Prof Klaus Fendler

Dr Sophie Lecomte

Prof Fouzia Boulmedais

Prof Michael Boersch

Prof Thomas Heitkamp

Dr Youssef

Dr Fatima

Ana F S Seiça

Cristina V Iancu

Gregor Madej

Jun-Yong Choe

Petra Hellwig The

Ana Filipa

Santos Seica

Luis A E Batista De Carvalho

M Paula

Christina V Iancu

H Ronald Kaback

Thomas Heitkamp

Michael Börsch

2-carboxyethyl) phosphine

I would like to

"I have forgiven mistakes that were indeed almost unforgivable. I've tried to replace people who were irreplaceable and tried to forget those who were unforgettable. I've acted on impulse, have been disappointed by people when I thought that this could never be possible. But I have also disappointed those who I love. I have laughed at inappropriate occasions. I've made friends that are now friends for life. I've screamed and jumped for joy.

Live! The best thing in life is to go ahead with all your plans and your dreams, to embrace life and to live every day with passion, to lose and still keep the faith and to win while being grateful. All of this because the world belongs to those who dare to go after what they want. And because life is really too short to be insignificant."

-Charlie Chaplin

INTRODUCTION

Dans cette thèse 2, différentes bandes marqueurs en infrarouge de la protéine ont été étudiées, la ν(C=O) Asp/Glu entre 1730-1750 cm -1 et la ν(SC≡N) entre 2175-2120 cm -1 .

Les sondes infrarouges ont été introduites dans les protéines membranaires de la chaîne respiratoire. Ainsi les grands mouvements des domaines protéiques gouvernant les mécanismes catalytiques ont été contrôlés par les spectroscopies vibrationnelles à l'aide d'une réaction induite.

La spectroscopie vibrationnelle est une technique analytique utilisée pour l'étude des protéines au niveau moléculaire et présente donc un outil puissant pour étudier la structure des protéines et les réactions protéiques. Il est possible d'observer des groupements moléculaires participant activement à une réaction catalytique à l'aide de la spectroscopie vibrationnelle en obtenant ainsi des informations sur la structure et l'environnement des acides aminés des chaînes latérales et sur le squelette de la protéine.

LA CHIMIE DE SURFACE

Avant d'étudier les marqueurs infrarouges, il était nécessaire de comprendre, d'une part, l'influence de la morphologie de la surface d'or nanostructurée et d'autre part, l'effet de la procédure d'immobilisation des protéines sur le facteur d'amélioration des signaux protéiques en infrarouge. Deux méthodes d'immobilisation ont été utilisées pour caractériser des cristaux de silicium après dépôt de nanoparticules métalliques à des temps de dépôt différents.

La sélection de la méthode d'immobilisation appropriée est un élément essentiel du processus d'immobilisation. La surface solide de silicium doit être modifiée chimiquement pour empêcher la dénaturation de la protéine lors de l'immobilisation.

Plusieurs études ont montré que les surfaces modifiées par la monocouche autoassemblée (SAM) sont efficaces pour l'immobilisation des protéines. Les SAMs peuvent protéger les protéines contre le contact direct avec les surfaces solides, ce qui réduit considérablement la possibilité de dénaturation des protéines adsorbées (Figure 1).

MARQUAGE DE PROTEINES DE LA CHAINE RESPIRATOIRE PAR UNE SONDE INFRAROUGE

L'autre protéine marquée par une sonde infrarouge est la F 1 F O -ATPase d'Escherichia coli, composée de deux parties distinctes reliées par deux tiges. La partie F 1 hydrophile est un complexe de cinq sous-unités α 3 β 3 γ 1 δ 1 ε 1 , dépassant d'environ 100 Ȧ la membrane et est responsable de la catalyse de la synthèse/de l'hydrolyse de l'ATP. La partie F O transportant le proton hydrophobe est composée de trois sous-unités a 1 b 2 c 10 et est ancrée dans la membrane. La sous-unité complexe γε est située au centre de la protéine et est liée à l'oligomère sous forme d'anneau des sous-unités 'c'. Par ailleurs, les sous-unités 'c' sont connectées au dimère périphérique b2 via la sous-unité 'a' liée à son tour au complexe α 3 β 3 γ 1 δ composant les tiges de la protéine.

Il y a des études révélant la participation de la sous-unité epsilon (ε) dans la production de l'ATP. Cette sous-unité a été observée en deux conformations différentes en fonction des conditions expérimentales, cependant, il n'y a aucune preuve directe. Les deux conformations identifiées sont des conformations contractées (εC) (Figure 4B) et étendues (ε) (Figure 4A). La structure γ-ε est en conformation de εX où les deux hélices sont étendues et entrelacées dans la structure de la sous-unité afin d'interagir avec l'hexamère 3. Dans l'autre structure, la conformation εC, les deux hélices de ε sont repliées ensemble sous forme de sandwich et sont présentes sur l'anneau c-sous-unité. Le signal thiocyanate est étudié en présence et en absence d'ATP dans les deux protéines mutantes situées dans différentes positions de la sous-unité ε.

Les sondes telles que le nitrile-et le thiocyanate (dérivé d'acides aminés) ont été déjà décrit dans la littérature pour les peptides et les protéines solubles. Elles révèlent ainsi des informations précises sur l'environnement local de la sonde, car leurs fréquences d'absorption infrarouge dépendent fortement des liaisons hydrogènes avec les molécules du solvant protique environnantes, le squelette ou les acides aminés. De plus, leur fréquence du mode d'élongation est observé dans une zone relativement claire du spectre IR ν(C≡N ~ (2100-2240 cm -1 ).

-Surface-Enhanced

GENERAL INTRODUCTION General Introduction

List of Figures

Figure 1: Scheme of a nitrile infrared probe introduced at three different positions of a helix, with distinct structural and electro-static environments (A) and position of the CN vibration in different water/tetrahydrofuran (THF) mixtures (B) (spectra adapted from [8] )………………………………………………………………………………………...6

Figure 2: Schematic representation of the respiratory chain in a mitochondrion. The following crystal structures described: Complex I (PDB: 4WZ7) oxidizes NADH to NAD + to reduce ubiquinone (Q) to ubiquinol (QH2). Complex II (PDB: 2WDV) oxidizes succinate to fumarate and reduces Q without proton translocation. The electrons are transferred from QH2 to cytochrome c via complex III (PDB: 2A06) accompanied by the translocation of two protons [9] ……………………...………………………..……..…….7 Image taken from ref [19] ...…………………………...……………….………...……..…..8

Ana Filipa Santos Seiça Domain movements, conformational changes, and protein dynamics play a crucial role in the catalytic reaction of enzymes. Highly diverse structural changes including rotor like movements (F1Fo-ATPase) [1] , drilling of helices (TIM/TOM) [2] , the switching of subunits between two sites (respiratory complex III or K + -ATPase) [3][4] or wavelike motions in transporters have been described. To obtain a comprehensive understanding of the functional role of these structural motifs and rearrangements, the movements need to be visualized.

Vibrational spectroscopy, an analytical technique that studies proteins at a molecular level is a valuable tool for the investigation of protein structure and protein reactions. It is possible to observe groups that actively participate in a catalytic reaction by obtaining information about the structure and environment of amino-acid side-chains, protein backbone and others [5] . In this thesis 2, different infrared markers bands in the protein were studied, the stretching vibration of thiocyanate [ν(SC≡N)] between 2175 -2120 cm - 1 and the ν(C=O) of Asp/Glu between 1730 -1750 cm -1 .

The visualization of the thiocyanate group in the protein was done by introducing infrared probes in large membrane proteins from the respiratory chain and reaction induced vibrational spectroscopies were used to monitor large domain movements that rules the catalytic mechanisms of the protein.

Several of these conformational movements (for example in ATPase) have been previously identified by large probes such as fluorophores, typically used for fluorescence or electron paramagnetic resonance (EPR) spectroscopies [6] however they tend to be large, relative to the size of amino acid side chains in proteins.

Other probes have been recently used such infrared probes, which are significantly smaller and thus less perturbative for the structures and the catalytic activity. Probes such as nitrile-and thiocyanate-derivatized amino acids have been first described for peptides and soluble proteins [7] . They have been found to give specific information on the local environment of the probe, because their infrared absorption frequencies are strongly dependent on the hydrogen bonding with the surrounding protic solvent molecules, backbone, or amino acids [7] (Figure 1).

To understand which residue is responsible for a high pKa of sugar-binding and if there are special residues that play a key role in the coupling mechanism symport, reactioninduced infrared spectroscopy was used. The pH and substrate-dependent conformational changes of immobilized transport protein on a modified gold surface were investigated by surface-enhanced infrared absorption spectroscopy (SEIRAS) in an attenuated total reflection (ATR) perfusion cell.

Ana Filipa Santos Seiça

CHAPTER I

The theory behind the techniques

Ana Filipa Santos Seiça Table 1: Absorbance bands of amide vibrations in the MIR range [13] …………..…........23

List of Figures

List of Tables

Table 2:

Principal amide I frequencies characteristic of protein secondary structure [14][15] …………………………………………………………………………..24 Table 3: Properties of ATR crystal materials [20]

CHAPTER I The Theory Behind the Techniques

-SPECTROSCOPY

Spectroscopy studies the interactions between matter and electromagnetic (EM) radiations, which is described as a particle and by Maxwell's laws as two oscillating fields perpendicular to each other on a unique plane. These oscillating fields are represented by sinusoidal functions that propagate at a constant velocity, the speed of light. The relation between the energy (E) associated with these radiations and their frequency (ν) is described by the Planck relation:

𝐸 = ℎ𝜈 = ℎ 𝑐 𝜆
where c is the speed of light and h is Planck's constant [1] . Electromagnetic radiation exists as a broad and continuous spectrum, which is subdivided into seven regions according to their energy level (Figure 1).

Figure 1: Electromagnetic spectrum

There are many different spectroscopic methods available for solving a wide range of analytical problems. The methods differ concerning to the species to be analyzed (such as molecular or atomic spectroscopy), the type of radiation-matter interaction to be monitored (such as absorption, emission, or diffraction), and the region of the electromagnetic spectrum used in the analysis. Those methods are very informative and widely used for both quantitative and qualitative analysis [2] .

Equation 1

Ana Filipa Santos Seiça Spectroscopy is a powerful tool for studying biological systems. It often provides a convenient method for analysis of individual components in a biological system such as proteins, nucleic acids, and metabolites. It can also provide detailed information about the structure and mechanism of action of molecules [3] .

1.1 -UV-VISIBLE SPECTROSCOPY UV/visible spectroscopy involves the absorption of ultra-violet (UV) or visible light by an atom/molecule causing the promotion of an electron from the ground state to an excited electronic state. Electronic transitions are detected in the near-ultraviolet between 190 to 800 nm and in the visible spectral regions from 400 to 800 nm.

The energy of the absorbed quantum light corresponds to the difference of the energy between the ground and excited states. Different types of electronic transitions are observed. The bonding π molecular orbital is occupied by two electrons each with a different spin. The non-bonding η orbital is occupied by two electrons. The transition occurs from the empty anti-bonding π* orbital, which has a higher energy level, and is described as π-π* transition and η-π* transition [4] .

The absorption spectrum of the matter is characterized by the molecular extinction coefficient ε at each wavelength. The Lambert-Beer law states that the absorbance A is directly proportional to the path length d of the sample and the concentration c.

𝐴 = 𝑙𝑜𝑔 𝐼 𝑜 𝐼 = 𝜀 × 𝑐 × 𝑑
The UV absorption is a common tool for the study of proteins in the range of 180 to 230 nm corresponds to π → π * transitions in the peptide bonds. Absorption in the range of 230-300 nm is mostly dominated by the aromatic side-chains of tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) residues, and also weak contributions by disulfide bonds near 260 nm [5] .

Equation 2

CHAPTER I The Theory Behind the Techniques 20

Ana Filipa Santos Seiça For an active Raman vibration, the polarizability must present a non-zero variation on the coordinated vibration, where Qi represents the coordinate i of the molecule vibration, equation 4.

