance enhancement in all investigated samples under low-intensity illumination (< 40 W/cm²). Photofatigue experiments comparing the performance of mutant and parent protein showed that the results vary a lot depending on the experimental environment. These results together with the absorption spectrum of the photofatigued sample confirm our previous hypothesis that the sulfoxidation of Met159 trapped the chromophore in a non-fluorescent state. Replacing the sulfur-containing residue by a sulfur-free residue avoids this trapping and improves the photoresistance of the protein (Fig16.).

Perspectives

After this thesis, we understand more about the photobleaching mechanism of RSFPs at the structural level. In the near future, we could use the same approaches to reveal the photobleaching mechanisms in Hydrozoan RSFPs and employ our findings to other proteins in order to generate new mutants with better photostability. However, there are still questions that remain unanswered that need to be investigated in the future. on this question by using QM/MM methods. This process could be simulated to see if this reaction pathway is valid or not. Likewise, the possible diffusion pathways of the triplet/singlet oxygen molecules inside and outside the FP molecule could be explored.

Crystallographic structures of IrisFP-M159A after low-and high-intensity illumination photobleaching

By using the same approaches as described in our first article, we tried to obtain the photobleached crystallographic structures of IrisFP-M159A. However, those attempts were not successful. The main reason is that the mutant is more photostable and requires much more photoswitching cycles to photobleach, which introduces damage to crystals by local heating of the illuminated area. To rescue the crystal's diffraction quality, compromise conditions will have to be found for photobleaching, such as slightly cooling the crystal in the presence of oxygen.

Mutating Met159 in monomeric RSFPs

Our studies demonstrated an enhancement of photoresistance by a single mutation at Met159. Therefore, such a mutation could be applied to other Anthozoan RSFPs. However, IrisFP, as most other "native" RSFPs, is tetrameric and hence not very suitable for serving as a biological marker. The mutation of Met159 thus should be applied to monomeric RSFPs such as NijiFP or mIrisFP. In this context, it is noteworthy that mutations of Met159 already do exist in Dronpa variants such as Dronpa2 (Met159Thr), Dronpa3 (Met159Ala) and bsDronpa (Met159Cys). However, their photoresistance was not systematically studied.

Further studies of photoresistance should be carried out. Substitution by other residues could be attempted at the Met159 position as well, to generate potentially more photostable mutants.

Mutant with enhanced photostability under high-intensity illumination

Although we have identified that residue Glu212 is involved in the high-intensity illumination photobleaching pathway within IrisFP, however, our E212Q mutant is not

Engineering of photostable mutants of Hydrozoan FPs

One possible photobleaching pathway in Hydrozoan FPs is that singlet oxygen diffuses to other residues more remote from the chromophore pocket and oxidizes them, which can result in photobleaching. Once the study of photobleaching mechanisms in Hydrozoan RSFPs progresses, key residues that are involved in photobleaching will be identified, and one could rationally design photostable mutants of Hydrozoan RSFPs like we did in Anthozoan RSFPs.

Long-lived dark state

In both articles, the photofatigue profiles display a clearly biphasic fluorescence amplitude decay suggesting that several photobleaching pathways occurs during photofatigue and that other photophysical process may be involved as well. One possible photophysical process is the establishment of a long-lived dark state with lifetime of several minutes. We speculate that, during the fast phase, a population of molecules enters in a long-lived dark state, creating a shelving effect and hence slowing down the photofatigue decay. Possibly, this long-lived dark state could be trapped in a protein crystal and visualized by X-ray crystallography.

Environmentally stable proteins

As shown in our second article (Duan et al., 2014), the photoswitching behavior and the photostability of IrisFP and the M159A mutant differ a lot in different environments.

Unlike the huge improvement that we found in solution, in some particular conditions, such as in PVA gel or live cells, the mutant showed less enhancement, possibly due to the complex external environment. To maintain the enhancement that we obtain within the isolated mutant, additional efforts are necessary.

The importance of the super-resolution techniques that can serve as very powerful

Moerner (image from internet)

In order to achieve a better spatial resolution of super-resolved fluorescence microscopy, the biological community demands new FPs particularly in the near-infrared range with better brightness and photoresistance. Our studies step forward to meet this demand.

  Martin Chalfie, Osamu Shimomura and Roger Y. Tsien were honored the 2008 Nobel Prize in Chemistry for their discovery and development of the green fluorescent protein (Fig4.).

Fig4.

  Fig4. The Nobel Prize in Chemistry 2008: Osamu Shimomura, Martin Chalfie, Roger Y Tsien (image from internet)

Fig7.

  Fig7. Photoswitching of Dronpa.

Fig8.

  Fig8. Photoswitching of Padron.

  Denra2-M159A, Dendra2-F173S (Adam et al., 2011), mIrisFP (Adam et al., 2011), pcDronpa2 (Moeyaert et al., 2014) and NijiFP (Adam et al., 2011).

Fig11.

  Fig11. Absorption and emission spectra of IrisFP displayed by solide line and dashed lines, respectively. (A) Irradiation by 488 nm light, decreases absorption at 488 nm as well as fluorescence at 516 nm and increases absorption at 390 nm. (B) Irradiation by 532 nm light, decreases absorption at 551 nm and fluorescence at 580 nm and increases absorption at 440 nm. Image from(Adam et al., 2008)

Figure 1 .

 1 Figure 1. Photofatigue fluorescence decay of crystalline IrisFP under high-intensity illumination by 488-nm (∼100 W/cm 2 , continuous) and 405-nm (∼1 W/cm 2 , on for 0.3 s every 2.3 s) laser light. Fluorescence was recorded at 512 nm. The crystal was submitted to 260 switching cycles during 10 min of illumination, which resulted in the loss of ∼75% of its initial fluorescence emission. The inset shows an enlarged view of the decay over the first 10 cycles. The fluorescence envelope can be fitted with a biexponential decay model (red trace).

Figure 2 .

 2 Figure 2. (A) Absorption spectra of crystalline IrisFP before (green) and after (blue) high-intensity photobleaching. Both anionic and neutral bands are largely decreased. (B) Raman spectra of crystalline IrisFP before (green) and after (blue) high-intensity photobleaching. The complete disappearance of the 1503, 1545, 1604, and 1564 cm -1 bands (inset, arrows) suggests the breakage of the chromophore methylene bridge πconjugation (C) NCS-averaged experimental difference electron density map F obs,bleached -F obs,native upon high-intensity photobleaching, overlaid on the crystallographic structure of intact IrisFP (PDB model 2VVH). The chromophore (orange) and the important surrounding residues (gray) in the chromophore pocket are shown. Positive electron density is shown in green (+5 σ) and negative electron density is shown in red (-5 σ). (D) Refined model of high-intensity photobleached IrisFP. The chromophore phenolate moiety is represented in dim color to highlight the disorder observed in the electron difference density map. H-bonds are represented with dashed lines, and water molecules as red balls.

Figure 3 .

 3 Figure 3. Photofatigue fluorescence decay of crystalline IrisFP under low-intensity illumination by 488-nm (∼10 W/cm 2 , continuous) and 405-nm (∼0.01 W/cm 2 , on for 4 s every 12 s) laser light. Fluorescence was recorded at 512 nm. The crystal was submitted to 460 switching cycles during 100 min of illumination, which resulted in the loss of ∼90% of its initial fluorescence emission. The inset shows an enlarged view of the decay over the first 10 cycles. The fluorescence envelope can be fitted with a biexponential decay model (red trace).

Figure 4 .

 4 Figure 4. (A) Absorption spectra of crystalline IrisFP before (green) and after (red) low-intensity photobleaching. The intensity of the anionic band at 488 nm decreased while that of the neutral band at 390 nm increased. (B) Raman spectra of crystalline IrisFP before (green) and after (red) partial low-intensity photobleaching. The decrease of the 1503 cm -1 and 1545 cm -1 bands and the increase of the 1604 cm -1 and 1564 cm -1 bands (inset, arrows) is consistent with a conversion from the anionic to a neutral state of the chromophore without the loss of π-conjugation (C) NCSaveraged electron difference density map F obs -F calc upon low-intensity photobleaching, overlaid on the crystallographic structure of intact IrisFP (PDB model 2VVH), as in Figure 2C. Positive electron density is shown in green (+5.3 σ) and negative electron density is shown in red (-5.3 σ, no feature visible). (D) Refined model of low-intensity photobleaching IrisFP. H-bonds are represented with dashed lines, and water molecules as red balls.

Figure 5 .

 5 Figure 5. (A) Lewis representation of the proposed doubly reduced/protonated chromophore CαN photobleached under high-intensity illumination conditions. This structure was used to model the bleached pBl red and Bl red states. (B) MD simulations: time-evolution of characteristic distances between atoms involved in the H-bond network around the IrisFP chromophore pocket, from the pBl red to the Bl red states. Blue: His194(NE2)-Glu144. Red: His194(ND1)-Arg66. Green: His194(NE2)-Ser142.

Figure 6 .

 6 Figure 6. Proposed photophysical scheme for a two-regime photobleaching in IrisFP. Yellow arrows represent chemical steps involving electron/ proton transfer or oxidation reactions. Blue arrows represent entry/exit of oxygen species.

S7(

  PNNL, Richland, WA). The intensities of the Mascot identified peptides corresponding to reduced and oxidized Met, Cys or Trp were compared in the two conditions. Averages and standard deviations of the percentages of oxidized forms were calculated from the triplicate experiments. (Figure S9). The Decon-2LS and Multi-Align softwares used for MS data processing were provided by the W. R. Wiley Environmental Molecular Science Laboratory, a national scientific user facility sponsored by the US Department of Energy's Office of Biological and Environmental Research, located at PNNL. (OMICS.PNL.GOV).

CαOII structure :

 structure No switch happens in the first two runs. Average values of τ and φ are 146° and -60°, values located in the large basin of the potential energy surface, close to the minimum energy point. Inspection of the trajectories lead to the same analysis as above: the movement of Arg66 is prevented by the phenolate. In the third run the switch is observed at the very beginning of the run, when the phenolate ring does not yet block the Arg66 displacement. An interesting feature of the CαOI and CαOII MD simulations is the significant weakening of the usually strong hydrogen bond between the imidazolinone oxygen O with Arg91, clearly due to the addition of the hydrogen atom on O.

Figure S2 .

 S2 Figure S2. Absorption spectra of IrisFP in the solution state, normalized at 280 nm: Prior to illumination (green); Under aerobic illumination: after 570 switching cycles at low-intensity (red), after 184 cycles at high-intensity in PVA gel (blue); Under anaerobic illumination: after 744 switching cycles at low-intensity (dark gray).

Figure S3 .