𝜕𝛼 𝜕𝑄 𝑖 ≠ 0

Raman spectroscopy is particularly sensitive to weak interactions. To understand this statement, it should be recognized that, in general, weak interactions are dispersion interactions, resulting from induced dipole -induced dipole interactions [8] .

Raman spectra have on the ordinates axis Raman intensities and on the abscissa, the Raman shifts, commonly expressed in wavenumbers (unit cm -1 ). The most important frequencies for identification of organic compounds occur in the region of 4000 to 1300 cm -1 . In this region, we know the functional groups present in the unknown substance.

The region of lower frequencies, 1500-500 cm -1 , is often called the fingerprint region, and the lines observed in the spectrum are the characteristics of the molecule as a whole.

The origin of these spectral lines is complex, involving vibrations of angular deformation, vibrations between the same or similar masses [9] .

There are many advantages and disadvantages in Raman Spectroscopy such as the possibility to selectively increase the vibration intensity of a particular group in the molecule. This is particularly advantageous in vibrational study of biological molecules that possess a chromophoric group. Since the diameter of the laser beam is normally 1-2 mm, only a small area of the sample is required to obtain Raman spectra [6] .

Since water is a weak diffuser, Raman spectra for aqueous samples can be obtained without much interference of water vibrations. Thus, Raman spectroscopy is ideally suited for biological studies of compounds in aqueous solution. Raman spectra of hygroscopic compounds and/or air-sensitive may be obtained by placing the sample in a sealed glass tube, the region of 4000 to 50 cm -1 can be withdrawn alltogether.

Despite its use, this spectroscopy has some disadvantages [6] . The use of a laser as a radiation source causes heating and/or photo-decomposition of the sample, especially in Raman Spectroscopy where the laser frequency is deliberately "tuned" on the absorption band of a molecule. Another disadvantage is the presence of fluorescence that obscures or hinders the observation of Raman bands [6] .

Equation 4

CHAPTER I The Theory Behind the Techniques

21

Ana Filipa Santos Seiça While Raman spectra are normally associated with vibrational transitions in liquids and solids, is possible to record a Raman spectrum associated with electronic transitions between the minimum and excited energy states. The diffusion of Resonance Raman occurs when the excited state involves intercepts energy of multiple excited electronic states. In liquid and solid, vibration levels are extended to produce a continuum whereas, in the gaseous state, there's a series scale of discrete levels [9] .

-INFRARED SPECTROSCOPY

Infrared spectroscopy (IR) is an absorption method in the wavelength region of 1 to 100 μm in that extends the region of the visible light to longer wavelengths and smaller frequencies/energies. The energy of infrared light is no longer sufficient to induce transitions of valence electrons. Instead, infrared radiation excites vibrational and rotational motions in molecules. The infrared spectral region is separated into three main parts, the near (NIR: 12500 to 4000 cm -1 ), middle or mid (MIR: 4000 to 800 cm -1 ) and the far-infrared region (FIR: 800 to 10 cm -1 ) [10] .

IR absorption positions are generally presented as either wavenumbers or wavelengths.

Wavenumber defines the number of waves per unit length. Thus, wavenumbers are directly proportional to frequency, as well as the energy of the IR absorption [11] . The absorption of IR radiation results in changes in the vibrational or rotational modes of molecules. For a linear or a non-linear molecule of N atoms, 3N-5 or 3N-6 normal vibrational modes exist, respectively. These normal vibrational modes are infrared active only if the dipole moment of a molecule changes during the vibration [12] .

For a molecule composed of two atoms, the vibrational frequencies can be predicted by comparing it to a harmonic oscillator. The atoms, of mass and, are considered as pointlike and the chemical bond is equated to a spring. The wavenumber of the stretching vibration can be described by equation 5, which is derived from Hooke's law. The most studied spectral region in protein is the mid-infrared spectroscopy, as it contains many indications of structural and functional relevance and is well-mastered from the technical point-of-view (Table 1). Three oscillators, amide A and amide I and II, belong to the backbone of the polypeptide chain and are therefore relatively intense and not independent of each other. Because of the formation of particular hydrogen bonding patterns in the peptide sequences of the protein that form secondary structures, they are coupled oscillators [13] .

The amide I band situated between 1700 and 1600 cm -1 , is hardly affected by the nature of the side-chain. It depends on the secondary structure of the backbone and is, therefore, the amide vibration that is most commonly used for secondary-structure analysis.

The amide II mode at 1550 cm -1 in the out-of-phase combination of the N-H in-plane bend and the C-N stretching vibration with smaller contributions from the C-O in-plane bend and the C-C and N-C stretching vibrations is hardly affected by side-chain vibrations.

The N-H stretching vibration gives rise to the amide A band between 3310 and 3270 cm -1 , exclusively localized on the N-H group and thus insensitive to the conformation of the polypeptide backbone.

Table 1:

Absorbance bands of amide vibrations in the MIR range [13] .

Designation Position / cm -1

Vibrational modes Amide A 3300 ν(N-H) Amide B 3100 ν(N-H), resonance with Amide II overtone

Amide I 1695 -1610 ν(C=O) 80%, ν(C-N) 10%, δ(N-H) 10% Amide II 1575 -1480 δ(N-H) 60%, ν(C-N) 40% Amide III 1320 -1220 δ(C-N) 30%, δ(N-H) 30%, ν(C=O) 10%, δ(N-C=O)

-Secondary Structure Analysis

The most common method of quantitation of protein secondary structure involves curve fitting of the amide I. Deconvolution or derivation is used to estimate the number and approximate position of the components of the amide I band.

Deconvolution is performed using the Gaussian profile of each component and consecutive optimization of the position, amplitude and width at half-height of each band. Afterward, the area of the individual band was used to calculate the relative contribution of each component of the secondary structure of the protein into the total surface area of amide I band. Positions of the different components of amide I band are summarized in Table 2.

Table 2: Principal amide I frequencies characteristic of protein secondary structures [14][15] .

There are many examples in the literature of the assignment of protein secondary structure based on the analysis of the amide I band. In general, α-helical structures have a band peak at wavenumbers 1650-1658 cm -1 , β-sheet structures tend to have bands between 1620-1640 cm -1 and 1670 -1695 cm -1 , random coil structures occur at around 1644 cm - 1 . It is even possible to assign more complex structures such as α-helix overlapped with random coil which can absorb IR radiation at 1654-1656 cm -1 [15] .

The study of aqueous protein samples is challenging because of the overlap of the water bending mode with the amide I band at 1600 cm -1 . This could be overcome through the although care should be taken to avoid over subtraction [14] .

Deuterated solvents are widely used in protein infrared spectroscopy since they allow us to distinguish between α-helical and random structures. The frequency of the amide II band decreases by about 100 cm -1 upon H/D exchange of amide protons. Therefore, this band is usually used to measure the extent of amide-proton exchange in proteins and peptides [14] .

-Fourier Transform Infrared Spectroscopy

Fourier transform infrared spectroscopy (FTIR) can provide fundamental information on the molecular structure of organic and inorganic components and is one of the most versatile analytical techniques for the non-destructive, chemical characterization of proteins. Fourier spectroscopy is a general term that describes the analysis of any varying signal into its constituent frequency components. Fourier-transform infrared spectroscopy is based on the idea of the interference of radiation between two beams to yield an interferogram [16] .

Fourier transform spectrometer uses polychromatic radiation in which the radiation emerging from the source is passed through an interferometer to the sample before reaching a detector. The radiation from the infrared light source is partially reflected and partially transmitted by the beamsplitter.

There are several advantages of using an FTIR spectrometer such as good quality spectra, rapidly, under-sampling conditions that are difficult or impossible for the dispersive spectrophotometer, a much higher signal-to-noise ratio of the FTIR systems.

Most commonly used FTIR techniques for bulk sample analysis are transmission FTIR (potassium bromide (KBr)-pellet FTIR), attenuated total reflection (ATR)-FTIR, and diffuse reflection infrared Fourier Transform (DRIFT) spectroscopy [17] .

CHAPTER I The Theory Behind the Techniques

26

Ana Filipa Santos Seiça

-Transmission Mode

In transmission mode, the sample is normally placed between two windows made of an IR transmitting material such as calcium fluoride (CaF2), and the IR measuring beam is passed through the assembly (Figure 4).

-Attenuated Total Reflection Spectroscopy

Attenuated total reflection spectroscopy utilizes the phenomenon of total internal reflection (Figure 5). A beam of radiation entering a crystal will undergo total internal reflection when the angle of incidence at the interface between the sample and crystal is greater than the critical angle, where the latter is a function of the refractive indices of the two surfaces [18] . When the angle of incidence is greater than the critical angle θc (where is a function of the refractive index of two media), all incident radiations are completely reflected at the interface, results in total internal reflection.

Ana Filipa Santos Seiça [19] The depth of penetration in ATR spectroscopy is a function of the wavelength, λ, the refractive index of the crystal, n2, and the angle of incident radiation, θ. The depth of penetration, dp, for a non-absorbing medium is given by equation 6, where n1 is the refractive index of the sample.

𝑑𝑝 = 𝜆 2𝜋𝑛 1 [𝑠𝑖𝑛 2 𝜃-( 𝑛 1 𝑛 2 ) 2 ] 1 2 ⁄
In ATR spectroscopy a crystal with a high refractive index and excellent IR transmitting properties is used as an internal reflection element (IRE) and is placed in close contact with the sample [19] . The crystals used in ATR cells are made from materials that have low solubility in water and are of a very high refractive index (Table 3). The surface of an IRE (or ATR crystal) can be easily modified making it well placed to study the effect of different surfaces on proteins without the need for additional labeling.

Different designs of ATR cells allow both liquid and solid samples to be examined. It is also possible to set up a flow-through ATR cell by including an inlet and outlet in the apparatus. This allows for the continuous flow of solutions through the cell and permits spectral changes to be monitored with time [20] .

There are advantages by using ATR like for example, faster sampling with no preparation, excellent sample-to-sample reproducibility and minimal operator-induced variations, however this technique also presents some disadvantages like, the ATR crystal absorbs Ana Filipa Santos Seiça energy at lower energy levels, if the sample does not have good contact with the crystal, the data will not be accurate and most of ATR crystals have pH limitations [21] .

Table 3: Properties of ATR crystal materials [20] .

-Polarized ATR FTIR Spectroscopy

Polarized attenuated total reflection Fourier transform infrared spectroscopy is currently one of the techniques widely used in membrane protein structural analysis. This method yields information about the polypeptide secondary structure as well as its orientation in the membrane [22] .

Perpendicularly polarized light traveling through the ATR crystal will result in an electric field on the surface of the ATR crystal with a component only on the surface of the crystal, 𝐸 0 = 𝐸 𝑦 . When light is polarized parallel to the incident plane, then 𝐸 0 2 = 𝐸 𝑥 2 + 𝐸 𝑧 2 . By combining spectral information using polarized light, information about protein orientation on surfaces is possible to obtain [23] . Polarize ATR FTIR spectroscopy can be used to determine the orientation of α-helices of membrane proteins.

Material

CHAPTER I The Theory Behind the Techniques

Ana Filipa Santos Seiça

List of Figures

Figure 1: AFM image of electroless deposition of an Au film on the silicon crystal [15] ………………………………………………………………………………..38 The objective of this project is to understand the influence of the gold nanostructure morphology and protein immobilization procedure on the enhancement factor of protein signals in infrared.

-SURFACE ENHANCED RAMAN SPECTROSCOPY

Surface-Enhanced Raman Spectroscopy was first documented in 1974 when Fleischann et al [1] demonstrated that the Raman signals of pyridine adsorbed on an electrochemical roughened silver electrode was many times greater than expected. Because the voltage applied to the electrode, in the described experiment, affected the signal level it was attributed to an increased surface coverage of pyridine on the electrode. A few years after, Van Duyne et al [2] quantified the phenomenon and found an enhancement or increase in Raman cross-section of the adsorbed pyridine molecules. This lead to the conclusion that the increase of the Raman signal was due to a unique electromagnetic enhancement present in the roughened silver in which pyridine was adsorbed [2] . Numerous other observations [3][4] [5] were made in the subsequent years to understand the electromagnetic explanation for stoke enhancement of Raman signals, which became universally known by the acronym SERS [6] , that stands for Surface-Enhanced Raman Spectroscopy. Since its discovery in 1974, SERS has become widely used, offering many advantages over other techniques such as FT-IR Spectroscopy, Ultra Violet-Visible-Near Infrared (UV-vis-NIR) Absorption, X-Ray Photoelectron Spectroscopy, Mass Spectrometry and others [7] .

The plasmon resonance of metal nanostructures is a core process in SERS. Silver (Ag) and gold (Au) metals are probably used as substrates because the surface plasmon resonance occurs in the visible region. Metals like Al, Ga, In, Pt, Rh, and alkali metals Ana Filipa Santos Seiça (Li, Na, K, Rb and Cs) can also be used as substrates in SERS but enhancement is low compared to Ag and Au [8] .

The use of some metal colloids allows to study the adsorption of many organic molecules.

The high sensitivity of SERS permits an accurate structural study of many molecules at very low bulk concentrations. The increase in intensity observed in SERS usually reach 10 5 -10 6 . Moreover, the SERS effect becomes even stronger if the frequency of excitation light (laser frequency) overlap the surface plasmon resonance and the v vibrational signal of the molecule [9] .

-SURFACE-ENHANCED INFRARED ABSORPTION SPECTROSCOPY

Surface-Enhanced Infrared Absorption Spectroscopy (SEIRAS) was first reported by Hartstein et al in 1980 [10] . Two mechanisms are responsible for SEIRAS: the electromagnetic enhancement mechanism (EM) and chemical enhancement mechanism (CM), which are also observed in SERS.

The EM mechanism is usually used to explain the near-field effect of SEIRAS. The absorption by the metal/adsorbate layer is considered to play an equally important role in the mechanism of enhancement. The dielectric properties of a metal film are altered, when dipoles of adsorbed molecules induce mirror dipoles in the metal substrate [11] , molecules studied are physically or chemically adsorbed on the surface of the metal can modulate the electromagnetic field.

The electromagnetic mechanism suggests that the resonance of the metal nanoparticle (now termed localized surface plasmon resonance) underpins the enhancement and it's independent of bonding between the analyte and the metal surface [11] .

Chemical enhancement accounts for 10-100 (relatively small, but still important) of the total enhancement factor and requires the molecule to be in direct contact (bonded) with the surface of the metallic nanostructure [11] . This is strongly dependent on the nature of the molecule, the metal and the particular experimental conditions, and can be envisaged as a resonant photoinduced electron transfer process between the metal and the molecule.

An important feature of SEIRAS is the so-called surface selection rule. Vibrational bands that arise from a change in dipole moment parallel to the normal surface are strongly enhanced while those comprising a perpendicular component are weakened [12] .

CHAPTER II Surface Chemistry

_____________________________________________________________________________

Ana Filipa Santos Seiça SEIRAS results in high surface sensitivity by enhancing the signal of the adsorbed molecule by approximately two orders of magnitude and has the potential to enable new studies, from fundamental aspects to applied sciences [12] . Another advantage of SEIRAS spectroscopy is that it is useful for in situ observations of metal surface reactions.

-PROPERTIES OF GOLD THIN FILMS FOR SEIRAS STUDIES

Gold is the plasmonic material mostly used nowadays due to its high chemical stability and electron mobility. Preparation of a nanostructured thin gold layer is important for a successful SEIRA experiment. Different properties can be easily affected by the experimental conditions, such as the protocol of preparation of the gold nanoparticles (AuNPs), the time of gold deposition, temperature and others. AuNPs layers should be stable over time and the interaction with the surface strong enough to ensure that the nanoparticles remain attached upon utilization [13] . A drawback of this method is the precise and controlled assembly of gold nanoparticles on the surface.

One of the methods to prepare metal island films appropriate for SEIRA spectroscopy is chemical deposition. The enhancement factor will depend on the morphology of the metal film, its ability to support surface plasmons with absorption bands that extend into the IR region and also is dependent on the size, shape, and particle density of the gold-island film [14] .

Prof. Masatoshi Osawa, University of Hokkaido, determined by Atomic Force Microscopy (AFM) that chemically deposited Au films have an island structure similar to that of vacuum evaporated Au films (Figure 1), with an average dimension of the islands about 300 nm for the chemically deposited film and an average thickness of 50nm for 60s deposition time [15] .

The enhancement factor can be calculated through the ratio of the absorption intensity with and without a metal film, however only the vibrational modes that present dipole changes perpendicular to the metal surface are SEIRA active.

The adhesion of the Au island films can be improved by removing the oxide layer and hydrogen-termination of the Si substrate before metal deposition, or by etching the surface during metal deposition [16] . [15] .

Ana Filipa Santos Seiça

-IMMOBILIZATION OF FUNCTIONAL PROTEINS ONTO SOLID

SURFACES

Selection of the appropriate immobilization method is a very crucial part of the immobilization process. The solid surface requires to be chemically modified, to prevent the denaturation of the protein upon immobilization [17] .

Immobilization methods are divided into two classes, chemical and physical methods.

Physical methods can be characterized by weaker, non-covalent interactions such as hydrogen bonds, hydrophobic interactions, Van der Waals forces, affinity binding, ionic binding of the enzyme with the support material, or mechanical containment of the enzyme within the support [18] .

With the chemical method, formation of covalent bonds achieved through ether, thioether, amide or carbonate bonds between enzyme and support material are involved [19 . There are four principal techniques for immobilization of enzymes, adsorption, entrapment, covalent and cross-linking.

Immobilization of membrane proteins on solid supports is intrinsically more difficult than water-soluble proteins. The amphiphilic character of membrane proteins and their low conformational stability easily influences their properties through interaction with the solid surface [20] .

Several studies have shown that self-assembled monolayer (SAM)-modified surfaces are efficient for protein immobilization [21] . SAMs can protect proteins from direct contact Ana Filipa Santos Seiça This technique has the advantage of following in situ the reduction and oxidation of the protein using transmission IR spectroscopy [31] and reflection modes [32] .

Moss and colleagues designed in the 1990s an electrochemical transmission cell for infrared spectroscopic measurements in aqueous solution [24] . The spectroelectrochemical thin-layer cell permits a fast, accurate, and reproducible control of the redox state of the protein. This electrochemical cell has the advantage to be used in the UV/vis [33] , fluorescence [34] , Infrared and Raman.

The disadvantage of this cell is that requires an amount of sample, about 20 μl, which is relatively high for biological samples. The solution found, was to use an electrochemical cell that works with a monolayer of proteins adsorbed on metal surfaces giving access to a direct electrochemical signal [35] [36] .

-Perfusion

Another approach to analyze ligand-protein, ion-protein interactions and redox changes of membrane proteins is perfusion-induced difference in the ATR-FTIR cell. It requires attachment of the protein on the surface, for instance in the Ni-NTA procedure described previously (Figure 4). The use of using this immobilization approach combined with perfusion allows us to study several proteins [37] .

In this method, several buffers, whit different composition can be changed, can be perfused over the protein film surface by switching between the different buffers (Figure 5). Difference spectra can be recorded over many cycles provided that the reaction is reversible. The time resolution of the perfusion method depends on the exchange time of a buffer solution in a sample chamber, which normally takes a few minutes because of the slow flow rate of a perfusion peristaltic pump [38] .

Ana Filipa Santos Seiça oxide layer and to terminate it with hydrogen and after the surface was rinsed with water and dried again.

The crystal and the plating solution were heated at 65 °C for 10 min together. The solution is a 1:1:1 mix (vol/vol/vol) of 15 mM NaAuCl4 + 150 mM Na2SO3 + 50 mM Na2S2O3 + 50mM NH4Cl, and 2% HF (wt/vol: 1 mL). The silicon prism was covered with the solution for a range of times between 5 to 100 s, and deposition was stopped by washing the crystal surface with water, followed by drying the surface with a stream of argon. The samples were imaged using a FEG-SEM (scanning electronic microscopy) (Hitachi SU8010) at 1keV.

-Immobilization Approaches

Two different methods were used to immobilize the soluble protein Cytochrome C from bovine heart, the glucose transporter from Staphylococcus Epidermidis (GlcPSe) and the sodium pumping NADH: quinone oxidoreductase (Na + -NQR) from Vibrio Cholerae.

-11-mercaptoundecanoic acid sam

The gold film was deposited on the silicon crystal, the background was recorded and used as a reference and the crystal was immersed in a solution containing 11-MUA with a concentration of 10 mM. The monolayer was allowed to self-assembled during 2.5 h.

The excess of 11-MUA was removed and the crystal was rinsed with ethanol and gently dried with an argon stream. Finally, the protein cytochrome c dissolved in 10mM KPi pH 8.0 with a final concentration of 2 mM was added to the modified surface and the absorbance spectrum was recorded.

-Ni-NTA SAM

After the formation of a gold layer on the silicon crystal, a nickel nitrilotriacetic acid selfassembled monolayer (Ni-NTA SAM) was deposited on the surface by adaptation to the procedure described in refs [32][38] .

First, 1 mg/mL of 3,3-dithiodipropionic acid di(N-hydroxysuccinimide ester) (DTSP) in dimethyl sulfoxide (DMSO) was allowed to self-assemble on the gold surface. After the monolayer was formed, excess DTSP was washed away with DMSO and the crystal was dried under an argon stream. Afterward, the modified surface was immersed in a solution of 100 mM Nα,Nα-bis (carboxymethyl)-L-lysine (ANTA) in 0.5 M K2CO3 at pH 9.8 for 3 h and then rinsed with water.

Finally, the surface was incubated in 50 mM nickel(II) perchlorate hexahydrate (Ni(ClO4)2) solution for 1 h. After washing with water, the protein GlcPSe and Na + -NQR, both dissolved in 50 mM phosphate buffer containing 0.05% n-dodecyl-beta-maltoside (DDM) pH 7.5, were drop-casted on the modified surface.

-Infrared Spectroscopy

The two immobilization approaches were followed by FTIR in transmission or attenuated total reflection mode using different types of silicon crystal (Figure 7).

The silicon crystal with 3-mm surface diameter (Figure 7A) was used as a single reflection ATR unit which allowed the possibility of a simultaneous acquisition of FTIR spectra in the ATR mode coupled to a perfusion of solutions with different compositions.

The hemicylindrical silicon crystal (Figure 7B) on which a glass cell was mounted was used to carry out ATR-electrochemical experiments. The cell contains 4 inlets, two of which were used to fit the platinum counter and Ag/AgCl reference electrodes, and the gold film acted as a working electrode.

Finally, a silicon wafer with a 1 cm diameter (Figure 7C) was used for the study of dry samples in transmission mode. After deposition of the gold surface, the crystal was sealed in a metal support between two Teflon joints and then the cell was transferred to the spectrometer.

CHAPTER II Surface Chemistry

_____________________________________________________________________________

The overlapping IR signature from both the 11-MUA layer and the protein layer are observed, making it difficult to distinguish between the two however, we can observe the strong amide I band signal at 1650 cm -1 . In the case of the ATR silicon crystal of 3mm, it was possible to subtract the spectrum of 11-MUA monolayer acquired before the protein deposition from the final spectrum, in this way only the IR signal associated with the protein remained.

ATR configurations require much longer timescales between background and sample acquisition, which results in a lower signal to noise ratio. The transmission mode is a robust method for the study of surface modifications and solid state-samples, but with lower sensitivity as compared to ATR setup.

Figure 9A shows the absorbance spectra in transmission mode of amide I band where we can see that the intensity of the band increases with the time of deposition, the maximum being reached at 40s, followed by a decrease of intensity. In the case of the silicon crystal for ATR, the protein is in solution, and the maximum intensity was obtained at 40s for the small crystal (3mm) and 60s for the larger ATR crystal (2mm) (Figure 9B andC).