 S3 Figure S3. Photobleaching of IrisFP in the absence of oxygen mainly proceeds via a two-subsequent-photon absorption process. The fast component of a bi-exponential fit of the photo-fatigue envelope kinetics was used as the photo-fatigue rate. In this doubly logarithmic presentation, a linear fit (red line) of this rate as a function of the incident power density of the 488 nm beam gives a slope of 1.8 ± 0.3. The inset shows the same data with a linear presentation. The error bars represent the confidence interval of the fitted time-constant to the photofatigue curves. Actual errors are expected to be larger due to additional experimental imperfections.

Figure S4 .

 S4 Figure S4. Visible-light-induced photobleaching at cryogenic temperature (100 K) shows a clear decarboxylation of Glu212 both in cis and trans conformations. (A) Photobleaching in trans conformation by 405-nm laser. NCS-averaged F obs -F calc difference electron densities are displayed in green (+6.5 σ) and red (-6.5 σ), respectively. (B) Photobleaching in cis conformation by 488-nm laser. NCS-averaged F obs -F calc difference electron densities are displayed in green (+4.5 σ) and red (-4.5 σ), respectively.

Figure S5 .

 S5 Figure S5. (A) Overall view of structural changes upon high-intensity-photobleaching of IrisFP. The structural modifications depicted by NCS-averaged electron density difference maps (± 6 σ) are all located around the chromophore. (B) Overall view of structural changes upon low-intensity-photobleaching of IrisFP. The major structural modifications depicted by NCS-averaged electron density difference maps (± 5.3 σ) are all located around the chromophore, near Met159 and Cys171. The negative densities in the back and in the front of the chromophore are due to disordered sulfate molecules.

Figure S6 .

 S6 Figure S6. (A-D) Structural changes in monomers A to D induced by low-intensity photobleaching. 2F obs -F calc (1 σ) and F obs -F calc (± 3.0 σ) electron density maps are shown as blue and green/red meshes respectively. No negative density is observed. (E-H) Surface view of monomers A to D of the IrisFP β-barrel. No access to the chromophore through the surface is visible in monomer C, which might explain the absence of significant sulfoxidation detected on Met159 in that monomer.

Figure S7 .

 S7 Figure S7. Structural modifications resulting from X-Ray-induced photobleaching. A NCS-averaged experimental electron difference density map is displayed. Positive density is shown in green (+6 σ) and negative density is shown in red (-6 σ), overlaid on the structure of the intact IrisFP chromophore pocket (PDB code 2VVH).

Figure S8 .

 S8 Figure S8. pH titration of the neutral absorption band of IrisFP solution samples, following low-intensity photobleaching. A Henderson-Hasselbalch fit (red curve) yields a pKa of 11.9 ± 0.2 for the oxidation-photobleached chromophore. Note that progressive protein denaturation makes the measurement of absorption values at pH >11 increasingly unreliable.

Figure S9 .

 S9 Figure S9. Mass spectrometry analysis of oxidation in non-illuminated IrisFP (green bar) and low-intensity-illuminated (red bar) samples. Only peptides identified in both non-oxidized and oxidized forms by the Mascot software are shown. Peptides displaying oxidation levels below 1% in both samples are not represented. Peptides containing Tyr78, Tyr211 and W86 fall in this category, although the data show a detectable increase in the oxidation level of these residues upon low-intensityillumination. Due to sample pre-treatment (heating and digestion) before injection into the mass spectrometer, some background oxidation of sensitive residues is generally observed on the intact, non-bleached IrisFP, as is commonly the case.19 The high scatter of oxidation state of the very sensitive Met159 from the reference sample is explained by the time dependent oxidation of the sample during the analytical process. The only way to evaluate Cys171 oxidation was through a low abundance miss-cleaved peptide. The non-oxidized form was not observed in any condition, but various oxidation states appeared in the illuminated sample. It was therefore impossible to derive a ratio for the corresponding peptide. For Cys195, the evaluation of the oxidation corresponded to the ratio of two oxidation states. Overall, these data clearly suggest that, upon low-intensity illumination, photo-oxidation occurs not only on residues close to the chromophore but also on more distant ones.

Figure S10 .

 S10 Figure S10. Lewis representation of the different chemical structures considered in this work. CαN, CαOI, CαOII describe reduced forms of the chromophore and differ by the two hydrogenated sites.

Figure S11 .

 S11 Figure S11. Potential energy surfaces (PES) for the different reduced forms of the chromophore. (A) CαN, (B) CαOI, (C) CαOII. Energies in kcal/mol.

Figure S12 .

 S12 Figure S12. A: Picture of the photobleached IrisFP crystal used for X-ray data collection. Due to the larger 405 nm laser beamsize as compared to the 488 nm laser a volume peripheral to the photofatigued region underwent green-to-red photoconversion. B: F calc,red -F calc,green map (± 9 σ) upon photoconversion in IrisFP: the difference density features are localized on the Phe61-His62 linkage.

Figure S13 .

 S13 Figure S13. Snapshots extracted from molecular dynamics simulations, showing the 3D organization of the chromophore and Arg66. (A) During CαOI simulation: τ=150° and ϕ=-80°. (B) During CαN simulation: τ=28° and ϕ=-85°. The arrow indicates the type of movement that the Arg66 side chain would undergo to reach its position in the high-intensity-photobleached structure. The phenolate hinders this movement in (A) but not in (B).

Figure S14 .

 S14 Figure S14. Characteristic distances of the H-bond network between Arg66, Glu144, His194 and Ser142, as a function of time in the four CαN simulation runs. Blue: His194(NE2)-Glu144, Red: His194(ND1)-Arg66, Green: His194(NE2)-Ser142.

Figure S15 .

 S15 Figure S15. Putative kinetic model representative of a two-regime photobleaching in IrisFP. Description of the rate constants is provided in the SI text. Yellow arrows represent chemical steps involving e.g. electron/proton transfer or sulfoxidation reactions. Blue arrows represent entry/exit of oxygen species.

Figure S16 .

 S16 Figure S16. Power-dependence simulation of the different bleaching pathways. The fractions of photobleached protein by the respective pathways Bl red (blue squares)and Bl oxy (red squares) are calculated as a function of excitation power density upon illumination for a time chosen so that 15% of molecules are bleached. Rate constants described in FigureS15were chosen as: k F =0.33×10 9 s -1 , k ex =5-50×10 3 s -1 , k T1,ex =0.15×k ex , k IS =0.3×10 6 s -1 , k 1 =4.0×10 3 s -1 , k 2 =3.9×10 6 s -1 , k 3 =1.2×10 5 s -1 , k T1,1 =5.0×10 3 s -1 , k T1,2 =1.2×10 6 Mol -1 s -1 , k 4 =3.0×10 4 s -1 , k 5 =1.0×10 4 s -1 , k 6 =1.0×10 1 s -

  PhotoTransformable Fluorescent Proteins (PTFPs) constitute an important subset of the FP family, displaying specific photophysical properties such as photoactivation, photoconversion and photoswitching. Because their fluorescence states are controllable by light, PTFPs have largely contributed to revolutionize the field of fluorescence imaging in recent years[1,2,3]. Amongst PTFPs,

  Photobleaching in fluorescent proteins is caused by the irreversible photodestruction of the chromophore or alteration of neighboring residues. However, the underlying photochemical mechanisms remain incompletely understood. A recent study of IrisFP[17], a tetrameric RSFP of Anthozoan origin, showed that two completely different photobleaching mechanisms can happen depending only on the intensity of the excitation light[18]. IrisFP is a single mutant of EosFP (EosFP-F173S), is both switchable and photoconvertible, and presents high sequence identity and structural similarity to well-known PTFPs such as mEos2, Dendra2 or Dronpa. Under relatively high-intensity illumination conditions (> 0.1 kW/cm², typical of single molecule localization based nanoscopy), photobleaching of IrisFP is caused by decarboxylation of the strictly conserved Glu212 (IrisFP numbering) coupled to an sp 2 -to-sp 3 hybridization change of the central carbon atom of the chromophore methylene bridge and an extensive rearrangement of the H-bond network surrounding the chromophore. In contrast, under low-intensity illumination conditions (~0.01 kW/cm², typical of standard widefield fluorescence imaging), Met159, a highly conserved residue in anthozoa PTFPs located next to the chromophore hydroxybenzylidene moiety, undergoes a sulfoxidation reaction that results in trapping the chromophore in a permanent nonfluorescent protonated state. Sulfoxidation of Met159 follows from the production of singlet oxygen within the chromophore pocket due to triplet-

(

  CrystaLaser, Reno, USA) gaussian-shaped laser beams with FWHM of ~20 µm at the focal plane. For low-intensity photofatigue experiments, continuous illumination at 488 nm (40 W/cm²) and alternating illumination at 405 nm (2.3 W/cm²) were performed with 15-ms frames recorded every 75 ms. Each cycle was 3-s long (off-to-on: 0.3 s, on-to-off 2.7 s). Each experiment consisted of 1000 switching cycles. For high-intensity photofatigue experiments, continuous illumination at 488 nm (400 W/cm²) and alternating illumination at 405 nm (10.8 W/cm²) were performed with 5-ms frames recorded every 25 ms. Each cycle was 250 ms long (off-to-on: 25 ms, on-to-off: 225 ms). Each experiment consisted of 600 switching cycles. Proper synchronization of laser timing and frame acquisition by the EMCCD camera (Evolve 512, Roper, USA) was realized with Labview.

Figure 1 .

 1 Figure 1. The mutation M159A in IrisFP only moderately affects its spectra but acidifies its chomophore. (A) Absorption (solid), excitation (dashes) and emission (bold) spectra of IrisFP (red) and IrisFP-M159A (blue) at pH 7.3. Absorption and excitation maxima peak at 488 nm for IrisFP and 484 nm for IrisFP-M159A. Emission spectra show a maximum at 516 and 513 nm for IrisFP and IrisFP-M159, respectively. (B) Titration curves of IrisFP (red) and IrisFP-M159A (blue), demonstrating a clear acidification of the mutant's chromophore. Measurement points (squares) are fitted by the Henderson-Hasselbalch equation (lines), which yields pKa values of 5.7 for IrisFP and 4.7 for IrisFP-M159A.

  methionine, a water molecule (Wat2) is found to fill up the cavity created by mutagenesis. Together with Wat2, two water molecules already present in the IrisFP structure (Wat1 and Wat3) are slightly moved to accommodate a new H-bond chain linking the phenolate moiety of the chromophore to Ser173. Ser173 is slightly shifted towards Ala159.

Figure 2 .

 2 Figure 2. Crystallographic structure of IrisFP-M159A (light blue) overlaid with that of IrisFP (gray). The chromophore and neighboring residues are shown in stick mode, water molecules are represented as balls, and dashed lines represent the newly formed H-bonds chain between the phenolate moiety of the chromophore and Ser173.

Figure 3 .