The enhancement factor was obtained from the ratio of the intensities of the spectra of amide I band deposited on a modified gold-coated silicon prism and a bare silicon prism for different deposition times.

The origin of the different enhancement observed for the two ATR crystals, 4 for the small and 8 for the big crystal, confirms that the coverage of the surface of the crystal contributes to the enhancement. The enhancement obtained with the silicon wafer (1cm) and the large ATR crystal are slightly similar, because the size of the crystal is relatively close and the coverage of the surface is bigger than the small ATR crystal (3mm).

to the surface (Figure 10B) and the corresponding SEIRAS spectrum was followed in situ during 3 hours.

Lysine reacted through its amine group with the succinimidyl ester group of the TSP forming an amide bond. The formation of this bond is evidenced by the presence of the amide I and amide II peaks at 1615 and 1565 cm -1 . The peaks at 1390 cm -1 correspond to the symmetric stretching mode of ν(COO -) of Nα,Nα-bis (carboxymethyl)-L-lysine (ANTA) carboxylate groups [42] .

Finally, the NTA surface was immersed for 1 hour with a nickel solution. Complexation of Ni 2+ with NTA surface led to small changes in the spectrum (Figure 10C) with the appearance of two bands at 1604 and 1402 cm -1 assigned to the asymmetric and symmetric stretching mode of the carboxylate groups of NTA that get deprotonated after complexation with Ni 2+ [41] .

Once the surface Ni-NTA was formed the protein GlcPSe and Na + -NQR were immobilized. Absorbance spectra of the amide I band were recorded to evaluate the dependence of the SEIRA enhancement factor with the Au deposition time (Figure 10D).

For the small ATR silicon crystal is possible to observe a maximum enhancement at 40s, the same as in transmission mode and for the crystal for the large ATR crystal a maximum enhancement is presented at 60s (Figure S2 (II) -Appendix II).

We observe that for the small silicon ATR, the amide I is visible, however the shape, position and intensity ratio are different, this can be explained due to the different orientation of the protein with respect to the surface.

Ni-NTA is a promising strategy to immobilized proteins ensuring fast binding and high specificity. The affinity of individual Ni-NTA to His-tags is usually low, however, when a support surface has a high density of immobilized Ni-NTA, multiple linkages by a Histagged protein provides a greater affinity and long-lasting association. The data obtained for Ni-NTA immobilization are consistent with the data obtained for the 11-MUA immobilization, showing that the enhancement of a protein in a gold layer on a silicon crystal is independent of the type of immobilization.

CHAPTER II Surface Chemistry

_____________________________________________________________________________ observe that, depending on the morphology of the structure, the enhancement of the electric field differs between the deposition times.

The map of the near electric field intensity distribution shown in Figure 11 confirms that the maximum enhancement is attained when the deposition time is around 40 seconds before and after immobilization which confirms the experimental results obtained for the silicon wafer in both immobilizations.

The difference in enhancement between the experimental results and the simulation is attributed first to the simulated random structures, which are similar to the real surface but not the same. Another reason for these differences can arise from the type of illumination beam in simulation and experiments as detailed in reference [43] . The difference of the enhancement of the electric field in nanoslits when illuminated by a plane wave and a Gaussian beam has been described.

As reported, the tightly focused Gaussian beams induce an intensity enhancement that can be one magnitude order below than the one created by a plane wave. This study was done to prove that the Gaussian beam, which resembles more the real shape of the incident beam in the experimental setup, will give us a clearer idea and more accurate values of the intensity enhancement of the electric field. So, in our simulation, it is obvious that the values of the electric field intensity should be divided by 10 to resemble more the reality of the enhancement shown in the experimental data of the Amide I band.

Figure 11 shows SEM images of gold nanoparticles on a silicon substrate obtained with a FEG-SEM (Hitachi SU8010). The particles have a nearly spherical shape and their average size is around 30 nm. SEM is a useful tool for characterization of the surface of the gold nanoparticles for different deposition times, giving us information regarding the size of the nanoparticles and the surface coverage, however in order to obtain more information of the surface other techniques need to be used, for example Atomic Force Microscopy (AFM) which give us information of the topography of the surface.

As indicated by

List of Figures

Figure 1 A) Overview of E. coli F1FO -ATPase (PDB 5t4o), subunits α in blue, β in red, γ in orange, ε in violet, c in green, a in grey, b in yellow, and δ in pink [14] 2) Substrate again binds to the open site, and another ATP is synthesized at the tight site (image taken from ref [15] ).…………………...……………..70 [START_REF] Fafarman | Site-Specific Conversion of Cysteine Thiols into Thiocyanate Creates an IR Probe for Electric Field in Proteins[END_REF] ……………………...………….…………...77 Figure 6. Reaction scheme of the coupled enzyme test in ATP hydrolysis [START_REF] Fischer | Comparison of ΔpH-and Δφ-driven ATP synthesis catalyzed by the H+-ATPases from Escherichia coli or chloroplasts reconstituted into liposomes[END_REF] ………..…79

-CYSTEINE CYANALATION OF MEMBRANE PROTEINS

As mentioned in General Introduction conformational changes play a crucial role in the catalytic reaction of enzymes. In this Chapter, we studied by using nitrile probes the conformational movements of the different residues of two membrane proteins from the respiratory chain: ATPase and Complex I.

-INFRARED PROBES

Infrared probes such as nitrile-and thiocyanate-derivatized amino acids have been found to give specific information on the local environment of the probe [1] .

Cysteine residues, one of the most conserved amino acids in proteins, are used in enzyme active sites and play structural roles which include iron-sulfur (Fe-S) assembly, heme, and zinc finger motifs [2] and are found on the surface of soluble proteins establishing contact with the surrounding aqueous environment [2] .

The formation of sulfide bond is governed by the pKa of cysteine which is influenced heavily by the surrounding microenvironment. Cysteines have a pKa of 8.3, and at neutral pH they are thus in a protonated and unreactive state [3] . Cyanylated alanine and cysteine residues are ideal IR probes that can be directly and easily incorporated into peptides and proteins to study electric field environments at enzyme active sites and to investigate protein folding and unfolding dynamics in realtime by studying the time-dependent change of the IR peak frequency [4] . The nitrile group is a particularly attractive probe because the frequency of the stretching mode is in a relatively clear region of the IR spectrum (νC≡N ~ 2100-2240 cm -1 ) [5] . The nitrile stretching vibration has recently emerged as a probe of the conformation and local environment of biological molecules due to its sensitivity to various factors, such as local electric field and hydrogen bonding interactions.

Ana Filipa Santos Seiça

The perturbation to the native conformation caused by introducing a nitrile group into the side chain of an amino acid should be minimal [6] . The nitrile group has a polarity between an amide group and a methylene, and thus it should be easily accommodated in the hydrophobic interior of a protein or a membrane as well as on the hydrophilic surface of a water-soluble protein.

-ATP SYNTHASES

FOF1-ATP synthases catalyze the synthesis of ATP during oxidative phosphorylation.

They are found in the plasma membranes of bacteria, the inner membranes of mitochondria, and the thylakoid membranes of chloroplasts [7] .

ATPase is a large protein complex sitting in the inner membrane of mitochondria with a membrane-embedded section FO and a soluble section F1-ATPase facing the matrix [9] .

They use can the proton motive force, built up by oxidative phosporylation or photosynthesis to generate ATP. There are different types of ATPases in diverse organisms, according to their function, structure, and localization:  F-ATPase also known as FOF1 ATP synthase, is found in bacterial plasma membranes, in mitochondrial inner membranes, and chloroplast thylakoid membranes. The role of this protein is to use the proton gradient to drive ATP synthesis by allowing the protons to flux across the membrane down their electrochemical gradient [7] .  V-ATPase, also called vacuolar-type H + -ATPase, is found in almost every eukaryotic cell. V-ATPase functions as an ATP-dependent proton pump. It couples the energy of ATP hydrolysis to proton transport across intracellular and plasma membranes of eukaryotic cells [8] .

 A-ATPase is called AoA1 ATPase. It is found exclusively in Archaea and has a similar function to F-ATPase. Structurally it is closer to V-ATPase [9] .  P-ATPase is also known as E1-E2 ATPase. They are found in bacteria and in some of eukaryotic plasma membranes and organelles. They transport a variety of different compounds, including ions and phospholipids across a membrane using ATP hydrolysis for energy [10] .

Ana Filipa Santos Seiça  E-ATPase is a type of membrane-bound cell-surface enzymes, they are found in most eukaryotic cells and hydrolyze nucleotide tri-and/or diphosphates in the presence of Ca +2 or Mg +2 [11] . 1A) is composed of two distinct portions connected by two stalks. The hydrophilic F1-portion, a complex of five subunits α3β3γ1δ1ε1, protrudes ~ 100 Ȧ from the membrane and is responsible for the catalysis of ATP synthesis/hydrolysis. The hydrophobic proton transporting FO-portion with three subunits a1b2c10 is anchored in the membrane [12] . The subunit γεcomplex is located centrally and bound to the ring-shaped oligomer of c-subunits and peripheral b2-dimer connecting subunit a to the α3β3γ1δ-complex compose the stalks.

3.2.1 -Structure of E. Coli F1Fo -ATPase F1FO -ATPase of Escherichia coli (E.coli) (Figure
There are three catalytic and three non-catalytic nucleotide-binding sites on the α3β3hexamer. Non-catalytic sites are involved in the regulation of the enzyme. During ATP synthesis or hydrolysis the three catalytic sites change their affinities to ATP, ADP and phosphate in a complex and co-operative manner (Figure 1B). The catalytic mechanism is realized by rotation of γεc10-15subunit complex (rotor) relative to the rest of the enzyme (stator) [13] .

During ATP synthesis the ion transport through FO powers the rotation of the ring-shaped c-oligomer, and the rotation is passed into F1 via γε-complex, where it drives the conformational transitions affinity changes. Vice versa, the binding and hydrolysis of ATP can induce the conformational transitions in α3β3-hexamer that result in rotation of subunit γ which is passed to the c-ring and drives the proton transport [14].

Ana Filipa Santos Seiça hydrolysis. The common structure of F1-ATPase contains five subunits, α, β, γ, δ and ε, with the stoichiometry of 3, 3, 1, 1 and 1 respectively [17] .

The α3β3 complex forms a cylinder that surrounds the central stalk. The three pairs of α/β subunits undergo a sequence of conformational changes leading to ATP synthesis (or hydrolysis), which are induced by rotation of γ subunit, which forms the central shaft that connects FO rotary motor to F1 catalytic core. δ subunit contributes to the assembly of the protein rotor by the connection of γ subunit and subunit c. Last, ε has an important role in the assembly of F1 and the incorporation of hydrophobic subunit c into the F1-c oligomer rotor structure of mitochondrial ATP synthase [18] .

FO is responsible for proton translocation during ATP synthesis and hydrolysis.

Reconstitution experiments have shown that all three subunits, a, b and c, are required for the formation of the proton channel, for proton translocation, and normal binding of F1.

Although subunit b may not directly participate in proton translocation, it is required for assembly of active FO and binding of FO to F1 [19] .

-Structure and Function of the ε Subunit

The three-dimensional structure of isolated subunit ε from E.coli enzyme was solved by NMR [20] [21] and by X-ray crystallography [22] showed a protein with molecular mass of 14kDa and two-domain structure consisting of a 10-stranded β-sandwich in the Nterminal domain (NTD) and two C-terminal αhelices that form a hairpin C-terminal domain (CTD).

In bacteria and chloroplasts, the subunit-epsilon plays an important role in the regulation of ATP synthase activity by affecting the efficiency of coupling, influencing the catalytic pathway and selectively inhibiting ATP hydrolysis activity [23] . ε's Nterminal domain binds to γ and directly connects to the c-ring of Fo. In bacteria and chloroplasts, the ε's C-terminal domain is thought to function as a mobile regulatory element that can change conformation in response to nucleotide conditions or PFM, or both. It was shown to be required for the functional binding of F1 and FO, and therefore involved in the energetic coupling of the F1FO enzyme. Responsible for inhibition of ATPase activity of both the F1-portion and the whole F1FO-ATP synthase [24] .