 3 Figure 3. Photofatigue of IrisFP and IrisFP-M159A in solution. A single switching cycle is shown (black circles) for IrisFP (A) and for IrisFP-M159A (B) along with their fits (red curves). A series of such switching cycles is recorded by integrating the fluorescence signal during the exposure of IrisFP (C) and IrisFP-M159A (D) samples to alternating 488-nm (0.04 W/cm²) / 405-nm (0.03 W/cm²) laser illuminations. At such low intensities in solution IrisFP-M159A demonstrates a >40-fold improvement in the achievable number of switching cycles as compared to its parent. Insets show absorption spectra before (dashed lines) and after (solid lines) photofatigue of the samples. IrisFP presents an absorption band at ~390 nm due to chromophore protonation consecutive to sulfoxidation of Met-159, while this feature is absent in the spectrum of IrisFP-M159A.

  sufficient time-resolution to follow individual switching cycles with sufficient accuracy, so that only the envelope of the fluorescence decay along the photofatigue profiles are reported. At low illumination intensity, individual switching cycles could be monitored and the available time resolution, depending on the investigated conditions (see below), was generally sufficient to achieve a coarse extraction of the on-and off-switching brightness.

Figure 4 .

 4 Figure 4. Photofatigue of IrisFP (black) and IrisFP-M159A (red) embedded in a PVA gel (A-C), in fixed bacteria (D-F) and in live bacteria (G-I) at low (~40 W/cm², left and central column) and high (~400 W/cm², right column) intensity 488 nm illumination. The insets show individual switching cycles. Green lines: model fits from which the switching yields reported in Table4were extracted.

Fig. 1

 1 Fig. 1 Enhanced green fl uorescent protein (EGFP). ( a ) The crystal structure (Protein Data Bank (PDB) ID 2Y0G) of a single chain is represented in gray cartoons . The green-glowing chromophore is represented in balls and sticks and stands in the center of the barrel. ( b ) Absorption spectrum of EGFP. Two bands are attributed to the neutral form (A-band) and the anionic form (B-band) of the chromophore. Lewis structures of the corresponding chromophore are represented as insets

Fig. 2

 2 Fig. 2 Various possible reactions in phototransformable fl uorescent proteins with typical examples of irreversibly photoactivatable fl uorescent proteins (PAFP), irreversibly photoconvertible fl uorescent proteins (PCFP), and reversibly switchable fl uorescent proteins (RSFP). Photoinduced reactions occurring at the chromophore level are represented for each case. Figure reproduced from D. Bourgeois, A. Regis-Faro and V. Adam (2012), Biochemical Society Transactions, 40 531-538, © the Biochemical Society, with permission from Portland Press Limited

  Abbreviations:λ max ex/em wavelength of maximum in excitation/emission spectrum; ε , molar extinction coeffi cient; Φ fl uo , fl uorescence quantum yield; Φ sw , photoswitching quantum yield; D, decoupled switching

  photobleaching mechanisms in IrisFP (Fig15.). Depending on the intensity of the excitation light, IrisFP can be photobleached by two different mechanisms. Under relatively intense excitation (~100W/cm² in this study, approaching that of PALM microscopy), photobleaching occurs via an oxygen-independent pathway. The photobleached structure shows that the chromophore and its surrounding residues are dramatically altered. The loss of chromophore fluorescence resulted from an sp 2 -to-sp 3 hybridization change of the Cα atom of the chromophore methylene bridge. Together with this hybridization change, decarboxylation of the highly conserved Glu212 (IrisFP numbering) occurs, followed by an extensive rearrangement of the H-bond network surrounding the chromophore. Under low excitation illumination (~10W/cm² in this study, typical of standard widefield fluorescence imaging), an oxygen-dependent photobleaching pathway dominates. In contrast to high-intensity illumination, there are very few structural modifications. The major modification is that next to the chromophore, Met159 -a highly conserved residue in Anthozoan PTFPs-becomes sulfoxided. The triplet excited state of the chromophore can interact with dissolved molecular oxygen within the chromophore pocket, forming singlet oxygen. This singlet oxygen in turn may diffuse to the nearby Met159 that undergoes a sulfoxidation reaction. The sulfoxided Met159 creates a new strong H-bond to the chromophore hydroxybenzylidene moiety and traps the chromophore in a permanent protonated and hence non-fluorescent state. Due to the very high sequence and structure similarity to other Anthozoan PTFPs such as Dronpa, EosFP

Fig15.

  Fig15. Proposed photophysical scheme for a two-regime photobleaching in IrisFP. Yellow arrows represent chemical steps involving electron/proton transfer or oxidation reactions. Blue arrows represent entry/exit of oxygen species.

Fig16.

  Fig16. Fluorescence decay during photofatigue of IrisFP (A) and IrisFP-M159A (B) in solution. Absorbance of IriFP (C) and IrisFP-M159A (D) before and after photofatigue.

1 .

 1 Computer simulation of the sulfoxidation reaction of Met159 in IrisFP under low-intensity illumination conditions and the diffusion of molecular oxygen.In our first article(Duan et al., 2013), we proposed a hypothesis of a possible sulfoxidation reaction pathway of Met159 and Cys171. Highly reactive singlet oxygen molecules are produced via a triplet-triplet energy transfer between chromophore and molecular oxygen, and then the singlet oxygen diffuses to nearby Met159, finally the sulfurcontaining Met159 gets sulfoxided(Pattison et al., 2012). As the product of the reaction, a hydrogen peroxide (H 2 O 2 ) is produced. This H 2 O 2 can oxidize the nearby Cys171[START_REF] Luo | Kinetics and mechanism of the reaction of cysteine and hydrogen peroxide in aqueous solution[END_REF]. Our collaborators Mikolaj FELIKS and Martin FIELD (Dynamo lab, IBS) are working

5 .

 5 suitable for biological applications. Because of the essential role of the Glu212 residue during the chromophore maturation process, the substitution of Glu by Gln can largely slow down the maturation of the chromophore. However, in the second paper(Duan et al., 2014) we still demonstrated enhanced photostability of IrisFP-M159A in bacterial cells under such illumination condition, which suggests that Met159 has unknown indirect effect to the high-3 Conclusion and Perspectives 123 intensity illumination photobleaching. Probably other neighboring residues are involved as well. Study of Hydrozoan RSFPs photobleaching mechanisms Although most RSFPs are originally from Anthozoan species, based on EGFP and EYFP, Jakobs's lab developed some Hydrozoan RSFPs with high switching performance such as rsEGFP, rsEGFP2 and Dreiklang, which were already successfully applied in RESOLFT nanoscopy. Despite high structure similarity between Anthozoan and Hydrozoan FPs, they share limited sequence identity. In Hydrozoan RSFPs, there is no sulfur-containing residue in the chromophore pocket. Thus the photobleaching caused by sulfoxidation of Met159 in low-intensity illumination pathway in Anthozoan RSFPs doesn't exist in Hydrozoan RSFPs. Furthermore, the decarboxylation of Glu212 (IrisFP numbering) at the origin of photobleaching in Anthozoan RSFPs probably does not to occur in Hydrozoan RSFPs either. PA-GFP is the best example displaying that the decarboxylation of Glu222 (GFP numbering) activates the chromophore and turns on the fluorescence. The Glu222 initially favors the protonation of the chromophore that hardly absorbs 488 nm light. Upon UV illumination, decarboxylation of this residue favors the deprotonation of the chromophore that emits fluorescence under excitation by 488 nm light. (Henderson et al., 2009).

  tools to investigate subtle cellular architectures or real life processes on the nanometer-scale was recently recognized. The Nobel Prize in Chemistry 2014 was awarded jointly to Eric Betzig, Stefan W. Hell and William E. Moerner for the development of super-resolved fluorescence microscopy (Fig.17

  ).

Fig17.

  Fig17. The Nobel Prize in Chemistry 2014: Eric Betzig, Stefan W. Hell and William E.

Table S1 .

 S1 Crystallographic data collection and refinement statistics.

		High-intensity	X-ray-induced	Low-intensity
		photobleaching	photobleaching	photobleaching
	PDB entry	4LJB	4LJC	4LJD
	Data collection
	Beamline	ID14-2	ID29	PXII3
	Wavelength, Å	0.933	0.997	0.999
	Space group	P 2 1 2 1 2 1	P 2 1 2 1 2 1	P 2 1 2 1 2 1
	Cell dimensions
	a, Å	86.09	86.64	85.99
	b, Å	96.41	96.81	96.53
	c, Å	140.03	140.69	140.19
	Resolution, Å	47.32-1.9 (2.0-1.9)	48.41-1.86 (1.96-1.86)	47.34-2.5 (2.64-2.5)
	*R sym , %	6.8 (42.2)	6.2 (42.5)	7.5 (49.4)
	Mean I/σ(I)	11.4 (3.2)	10.4 (2.5)	11.8 (2.7)
	Completeness, %	99.2 (99.2)	99.3 (98.6)	97.8 (97.5)
	Redundancy	4.2 (4.1)	3.3 (3.2)	3.4 (3.5)
	No. of unique reflections	91193 (13214)	99096 (14169)	40095 (5752)
	Wilson B factor, Å 2	34.9	24.8	55.6
	Refinement
	# R work /R free	0.34/0.38	0.20/0.24	0.20/0.25
	Average B factor, (Ų)	27.9	27.0	29.0
	Rmsd
	Bond length, (Å)	0.009	0.012	0.006
	Bond angles, (°)	1.52	1.63	0.93
	Ramachandran statistics (%)
	Favored	99.4	100	99.1
	Allowed	0.6	0	0.9
	Outliers	0	0	0

*R sym = ΣjΣh|I h,j -〈Ih〉|/ΣjΣh I h,j # R work = Σh|F obs -F cal |/ΣhF obs, R free is calculated with a small fraction (5 %) of reflections chosen to be part of a test group. Values in parentheses refer to the highest resolution shell.

Table S2 .

 S2 Mass spectrometry analysis of IrisFP decarboxylation.

		Decarboxylation
		(E212)
	#Native IrisFP	0%
	#Low energy solution	3.4%
	*Low energy solution (anaerobic)	18.8%
	¤High energy in crystallo	26%
	#,*,¤ : Different digestion times used. (Due to the amount of sample necessary for mass-spectrometry
	analysis, high-energy experiments in the solution state (requiring highly focused laser beams) could
	not be successfully conducted.)

Table 1 .

 1 Photophysical properties of IrisFP and IrisFP-M159A.

Table 2 .

 2 Crystallographic data collection and refinement statistics.

	PDB entry

*R sym = ΣjΣh|I h,j -〈Ih〉|/ΣjΣh I h,j # R work = Σh|F obs -F cal |/ΣhF obs, R free is calculated with a small fraction (5 %) of reflections chosen to be part of a test group. Values in parentheses refer to the highest resolution shell.

Table 3 .

 3 Photoswitching behavior in solution. Calculated over the first halftime of the photofatigue decay b Calculated from the ratio of the integrated photofatigue decays × extinction coefficients at anionic peaks taken from Table1. c Values between parentheses are normalized to IrisFP.

a Photofatigue experiments were then performed on IrisFP and IrisFP-M159A, for about 8000 cycles with moderate time resolution (20 points/cycle, 0.04 W/cm² at 488 nm). The photofatigue kinetics are shown in Figure

Table 4 .