Ana Filipa Santos Seiça

The εCTD has three regions of contact with other F1 subunits. in region 1, ε's loop1 and helix1 contact only γ; εloop1 forms a salt bridge with the C-terminal α-helix of γ (γCTH), and εhelix1 packs mainly against γ's Rossmann fold. In region 2 εloop2 and εhelix 2 contact five other chains (α1, α2, β1, β3, γ), with εhelix2 inserted into the central rotor cavity. εhelix2 and γNTH form an antiparallel coiled-coil, with substantial burial of hydrophobic residues, that is also stabilized by hydrogen bonds at both ends and by electrostatic contacts [17] .

Previous work on the ε subunit has identified two different conformations during catalysis [25] [26] . Trypsin cleavage has shown that these conformational changes in ε are ligand-dependent. Because ε subunit together with γ is involved in binding of F1 to FO, these conformational rearrangements are probably coupled between events at catalytic sites and proton translocation. The ε subunit interacts with the β subunit in the hydrolysis inactive state while in the active state, induced by ATP binding, ε subunit interacts with the c subunit [27] .

The two conformations identified are contracted (εC) (Figure 2B) and extended (εX) (Figure 2A). The γ-ε structure is in εX conformation where the two helices are extended and entwined in the coiled-coil structure of the γ subunit to interact with the α3β3 hexamer.

In the other structure, εC conformation, the two helices of ε are folded together and adjacent to the β-sandwich and are present on the c-subunit ring [17] .

The transition of subunit εC-terminus from contracted hairpin conformation to the extended one is responsible for the inhibition of ATP hydrolysis. It was shown by crosslinking that in the presence of ATP subunit ε adopts the contracted conformation, while ADP promotes an extended one [27] .

The two conformations of subunit ε might have different physiological roles, the extended conformers being responsible for ATP synthesis and the contracted for the maintenance of the ΔμH + at the expense of ATP hydrolysis. However, it is not determined whether direct binding of ATP to subunit ε or whether the overall conformation of the F1 induced by nucleotide binding to catalytic sites plays a critical role in the C-terminus transitions [28] .

CHAPTER III Cyanide label of proteins from the respiratory chain

___________________________________________________________________________

The peripheral arm encloses the subunits NuoB, CD, E, F, G, and I, contains all the prosthetic groups, including one flavin (FMN) at the NADH binding site, and seven to eight Fe-S clusters [35] . Oxidation of NADH to NAD + occurs at the flavin binding site at the distal end of the peripheral arm, and the reduction of ubiquinone occurs at the ubiquinone binding site located 20-25 Å above membrane surface [36][37] [38] .

Complex I reaction starts with NADH. The primary electron acceptor of the complex, is located in a pocket formed by the subunits NuoEF and contains the first Flavin mononucleotide (FMN). NADH give the FMN two electrons in a form of a hybrid. The two electrons are transferred through the seven Fe/S-clusters to the ubiquinone-binding site, which is located at the interface of the subunits NuoBCDH. The quinones are reduced by the cluster chain N2. The Fe-S clusters only transfers one electron at a time.

Conformational changes are transmitted to the membrane arm over a distance of approximately 180 Å due to the reduction of N2 and ubiquinone [40] .

The hydrophobic arm from E.coli has been visualized at low resolution by cryoelectron microscopy [41] and higher resolution by X-ray diffraction [42][43] allowing identification and determination of the subunits in the membrane arm. The membrane arm has a beanshaped and is about 180 Å long and comprises the subunits Nuo L, M, and N, which are involved in proton translocation and are homologous to transporter subunits [44] . Each of the subunits contain 14 transmembranous (TM) helices [45] . An 11-helix bundle of subunits NuoA/J/K separates the peripheral arm from the three membrane antiporter-like subunits NuoN/M/L [41] .

The NuoL subunit is located at the distal end of the membrane arm in relation to the peripheral arm. It contains a unique long amphipathic helix ~ 110 Å, parallel to the membrane [44] . The horizontal helix between TM15 and TM16 of subunit L was first revealed with crystallographic analysis by Efremov et al. in 2010 [42] . The amphipathic helix, termed helix HL, links the antiporter-like subunits and also can act as a putative coupling element [44] .

It has been hypothesized that long-range conformational changes triggered by redox energy, drives proton translocation through these antiporter-like subunits. Studies on the role of the amphipathic helix have been performed [46] [47] , investigating if the electron transfer reaction induces a piston-like movement of the horizontal helix, leading to an CHAPTER III Cyanide label of proteins from the respiratory chain ___________________________________________________________________________ 76 Ana Filipa Santos Seiça opening and closing of proton translocation channels in the antiporter subunits of NuoL, M and N. However, how the conformational changes are transmitted into the antiporter domains in order to activate the proton pumps is still under debate. Zhu et al proposed that the horizontal helix might transmit conformational changes by opening the quinone binding site [START_REF] Zimmermann | Movements of the ɛ-subunit during catalysis and activation in single membrane-bound H+-ATP synthase[END_REF] .

In this study respiratory complex I, was labeled with SCN residues and the changes of the probe by addition of NADH was studied. Mutants used for the labeling have been suggested based on the structural studies previously reported [49] .

-MATERIAL AND METHOD

-Sample Preparation and Experimental Conditions of F1-ATPASE

-Construction of Mutants of F1-ATPase Synthase from Escherichia Coli

For the overexpression of H + -ATP synthase from E. coli (EF1FO) the strain RA1 [50] was used. All plasmids are based on the pRA100 plasmid [START_REF] Aggeler | Introduction of reactive cysteine residues in the epsilon subunit of Escherichia coli F1 ATPase, modification of these sites with (azidotetrafluorophenyl) maleimides, and examination of changes in the binding of the epsilon subunit when different nucleotides are in catalytic sites[END_REF] which includes the unc operon.

The plasmid pMb3.1 derived from pRAP100 (F1FO-εH56C) [START_REF] Zimmermann | Movements of the ɛ-subunit during catalysis and activation in single membrane-bound H+-ATP synthase[END_REF] and harbors an εH56C substitution, a N-terminal His6-tag at β and the substitution of the wildtype cysteines in δ by alanine's. In plasmid pRAP101 the tyrosine at position 114 of the ε subunit was replaced by a cysteine. Isolation of EF1 from E.coli cells were performed according to established protocols [52] [53] . The mutants were provided by Dr. Heitkamp from the University of Jena, Germany

-Determination of Labeling Efficiency

-Fluorescence Labeling of F1-εH56C and εY114C

Fluorescence labeling of the genetically introduced cysteine in the ε-subunit was performed according to the protocols described in Heitkamp et al [START_REF] Heitkamp | Single Molecule Spectroscopy and Superresolution Imaging[END_REF] . Briefly, the maleimide group of the fluorophore reacts specifically with the sulfhydryl group of the cysteine at pH between 6.5 and 7.5, resulting in an irreversible formation of a stable thioether bond (Figure 4). The cysteine in the ε subunit was labeled with ATTO 594 maleimide (ATTO-TEC, Germany). Labeling was carried out on ice in a total volume of 100 μl using a protein concentration of 14 μM. A fivefold molar excess of Tris (2-carboxyethyl) phosphine (TCEP) to F1 was used to convert the cysteines to the reactive reduced form in a ratio of F1 to dye 1:1. The reaction was stopped after 4 minutes by three consecutive self-packed Sephadex G50 medium columns. The labeling efficiency was determined by absorption spectroscopy and the specifity was checked by SDS-PAGE with subsequent fluorography.

Ana Filipa Santos Seiça

-Cyanide Labeling of F1-εH56C and εY114C

The strategy for converting cysteine thiols into thiocyanates (Figure 5), is routinely employed as the first step in the selective cleavage of peptide bonds at cysteine residues.

The protein reacts with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB or Ellman's reagent) to form the mixed protein-thionitrobenzoic acid disulfide (PS-TNB), followed by displacement by cyanide (CN -), to form the protein-thiocyanate (PS-CN) [START_REF] Fafarman | Site-Specific Conversion of Cysteine Thiols into Thiocyanate Creates an IR Probe for Electric Field in Proteins[END_REF] .

Ana Filipa Santos Seiça Figure 5: Cysteine cyanylation scheme [START_REF] Fafarman | Site-Specific Conversion of Cysteine Thiols into Thiocyanate Creates an IR Probe for Electric Field in Proteins[END_REF] .

A protein solution with a concentration of 5.21 µM was incubated at 4°C with dithiothreitol (DTT) for 30 min, followed by removal of the excess of DTT with two consecutive Sephadex G50 medium columns [START_REF] Heitkamp | Single Molecule Spectroscopy and Superresolution Imaging[END_REF] . After the protein was incubated at 4°C with a forty fourfold molar excess of DTNB during 1hour and the excess of DTNB was removed with two Sephadex G50 medium columns. Finally 6mM potassium cyanide (KCN) was added to the protein and incubated for 2h at 4°C. The labeling efficiency of KCN was determined by UV-Visible spectroscopy by following the absorption band of TNB (412 nm) release after addition of potassium cyanide.

-Measurement of ATP Hydrolysis of F1-εY114C

The measurement of the ATP hydrolysis was carried out according to Fischer's one-step method [START_REF] Fischer | Comparison of ΔpH-and Δφ-driven ATP synthesis catalyzed by the H+-ATPases from Escherichia coli or chloroplasts reconstituted into liposomes[END_REF] (Figure 6). The continuous measurement of ATP hydrolysis was carried out in a coupled enzymatic assay in a spectrophotometer at 21°C. Briefly, 2.5 to 10 nM of the purified protein was added to 1 ml of the reaction buffer (100 mM Tris-HCl pH 8.0, 25 mM KCl, 4 mM MgCl2, 2.5 mM phosphoenolpyruvate, 18 units pyruvate kinase, 16 units lactate dehydrogenase, 2 mM ATP and 0.4 mM NADH). ATP hydrolysis was detected by the decrease of nicotinamide de adenine dinucleotide (NADH) absorbance.

The reaction buffer was pre-heated to 37°C, placed in the spectrophotometer and the baseline was recorded at 340 nm. After adding the solubilized F1-ATP synthase, a decrease of absorbance was recorded. The rate of ATP hydrolysis was calculated from the linear slope using an extinction coefficient of 6,220 L•mol -1 •cm -1 for NADH. The Ana Filipa Santos Seiça activation of F1-ATP synthase was proven by adding 20 μl of a 30 % (v/v) N,N-dimethyln-dodecylamine N-oxide (LDAO) solution to the assay mixture, corresponding to a final concentration of 0.6 % (Table 1 and2). [START_REF] Fischer | Comparison of ΔpH-and Δφ-driven ATP synthesis catalyzed by the H+-ATPases from Escherichia coli or chloroplasts reconstituted into liposomes[END_REF] .

Table 1: ATP hydrolysis of F1-εY114C, before and after label with the fluorescence probe ATTO 594 in presence or absence of ATP at 37°C. The concentration of F1 during the measurement was 3, 10 and 20nM (results provided by Dr. Heiktamp from Jena University). Ana Filipa Santos Seiça Table 2: Activation factor calculated from the ratio of ATP hydrolysis with and without 0.03% (v / v) LDAO (results provided by Dr. Heiktamp from Jena University).

-UV / VIS Spectroscopy

The concentrations of the F1 solution, fluorophore solutions, fluorescent and infrared labeled proteins were determined by absorption spectroscopy (Lambda 2, Perkin Elmer). The measurements in the ultra-violet (UV) were carried out in a quartz cuvette (Hellma QS 1,000, layer thickness 1 cm, minimum volume 75 μl). For measurements in the visible range, plastic cuvettes (Plastibrand 1.5 ml semi-micro, layer thickness 1 cm, minimum volume of 800 μl) were used. The solution of F1 was obtained after washing with a solution of ammonium sulfate (NH4)2SO4 saturated.