 4 Conditions and parameters for IrisFP and M159A photofatigue experiments in vitro and in cellulo.

  intensity exceeds that of IrisFP by a factor of ~6.1. As in PVA, individual switching cycles were best fitted with a kinetic model assuming fixed dipole orientations of the fluorophores. This is consistent with the hypothesis that cross-linking by paraformaldehyde in fixed cells may prevent tumbling of macromolecules.

	under low illumination
				Off-to-on	On-to-off		Photo-	Photo-
	Matrix	Power density a	Protein	switching brightness b	switching brightness b	Switching contrast c	fatigue half decay	resistance enhance
				[cm -1 .M -1 ]	[cm -1 .M -1 ]		[cycles]	ment d
	PVA	Low	IrisFP IrisFP-M159A 820 (0.20) e 440 (0.11) e	≈ 52 (0.20) e 18 ≈ 25 (0.10) e 10	25 45	1.9
	gel	High	IrisFP IrisFP-M159A ND ND	ND ND	ND ND	31 17	ND
	Fixed	Low	IrisFP IrisFP-M159A 880 (0.21) e 1900 (0.46) e ≈ 36 (0.14) e 16 ≈ 56 (0.21) e 22	33 248	6.1
	cells	High	IrisFP IrisFP-M159A ND ND	ND ND	ND ND	35 115	ND
	Live	Low	IrisFP f IrisFP-M159A f ND ≥ 1400 (0.34) e ≥ 52 (0.20) e 14 ND 8	178 315	2.0
	cells	High	IrisFP IrisFP-M159A ND ND	ND ND	ND ND	54 > 600	ND

Table 1 Properties of well-characterized RSFPs developed to date Class

 1

		λ max ex/	em a
	Chromo	phore	triad
			Source organism
		Parent protein	[wild-type]
		Protein	(oligomerization)
		Switch-	ing

  reversibly switchable red fl uorescent proteins for far-fi eld fl uorescence nanoscopy. Biophys J 95(6):2989-2997. doi: doi:biophysj.108.130146 [pii] 10.1529/biophysj.108.130146 78. Subach OM, Malashkevich VN, Zencheck WD, Morozova KS, Piatkevich KD, Almo SC, Verkhusha VV (2010) Structural characterization of acylimine-containing blue and red chromophores in mTagBFP and TagRFP fl uorescent proteins. Chem Biol 17(4):333-341. doi: S1074-5521(10)00086-4 [pii] 10.1016/j. chembiol.2010.03.005 79. Adam V, Carpentier P, Violot S, Lelimousin M, Darnault C, Nienhaus GU, Bourgeois D (2009) Structural basis of X-ray-induced transient photobleaching in a photoactivatable green fl uorescent protein. J Am Chem Soc 131(50):18063-18065. doi: 10.1021/ja907296v 80. Roy A, Field MJ, Adam V, Bourgeois D (2011) The nature of transient dark states in a photoactivatable fl uorescent protein. J Am Chem Soc 133(46):18586-18589. doi: 10.1021/ ja2085355 81. Lukyanov KA, Fradkov AF, Gurskaya NG, Matz MV, Labas YA, Savitsky AP, Markelov ML, Zaraisky AG, Zhao X, Fang Y, Tan W, Lukyanov SA (2000) Natural animal coloration can be determined by a nonfl uorescent green fl uorescent protein homolog. J Biol Chem 275(34):25879-25882 82. Abbruzzetti S, Grandi E, Viappiani C, Bologna S, Campanini B, Raboni S, Bettati S, Mozzarelli A (2005) Kinetics of acid-induced spectral changes in the GFPmut2 chromophore. J Am Chem Soc 127(2):626-635. doi: 10.1021/ ja045400r

Chenxi Duan et al.

Photoswitching in Fluorescent Proteins

Numbers in parenthesis are the standard deviations in the MD simulations. The different switching times and dihedral angles values report results obtained for the different MD simulation runs. For crystallographic structures the interval covered by the values taken in chains A, B, C and D is given.

Abstract of article 2

Based on our previous results on the IrisFP photobleaching mechanism, we have identified that Glu212 is importantly involved in the high-intensity illumination photobleaching pathway and Met159 is importantly involved in the low-intensity illumination photobleaching pathway. Therefore, thanks to Xavier HENRY (Pneumo lab, IBS), we obtained IrisFP-E212Q and IrisFP-M159A mutants with the intent to improve the resistance against photofatigue under high-or low-intensity illumination, respectively. It is well-known that Glu212 has a very important role during the chromophore maturation. During the dehydration step, the carboxyl group of this Glutamate serves as a proton donor to the hydroxyl leaving group. Replacing Glu212 by Gln hindered chromophore maturation, leaving us too little protein to perform complicated experiment. This phenomenon was already observed before in Hydrozoan EGFP [START_REF] Sniegowski | Base catalysis of chromophore formation in Arg96 and Glu222 variants of green fluorescent protein[END_REF]. Therefore we turned our focus to the IrisFP-M159A mutant and investigated it. We transformed plasmidic DNA into E.coli cells for expression and then we were able to purify the IrisFP-M159A protein. The native crystallographic structure was resolved; photophysical characterization and comparative photofatigue experiments to its parent protein in vitro and in vivo were performed.