-SAMPLE PREPARATION AND EXPERIMENTAL CONDITIONS OF COMPLEX I

-Construction of Mutants of NADH: Ubiquinone Oxidoreductase from

Escherichia Coli

Complex I wild type and mutants were produced by Pr. Dr. T. Friedrich's group at the Albert-Ludwigs University Freiburg. Complex I was prepared as previously described in Steimle et al. [47] . The cysteine mutations at positions K551 L and Y590 L Ana Filipa Santos Seiça from the strains described in [49] . Samples were purified and stored in 50 mM MES/NaOH, 50 mM NaCl, 5 mM MgCl2 and 0.05% MNG-3, pH 6.0.

The E. coli cells Strain BW25113Δnuo [START_REF] Datsenko | One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products[END_REF] (derivative of E. coli strain BD792) were transformed with pBADnuo/nuoFhis. The expression of the nuo-operon was induced with 0.2% w/v L-arabinose after 1 h. Cells were harvested until they entered the stationary growth phase by continuous flow centrifugation at 17000 g at 4 °C, shock frozen in liquid nitrogen and stored at -80 °C.

-Cyanide Labeling of Complex I

The same labeling protocol used for labeling the residues of F1-ATPase was applied to the residues of Complex I: The mutants with a concentration of 10 M were incubated with DTT to reduce the cysteines follow by addition of DTNB and KCN.

-UV/VIS Spectroscopy of Complex I

To quantify the concentration of the Complex I and to determine the label efficiency of the variants of Complex I, an UV-Vis spectrum of the sample diluted in buffer (MES 50 mM, NaCl 50 mM, 5 mM MgCl2, 0.05% MNG-3) were recorded in a 1 cm -1 path length quartz cuvette via a Diode-Array UV-Vis Spectrophotometer (TIDAS II, J&M). The spectrum of the buffer was previously recorded and subtracted from the sample spectrum.

-INFRARED SPECTROSCOPY

A configuration allowing the acquisition of Fourier transform infrared (FTIR) spectra in the ATR mode with was employed, including a silicon crystal with a 3-mm surface diameter as a single reflection ATR unit. IR spectra have been measured on a Bruker Vertex 70 FTIR spectrometer (Globar source, KBr Beamsplitter, mercury cadmium telluride detector) with, 40-kHz scanner velocity, 256 scans and 4 cm -1 resolution.

Typically 12 spectra have been averaged.

A solution of 30mM KSCN was prepared in the following solvents, respectively: DMSO, THF, ethanol, methanol and in MES buffer pH 6.0. The spectra were normalized to the same intensity.

CHAPTER III Cyanide label of proteins from the respiratory chain ___________________________________________________________________________

-Surface-Enhanced Infrared Absorption Spectroscopy

The residues of F1-ATPase before and after labeling were incubated with 1mM ATP for 2 hours on ice at 4°C before immobilization of the protein in a self-assembled monolayer.

Initially, a gold layer was deposited on a 3-mm silicon crystal (as described in Chapter II) [START_REF] Ataka | Use of surface enhanced infrared absorption spectroscopy (SEIRA) to probe the functionality of a protein monolayer[END_REF] . Once the gold surface was formed the spectrum of the prism was recorded as background. Then a 1:1 mixture of cysteamine (CEA) and 6-mercaptohexanol (6-MCH) was added to a final concentration of 2mM. The crystal was rinsed to remove the excess of thiols and dried under a stream of argon. The monolayer was recorded as background and 3μl of a final concentration of F1-ATPase residues label was added to the surface and the spectra were recorded for 1 hour. The same experiment was repeated 2 times for reproducibility. A baseline correction was performed.

Complex I wild type and variants were immobilized in a Ni-NTA surface [START_REF] Kriegel | Biomimetic environment to study E. coli complex I through surface-enhanced IR absorption spectroscopy[END_REF][60] [START_REF] Grytsyk | Arg302 governs the pKa of Glu325 in LacY[END_REF] as described in Chapter II. The absorbance spectrum of the mutants label incubated with 50μM NADH was also recorded. A baseline correction was performed.

-RESULTS AND DISCUSSION

-Cyanide Labelling -Cyanide Label of F1-ATPase Residues

The thiocyanate moiety is produced by chemical modification of the free thiol of cysteine, and can thus be inserted at any cysteine site resulting from mutagenesis. The effect of the environment on the vibrational mode of potassium thiocyanate (KSCN), including hydrogen bonding and polarizability in different solvents is presented in Figure 7.

The frequency mode of the thiocyanate was shown to be blue shifted in the presence of water, in comparison with those in aprotic solvents (Figure 7). The lone pair electrons of the terminal N-atom can form a single hydrogen bond with a water molecule, which in turn makes the CN triple bond length short and the CN stretch force constant increase, resulting in a blue shift [START_REF] Van Wilderen | Vibrational dynamics and solvatochromism of the label SCN in various solvents and hemoglobin by time dependent IR and 2D-IR spectroscopy[END_REF] .

The study in different solvents helps to evaluate the effect of a more or less hydrophilic environment on the probe, when comparing for example two states of a protein during a reaction cycle [START_REF] Van Wilderen | Vibrational dynamics and solvatochromism of the label SCN in various solvents and hemoglobin by time dependent IR and 2D-IR spectroscopy[END_REF] . An IR spectrum of the CN stretching frequency contains the balance of

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Ana Filipa Santos Seiça

The Figure 11.A and 11.C shows the deconvoluted spectra for εH56C and εY114C in absence of the nucleotide. The bands at 1634-1638 cm -1 and 1677-1682 cm -1 were assigned to β-sheets [START_REF] Yang | Obtaining information about protein secondary structures in aqueous solution using Fourier transform IR spectroscopy[END_REF] . In particular, the presence of a strong peak at 1634-8 cm -1 in conjunction with a weak one at 1677-82 cm -1 is indicative of an anti-parallel β-sheet conformation [START_REF] Yang | Obtaining information about protein secondary structures in aqueous solution using Fourier transform IR spectroscopy[END_REF] . The band at 1624 cm -1 corresponds to β-structures, generally attributed to the formation of intermolecular hydrogen bonds, finally the band at 1667 cm -1 can be assigned to turns [START_REF] Barth | Infrared spectroscopy of proteins[END_REF] . The results obtained are in agreement with the crystal structure of F1-ATPase with a 2.8 Ȧ resolution [16] .

To determine whether the binding of different ligands promotes structural changes in the F1 moiety, ATP was added to the complex. Small changes in the amide I band (Figure 11.B and 11.D) were seen. Addition of ATP to F1 induced local conformational changes originating a decrease in the βstructure content with a parallel formation of reverse turns and α-helical structures [START_REF] Sala | Effect of neutral and acidic phospholipids on mitochondrial ATP synthase secondary structure[END_REF] .

Control experiments were done to the residues labeled and the spectra of the secondary structure was studied in which showed that the use of the infrared probes did not affected the structure of the protein (Figure S4 (III) -Appendix IV).

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Cyanide label of proteins from the respiratory chain ___________________________________________________________________________ 92 Ana Filipa Santos Seiça

-Labeled Cysteines

The introduction of nitrile probes at variants εH56C and εY114C led to a complex spectral signature pointing to the presence of several cysteines. The residue F1 εH56C contain 15 cysteine residues (4 in each α subunit, 2 γ subunit, and the one introduced by site mutagenesis in ε subunit). The two cysteines present in δ subunit and the one cysteine in β subunit in the wild type protein, were substituted by alanines and the residue F1-εY114C has all the native cysteines residues. In the data seen in Figure 13 and Figure 14, signals at 2139, 2129, 2115 cm -1 can be distinguished that involve at least three SCN groups, however, is difficult to reassigned this three SCN groups to a specific cysteine label.

-Interpretation of the Observed Shifts of the Labeled Cysteines

Previous studies on the influence of the microenvironment on the signature of the SC≡N stretching frequency in proteins, showed typical signals at about 2160 cm -1 for example for labeled Ribonuclease S [START_REF] Dalton | Cyanylated Cysteine Reports Site-Specific Changes at Protein-Protein-Binding Interfaces Without Perturbation[END_REF] and the protein of the calmodulin [START_REF] Bagchi | Ribonuclease S Dynamics Measured Using a Nitrile Label with 2D IR Vibrational Echo Spectroscopy[END_REF] . The relative effect of the environment, including factors like hydrogen bonding and polarizability, can be seen from the data obtained for KSCN in different solvents (Figure 7).

When bound to the cysteine residue the SCN vibrational mode upshifts in direct comparison to KSCN in different solvents by up to 100 cm -1 . Here we observe the shifts of 1-5 cm -1 for the two variants of F1 ATPase, which revealed a complex signature, perturbed in the absence and presence of ATP respectively.

The residue H56C located in a β-sheet in the loop connecting the sixth and seventh strands of the N-terminal domain was suggested to be non-interacting and completely exposed residue, and to be far from the γ subunit (Figure 12). Accordingly, to Zimmermann et al [START_REF] Zimmermann | Movements of the subunit during catalysis and activation in single membrane-bound H + -ATP synthase[END_REF] , there is no evidence for an extended or contracted conformation of epsilon subunit in the ε56-region relative to the b subunit, when the protein switches between ATP synthesis and ATP hydrolysis. However, it is not possible to exclude that switching between both modes of catalysis, might be connected with the movement of other parts of the epsilon subunit.

CHAPTER III Cyanide label of proteins from the respiratory chain

___________________________________________________________________________

The residue εTyr114, found close to loop 2 interacts with multiple subunits (α, β, and γ) and is involved in F1 inhibition. Futai et al [73] predicted that substituting the amino acid by alanine and examined the effect on F1-ATPase activity, the inhibition of εY114A mutant decreased to 41±3.1%, suggesting that this residue in helix 2 is involved in the ε subunit function.

This residue is located near the amino acid G85 of γ subunit, the hydroxyl group of tyrosine could form a hydrogen bond with the amide group of the main chain of glycine (Figure 12). The interaction between the two amino acids εTyr114 and γGly85 are possibly important for holding the ε subunit in a certain conformation for F1 inhibition [73] , which led us to conclude that by substituting the amino acid tyrosine for a cysteine, the interaction with γG85 is prevented and the ε subunit is found in a different conformation.

In absence of ATP the CTD of ε subunit is elongated and reaches in the α3β3γ interface interacting with other subunits, possibly with α and β, and inhibiting ATPase activity.

Through these interactions, the ε subunit is maintained in an extended conformation, and thereby interferes with smooth rotation of the γ subunit. Once ATP is added to the protein subunit ε adopts the contracted conformation, and epsilon is no longer close to the stalk part of gama or to the α3β3γ interface.

Finally, the use of a small infrared probe allowed us to label the residues in NTD and CTD of the epsilon subunit with a higher labeling efficiency as compared with big fluorescent probes without perturbing the secondary structure.

-Cyanide Label of Complex I Residues

-Deconvolutionof the amide I band of Complex I Residues After Labelling

A stable monolayer with Complex I immobilized through a C-terminal His-tag to a modified gold layer was obtained as described for the labeled samples [START_REF] Kriegel | Biomimetic environment to study E. coli complex I through surface-enhanced IR absorption spectroscopy[END_REF] . Figure 15 shows the absorbance spectrum of nuoL K551C after reacting with DTNB and KCN to form the protein-thiocyanate. The spectrum show characteristically signals for amide I, amide II and the thiocyanate bond.

The amide I signal of the protein was used to confirm the structural integrity of the complex after labelling and immobilization. Deconvolution was performed after Ana Filipa Santos Seiça calculation of the fourth derivative to estimate the number and position of the components of amide I band, followed by deconvolution using the Gaussian profile of each component. Figure 16 shows the spectrum of Complex I wild type and mutants after labeling as well as each of the decomposed bands. The deconvolution served as a control of the structural integrity of the complex after labeling and immobilization.

The amide I of complex I from E.coli appears at 1653 cm -1 , indicating a predominantly α-helical structure. The deconvolution indicate a 46% α-helix, 34% βsheet and random coils and 24% β-turns, being in agreement with the crystal structure of complex I [START_REF] Baradaran | Crystal structure of the entire respiratory complex I[END_REF] .

The results obtained in Figure 16 show that the amide I band of all samples studied was highly comparable, confirming that there are no detectable differences in the conformation of wild type and mutants, the predominately alpha-helical contribution is maintained. The labeling protocol did not affect the integrety of the protein. The placement of nitrile labels to cysteine residues of E. coli respiratory complex I allowed to monitor minor movements of the amphipathic horizontal helix of NuoL in the membrane arm upon reduction by NADH. Previous EPR and fluorescence spectroscopy studies by Steimle et al [49] pointed to a nearly imperceptible movement of the helix upon reduction by NADH.

Even if the studied residues are pointing away from the protein in the oxidized state, the reduction may induce a change in position, which would be hindered by a too large label. So in contrast to these studies, the nitrile label shows a higher sensitivity to the conformational changes due to the smaller size of the label.

When subtracting the spectra before and after addition of NADH differences of 15 to 18 cm -1 for the signals of the label visualize experimentally a reorganization of distinct positions within the helix towards a more hydrophobic environment.

The significance of the shifts is derived from comparison to similar experiments on other proteins. Indeed, shifts below 5 cm -1 have been found to be relevant [START_REF] Tucker | A New Method for Determining the Local Environment and Orientation of Individual Side Chains of Membrane-Binding Peptides[END_REF] . Tucker et al studied the membrane-binding amphipathic peptide, mastoparan x (MPx peptide) by infrared spectroscopy using a PheCN probe. The peptide MPx-CNy in water showed a CN stretching vibration at 2235 cm -1 . When bound to POPC phospholipid bilayers, the CN stretching vibration shifted by 6 cm -1 to 2229 cm -1 indicating a more hydrophobic and less solvent accessible environment for the CN probe [START_REF] Tucker | A New Method for Determining the Local Environment and Orientation of Individual Side Chains of Membrane-Binding Peptides[END_REF] . A change of the hydrogen bonding environment, or the presence or absence of water molecules after addition NADH may also influenced the spectral signature. So the shifts observed here correspond to a movement towards a significantly more hydrophobic environment or an environment with weaker hydrogen bonds upon NADH addition. It was proposed that the enzyme cycles between an "open" conformation (NADH-reduced state) and a "closed" conformation (oxidized state) The three antiporterlike subunits (NuoL, M, and N) are involved in proton translocation most likely driven by a re-arrangement of the hydrophilic loops and the position of TM helices within the membrane [START_REF] Mamedova | Substrate-induced Conformational Change in Bacterial Complex I[END_REF]. Mamedova et al predicted that upon NADH binding, the peripheral arm of the complex adopts a more open conformation, with increased distances between subunits, and single-particle analysis showed that the membrane domain also expands [START_REF] Mamedova | Substrate-induced Conformational Change in Bacterial Complex I[END_REF] .

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Cyanide label of proteins from the respiratory chain ___________________________________________________________________________ 99

Ana Filipa Santos Seiça

The residues studied in the membrane arm are clearly sensitive to the added NADH, however, this does not mean that the rearrangement of the helix is of functional significance, the movement of the positions on the amphipathic helix is an indirect effect that results from a structural compensation of the movement of the membrane arm during reduction [77] , that would be in line with the conformational change suggested above to occur after NADH addition.

These results are in agreement with the data obtained by electron paramagnetic resonance (EPR) where the same residues were labeled using a fluorescent probe to study the mutants in the oxidized and reduced states. Slight conformational changes at distinct positions of the helix were possible to observe with fluorescent probes [49] . LacYww/D240A (orange), LacYww/K319L (cyan), and LacYww/H322Q (gray).......144 Table 1:

Ana Filipa Santos Seiça

List of Figures

List of Table

-MEMBRANE TRANSPORT PROTEINS

In this Chapter SEIRAS approach was combined with a perfusion cell and used in order to study the reaction mechanism of transport proteins Staphylococcus epidermidis (GlcPSe) and E.coli Lactose Permease (LacY)

With this approach, we address the function of the protein, and the role of individual residues that are crucial for a high pKa, more specifically residues responsible for the coupling mechanism of symport have been monitored.

-MECHANISM OF THE TRANSPORTERS

-Carrier or Channel Proteins

There are two general classes of transport proteins: carriers or channels (Figure 1). Carriers (also named transporters or permeases) are polyprotic transmembrane proteins that function as topological enzymes, with the ability to cycle between conformational changes. They catalyze the translocation of substrates from one side of the membrane to the other [1] . A solute binding site is accessible on either side of the membrane, depending on the respective conformation of the membrane protein. A possible intermediate will also exist, in which the bound solute is inaccessible to both. A carrier protein transport either a single or a defined number of solute molecules per conformational cycle and it does not contain a continuous open-pore accessible from both sides of a membrane [2] .

Channel proteins are intrinsic proteins, spanning over the entire cell membrane. One side of the protein is exposed to the extracellular fluid while the other side is exposed to the cytosol. The two exposing domains of the protein are hydrophilic. The hydrophobic channel is embedded in the lipid bilayer [3] .

The control of the channel gating, between the open or closed conformation, can be regulated by: voltage, binding of a ligand regulatory molecule, or by mechanical means such as membrane stretch (i.e. under stress) [4] .

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Perfusion-SEIRAS approach for the study of membrane transport proteins ___________________________________________________________________________ 117 Ana Filipa Santos Seiça

-Uniport

Carrier proteins that mediate transport of a single solute are classified as a uniporter. The uniporter carrier protein allows transport of the non-diffusible solute across the membrane barrier at a rate that is much higher than passive diffusion in which the solute molecule is never in contact with the hydrophobic core of the membrane [7] .

These transport proteins facilitate the mode of diffusion by accelerating a reaction that is already thermodynamically favored, providing a carrier-mediated solute transport down the concentration gradient.

An example is the GLUT1 glucose carrier (Figure 2), found within the plasma membrane of various mammalian cells of blood glucose as the major source of cellular energy.

GLUT1 is a large, 12 α-helical membrane protein that alternates between two conformational states: a glucose-binding site that faces the outside of the membrane and a glucose-binding site facing the inside, permitting the facilitated diffusion of a single glucose molecule per cycle into the cytosol of the cell [8] .

Figure 2: Mode of transport for the uniporter transport protein GLUT1 [9] 4.2 -MAJOR FACILITATOR SUPERFAMILY OF TRANSPORTERS A large number of transport proteins mediate the exchange of ions, sugars, lipids, amino acids, peptides and also play a role in signal transduction, energy conversion, catalysis, mechanosensing, and structural integrity. The cellular uptake of monosaccharides, polyols, and other small carbon compounds across the membranes of eukaryotic cells is CHAPTER IV Perfusion-SEIRAS approach for the study of membrane transport proteins ___________________________________________________________________________ an essential physiological process. The transport is mediated by specialized transporters that are members of the GLUT family (SLC2 gene family), which belong to the major facilitator superfamily (MFS) [10] .

MFS includes over 10 000 sequenced members of membrane transporters, in which 76 subfamilies are distributed in bacteria, archaea, and eukaryote, yielding the largest known superfamily of secondary transporters. These membrane transporters have the capability to catalyze uniport, symport and/or antiport transport [11] .

The members of the MFS are typically comprised of 12 transmembrane (TM) helices, where the N and C termini are located on the cytoplasmic side of the bilayer and transmembrane segments 1, 4, 7 and 10 are positioned in the center of the transporter and contribute to the majority of residues that are important for substrate transfer and proton coupling. The other TM segments participate in substrate binding and proton coupling as well as structural integrity [12] .

-Protein transporter GlcPSe from Staphylococcus Epidermidis

Glucose transporters (GLUTs) are implicated in several diseases including cancer [12][13] [14] and diabetes [15] . In plants, monosaccharide and sucrose transporters play a fundamental role in stress responses and developmental processes including seed germination and balanced growth [16][17] . Yeast glucose transporters are important for glucose uptake and metabolism during sugar fermentation and alcohol production [17] .

Staphylococcus epidermidis (GlcPSe) is a common constituent of the healthy human skin and mucosal surfaces, with a low pathogenic potential. The crystal structure of the Staphylococcus epidermidis glucose/H + symporter, a bacterial homolog of GLUTs, was in the inward-facing conformation [18] (Figure 3A). GlcPSe shares high sequence identity (27-34%) and homology (49-58%) with the human GLUTs. It is highly specific for glucose, and it is inhibited by the well-characterized inhibitors of human GLUTs phloretin, cytochalasin B, and forskolin [18] .

Most glucose transporters have an uncharged amino-acid, alanine or asparagine, in the H + binding site, which obviously makes them unable to perform H + coupled transport, due to the missing H + binding site, however there are exceptions: Glut 2, 10, 12 and 13 Ana Filipa Santos Seiça

-GLCPSe

The same protocol to monitor pH-induced difference spectra as described above was used for GlcPSe. Firstly, the system was equilibrated with 50 mM KPi, 200 mM NaCl, 0.05% (w/v) DDM) at a pH 5.5 for 20 min, then a spectrum of the protein was recorded as background and the perfusion solution was changed to the second solution (pH range 7.5-10.5). After 20 min, (pH 7.5-10.5) minus pH 5.5 difference spectra were recorded.

The spectrum of the protein in a new state was recorded as background, and the solution was changed to pH 5.5. Again after 20 min, the pH 5.5 minus (pH 7.5-10.5) difference spectra were obtained. The same experiments that have been performed in the absence of glucose were performed in the presence of glucose. Glucose was dissolved in 50mM KPi, 200 mM NaCl, 0.05% (w/v) DDM (pH 5.5) to a final concentration of 100 μM. Firstly, the residue Asp22 (D22) of the protein GlcPSe was identified. Figure 6 shows the reversible perfusion-induced difference spectra obtained by subtracting data from samples equilibrated at pH 5.5 from samples obtained at a pH that completely or almost completely deprotonates Asp22 (pH 8.5 and 9.5), in the absence of glucose of GlcPSe WT and for the mutant Ala22 (D22A). Difference spectra were measured in order to obtain information on protonation/deprotonation reactions and coupled conformational changes.

The red line in Figure 6 shows the inverse reaction and thus the high degree of reversibility of the reaction. In the spectrum is possible to observe the COOH vibrational mode of protonated acidic residues typically found at signals around 1750 cm -1 [34][35] . The signal can be seen for the wild type (Figure 6a) while for the mutant D22A (Figure 6b) there is no contribution of the band.

CHAPTER IV

Perfusion-SEIRAS approach for the study of membrane transport proteins ___________________________________________________________________________ 126 Ana Filipa Santos Seiça The spectra of the mutant D22A and WT (Figure 7), is characterized by signals in the socalled amide I region between 1690 and 1620 cm -1 that involve backbone contributions, as well as contributions from individual side chains [37] . The position of the amide I backbone signal is specific for the type of secondary structure, and signals at ~1650 cm -1 correspond to α-helices and disordered structures observed at ~1642 cm -1 [38] .

The bands observed at ~1636 and 1670 -1690 cm -1 are typically assigned to β-sheets.

However, this type of difference spectra may also shift due to a change in the environment of only a part of the peptide backbone. The so-called "doorway" modes have been CHAPTER IV Perfusion-SEIRAS approach for the study of membrane transport proteins ___________________________________________________________________________ 127 Ana Filipa Santos Seiça described in KcsA K + channels [39] where α-helices, which reorganize during ion transport, show shifts to positions usually assigned to β-sheet structures.

In order to probe the amide region, H/D exchange was performed (Figure S7 (IV) -Appendix VII). In the amide II region near 1570 cm -1 , where the contribution from the protein backbone includes the in-plane N-H bending (40-60%) coupled to the ν(C-N) (20-40%) vibrational mode, characteristic signals are seen around 1509 cm -1 [37] . Upon H/D exchange Figure S7 (IV). the amide II band intensity decreases, the in-plane N-H (N-D) bending mode uncouples and appears in the 940 -1040 cm -1 region, and the ν(C-N) moves to 1488 and 1461 cm -1 , mixing with other modes to form a new band called amide II*.