As expected, the IrisFP-M159A mutant displays an enhanced photoresistance under low-intensity illumination under various conditions: in solution, in PVA gel, in fixed and live E.coli cells. This enhanced photoresistance by the single mutation of Met159 confirms our finding in the previous paper that under low-intensity illumination, the sulfoxidation of nearby sulfur-containing Met159 is the predominant reason of photobleaching.

The same experiments were also carried out in PVA gel and in E.coli cells under highintensity illumination. We also observed a photoresistance enhancement in fixed and live cells, which suggests that Met159 plays an unknown role in high-intensity illumination photobleaching. From this study, we cannot completely understand the role of Met159 under high-intensity illumination conditions. Further investigation should be carried out.

Supplementary information

Rational Design of Enhanced Photoresistance in a Photoswitchable Fluorescent Protein

Chenxi Duan

Description of the fitting model

A simple kinetic model involving one-to-one reversible conversion between the on and off states was used to extract the on-and off-switching quantum yields:

Where k 1 and k -1 are the excitation rate constants during the switching-off and switching-on processes, respectively, and Ф OFF and Ф ON are the quantum yields for off-switching and onswitching. A proper evaluation of the dipole-orientation-dependent molecular excitation rates is necessary.

Case of tumbling molecules:

The excitation rate [s -1 ] of a rapidly tumbling molecule is given by:

where ε [M -1 cm -1 ] is the extinction coefficient of the considered species (ON or OFF molecules) at the excitation wavelength λ [nm], P [Wcm -2 ] is the laser power-density ON OFF

Abstract of article 3

This publication is a book chapter published in "Methods in Molecular Biology" (Duan et al., 2014a)

Résumé Etude structurale des mécanismes de photoblanchiment des protéines fluorescentes photocommutables

La découverte des Protéines Fluorescentes Phototransformables (PTFPs) issues d'espèces anthozoaires a ouvert, grâce à leurs propriétés photophysiques particulières, un vaste champ d'investigation pour l'imagerie biologique de fluorescence. L'un des sousgroupes des PTFPs est formé des protéines fluorescentes réversiblement photocommutables (RSFPs), qui peuvent être commutées réversiblement entre des états non-fluorescent et fluorescent. Le photoblanchiment est la perte définitive d'émission de fluorescence sous excitation et est un phénomène commun à toutes les molécules fluorescentes. Le photoblanchiment a un impact important sur la qualité des images de microscopie, notamment en imagerie de super-résolution. Les RSFPs ont tendance à perdre de leur performance à chaque cycle de commutation, un processus dénommé "photofatigue". Notre intérêt est centré sur l'étude des mécanismes de photofatigue des RSFPs.

Nous avons rapporté les structures cristallographiques d'IrisFP photoblanchie par une forte et une basse intensité d'illumination à température ambiante ainsi que les modifications spectroscopiques associées. Nos résultats démontrent que différentes intensités d'excitation peuvent donner lieu à différentes voies de photoblanchiment. Sous faible intensité d'excitation, une voie de photoblanchiment dépendante de l'oxygène a été mise en évidence. Les modifications structurales induites par la production d'oxygène singulet à l'intérieur de la poche du chromophore ont révélé l'oxydation de deux résidus soufrés, Met159 et Cys171, piégeant le chromophore dans un état protoné non-fluorescent. Sous haute intensité d'excitation, une voie de photoblanchiment oxygène-indépendante totalement différente a été trouvée. Le Glu212, strictement conservé, subit une décarboxylation associée à un important réarrangement du réseau de liaisons hydrogènes autour du chromophore, et un changement d'hybridation sp 2 vers sp 3

Abstract Structural insight into photobleaching mechanisms of reversible photoswitchable fluorescent proteins

The discovery of phototransformable FPs (PTFPs) from Anthozoa species, thanks to their photophysical properties, has opened a large field in biological fluorescence imaging. One of the PTFPs' sub-groups consists of Reversible Photoswitchable Fluorescent Proteins (RSFPs), which can be reversibly switched between nonfluorescent and fluorescent states. Photobleaching is the permanent loss of the fluorescence-emitting capacity under excitation, which is a common phenomenon among all the fluorescent molecules. Photobleaching has a large impact on the microscopy image quality, notably on super-resolution imaging. Photoswitchable fluorescent proteins have a tendency to lose performance within every switching cycle, a process referred to as "photofatigue". Our interest of study is focused on the photobleaching mechanisms of RSFPs.

We have reported the crystallographic structure of photobleached IrisFP under high and low illumination intensity at room temperature as well as its spectroscopic modifications. We found that different illumination intensities can result in different photobleaching pathways. Under low illumination intensity, an oxygen-dependent photobleaching pathway was evidenced. Structural modifications induced by singlet-oxygen production within the chromophore pocket revealed the oxidation of two sulfur-containing residues, Met159 and Cys171, locking the chromophore in a nonfluorescent protonated state. Under high illumination intensity, a completely different, oxygen-independent photobleaching pathway was found. The conserved Glu212 underwent decarboxylation concomitantly with an extensive rearrangement of the H-bond network around the chromophore, and an sp 2 -to-sp 3 hybridization change of the carbon atom bridging the chromophore cyclic moieties was observed. As Met159 is the key residue involved in low-intensity illumination photobleaching, we have then mutated Met159 into Alanine in order to avoid sulfoxidation. We found that the IrisFP-M159A mutant display an enhanced photostability in solution, in PVA gel and in E.coli cells.

Keywords: fluorescent proteins, RSFPs, photobleaching, crystallography, spectroscopy rational design