The changes in the spectra upon H/D exchange, confirm that most of the signals in the difference spectra at positions lower than 1700 cm -1 originate from the protein backbone.

Figure 7 A shows the negative signal at 1750 cm -1 which starts to appear at pH 7.5 and increases in intensity with the increase of pH, pointing towards the deprotonation of the amino acid D22. Signals from deprotonated acid residues are expected between 1580 and 1520 cm -1 for the v(COO -)as and between 1480 and 1320 cm -1 for v(COO -)s.

It is also possible to observe that a change in the environment happens from pH 7.5 to 8.5. The signal at 1650 cm -1 present at pH 7.5, is shifted to higher wavenumbers and with increasing pH the shift becomes more sharper pointing towards a conformational change related to the increase of pH.

-Deconvolution of Amide I band of GLcPSe Asp22 and Ala22

The deconvolution of the amide I band of the absorbed protein (Figure 8) immobilized via its His-Tag located on the C-terminal domain of the protein on a silicon crystal, covered with a gold nanostructure, give us information about the integrity of the protein after immobilization or mutation.

CHAPTER IV

Perfusion-SEIRAS approach for the study of membrane transport proteins ___________________________________________________________________________ 128 Ana Filipa Santos Seiça The amide I band is sensitive to protein secondary structure [38] . The frequency centered at 1654 cm -1 is consistent with the presence of α-helices and disordered structures. After deconvolution of the amide I band we observe 5 bands, 1691, 1680, 1629 and 1613 cm -1 suggesting the presence of parallel β-sheets and the band component at 1691 cm -1 assigned to β-turns and amino acid side chains [38] .

The spectrum of the mutant (Figure 8B), after deconvolution of amide I band reveals to have the same profile as the WT, confirming that the mutation of Asp to Ala did not affected the integrity of the protein.

The spectrum of the glucose transporter gives evidence of a predominant α-helical structure with 70%. Part of the α-helical structure of the protein is buried in a hydrophobic portion of the membrane, but mostly the structure is freely accessible to the solvent. The structural integrity of the protein after immobilization remains stable during the experiments. Ana Filipa Santos Seiça These results are in accordance with the results obtained for the structure of lactose permease, a protein of similar molecular weight and related function to the glucose transporter [25] .

The orientation of the amide I mode was studied with polarized IR light after immobilization. The results showed that at pH 7.5 the protein is oriented perpendicular to the ATR crystal and at pH values higher and lower the protein shows some inclination, most likely due to the charges on the surface of the protein (Table 1).

Table 1: IR dichroic values of GlcPSe WT prepared in 50mM KPi/0.05% DDM pH 7.5, 8.5 and 5.5 and pD 7.5 prepared in 50mM KPi/0.05% DDM adsorbed on the Ni-NTA modified gold surface.

-Effect of Glucose on the Residue Asp22

Figure 9 show the difference spectra of WT in presence of glucose. In all spectra, the step to pH 8.5 and 9.5 are very similar, whereas a major difference can be found for the step between pH 8.5 and 7.5. The presence of glucose does not seem to influence the signal at 1750 cm-1, however major structural changes occur in the amide I and amide II regions indicating differences in the pH-dependent conformational reorganization of the protein in the presence of glucose. The result obtained for D22 in presence of glucose is very different from the LacY data [25] where the presence of sugar did not influenced the conformational changes pointing to a different mechanism of sugar transport.

GlcPSe

Translocation of sugar after binding is normally affected by a conformational change between the inward and outward-facing conformation. However, how the transport of sugar leads to the conformational change that occurs during the process of transport between the outward-and the inward-facing conformations is still unclear. Once confirmed that the residue D22 is responsible for the band at 1750 cm -1 , the pKa of D22 in wild type in presence and absence of glucose was determined by plotting the difference in signal intensity at 1750 cm -1 versus pH (Figure 10).

From the ΔIR fit observed (red and black correspond to the WT, with and without sugar respectively), D22 has a pKa of 8.5 ± 0.1, a value that agrees well with the pKa obtained previously by Bazzone et al. with a different approach. The presence or absence of glucose did not affect the pH titration [19] . For the mutant D22A no absorbance change of the COOH group is observed (blue line). Harris and Turner studied several membrane proteins that has residues with perturbed pKas in important mechanistic positions [40] and concluded that the pKa is regularly modulated by a combination of several types of interactions such as long-range and local electrostatic effects. The protein cytochrome c oxidase showed a high pKa. The residue E278 (Paracoccus denitrificans numbering), localized in the membrane, was found to have a pKa higher than 11 [41] . First, the amide I band of I105S, A167E and I105G-A167E was measured in order to confirm if the mutation of the amino acid affected the integrity of the protein (Figure 12). Amide I band is centered at 1655 cm -1 and five bands, 1690, 1679, 1631 and 1616 cm -1 were used to deconvoluted the amide I band of the mutants. Figure 12 shows that the major secondary structure is α-helices which agrees with the results obtained for the GlcPSe WT. The structure was not affected by the mutations however, the band profile of the mutant A167E is slightly different from the other residues. This can be due to a small CHAPTER IV Perfusion-SEIRAS approach for the study of membrane transport proteins CHAPTER IV Perfusion-SEIRAS approach for the study of membrane transport proteins ___________________________________________________________________________ Ana Filipa Santos Seiça Mutations of residues in critical positions for glucose binding or proton transfer, have been studied for different pH values. Figure 13 shows the data obtained for pH 7.5, 8.5 and 9.5 with (a-c) and without glucose (d-e). Whereas glucose does not seem to influence the signal at 1750 cm -1 , major changes occur in the amide I and amide II regions indicating differences in the pH dependent conformational reorganization of the protein in the presence of glucose.

The I105S mutation located in close vicinity to the H + -binding site and analogous to the eukaryotic glucose uniporter GLUT2 [18] does not perturb the signal at 1750 cm -1 , though some changes in the amide I and II region suggest smaller structural changes (Figure 13A). The signal at 1750 cm -1 also appears in the presence of glucose and the intensity increases with the pH, showing that sugar translocation is independent of the H + binding site.

Interestingly, in the case of the A167E mutation (Figure 13B) located in the TM 4 show a signal at 1750 cm -1 and the signal at 1724 cm -1 are lost in the absence of glucose and restored 2 cm -1 lower position in the presence of the substrate. The introduced A167E clearly changes the environment of D22 and point towards an interaction between D22 and the introduced E167, eventually introduced by a conformational change.

Finally, the double mutation I105G-A167E was investigated (Figure 13C) pointing to major structural changes. The negative signal at 1750 cm -1 is lost in all cases and replaced by a positive feature at 1732 -1720 cm -1 . There is no easy explanation for this signal that is located in the spectral region typical for protonated acidic residues and the residue would clearly be protonated at very alkaline pH values.

CHAPTER IV

Perfusion-SEIRAS approach for the study of membrane transport proteins ___________________________________________________________________________ 135 Ana Filipa Santos Seiça The pKa was determined with the same experimental protocol as for the WT. The absorbance intensity at 1750 cm -1 was plotted against the pH and the titration curve was obtained. The mutant I105S GlcPSe has a pKa around (8.5 ± 0.1), similar to that of WT GlcPSe (Figure 14) having the same behavior to WT showing that D22 is not interacting with this residue. The comparison of the data with and without glucose reveals in addition that the pKa of D22 and of I105S does not depend on the presence of glucose. I105S can bind sugar in the protonated and deprotonated form like WT [19] . Serine in the position 105 has little effect on H + and sugar-binding, however was reported to act in different steps of the reaction cycle, for example in the cytoplasmic H + release [19] . For the mutant A167E located in the TM4, the scenario is more complex. Whereas a pKa of 8.5 is found in the presence of sugar, there is no signal from the D22 at all in the absence of sugar (green circle line) pointing to a major structural reorganization in the environment of the residue inducing a drop of the pK below a value of 6 (lowest pH Ana Filipa Santos Seiça measures here). In the I105G-A167E mutant, the signal of the acidic residue is lost in both cases indicating clearly that the acidic residues are protonated at very alkaline pH values (Figure 13C).

One feature that distinguishes the GlcPSe from the classical E. coli sugar transporters e.g. LacY classified as symporter, is that the affinity towards sugar decreases with higher H + concentration (lower pH). In view of the universal eight-state kinetic model, high affinity to sugar of the deprotonated and low affinity to sugar of the protonated transporter are characteristic for antiporter [19] .

The wild type GlcPSe generates a pH dependence of the transient currents after a sugar concentration jump [19] , the double mutant I105G-A167E shows a very similar behavior to wild type with the pKa shifted to the acidic region at pH 6.8 (Figure 15). The GlcPSe mutant I105G-A167E shows a comparable Kd of 11.4 ± 3.3 mM at alkaline pH 8.5. This observation is in line with the results from the IR spectroscopy (Figure 13C) that the residue D22 would be protonated at very alkaline pH in the I105G-A167E mutant (Figure 15b).

CHAPTER IV

Perfusion-SEIRAS approach for the study of membrane transport proteins ___________________________________________________________________________ 138 Ana Filipa Santos Seiça Our results revealed that D22 has a pKa of 8.5 ± 0.1, a value that corroborates with the pK of glucose transport [19] . A neutral replacement of the negatively charged D22 led to positive charge displacements over the entire pH range, suggesting that the polarity change of the wild type reflects the protonation state of D22.

The substitution of the residue Ile105 for a serine led to a change in the microenvironment of this residue, but the pKa of D22 corresponded to wild type values. Finally, an additional protonable site was introduced in analogy to the XylE protein, A167E, and studied together with the double mutation G105-E167, we found a strong perturbation of the protonation site of D22, indicating that the carboxyl group at position 22 is deprotonated at lower pH. This perturbation may be because of the change of charge distribution when alanine group at position 167 was substituted to glutamic acid and with consequence perturbing the surrounding environment in the H + binding site.

-Perfusion-Induced Difference Spectrum of Mutants of Glu325 from

Lactose Permease

-Study of the Microenvironment of Glu325

A systematic study on the effect of potentially important mutations on the SEIRAS difference spectrum of Glu325 (E325) of the transporter protein LacY was undertaken (Figure 16B) in order identify which residues in the side chains sufficiently close to Glu325 can perturb the pKa. Part of the results presented here were done by a former PhD student of the laboratory, Dr. Natalia Grytsyk.

Each mutant was introduced into the G46W/G262W background (LacYww) to ensure high stability at alkaline pH [25] . Difference spectra between pH 7.0 and pH 11.0 in which Glu325 is essentially completely deprotonated were carried out with each mutant (Figure 16A): (i) pseudo-WT LacYww, (ii) LacYww/ E325D, (iii) LacYww/R302K, (iv) LacYww/R302A, (v) LacYww/ H322Q, (vi) LacYww/K319L, and (vii) LacYww/D240A.

CHAPTER IV

Perfusion-SEIRAS approach for the study of membrane transport proteins ___________________________________________________________________________ 140 Ana Filipa Santos Seiça The amide I/II region does not change significantly in the difference spectra of the mutant, arguing against a change in the structural integrity of the protein. With mutations R302K and R302A, only small shifts are observed for the signal at 1745 cm -1 . However, with mutation R302K, the difference signal is significantly broader, an effect that may be due to a higher degree of freedom of Glu325 at this position. Mutation of residue H322Q (Figure 16A) causes a shift in the Glu325 COOH vibration to 1737 cm -1 , which suggests stronger hydrogen bonding in the immediate environment of the residue without significant perturbation of the overall structure.

In the spectrum of the K319L mutant, the signal discussed is found at 1750 cm -1 , similar to the D240A mutation (Figure 16A). The amide signature for these two mutants is clearly changed compared with the pseudo-WT. While there is a broad positive peak at 1674 cm - 1 with a shoulder at 1722 cm -1 and a negative signal around 1625 cm -1 for the pseudo-WT and the mutants discussed above, the signals are inverted here, suggesting a strongly modified change in conformational reorganization that takes place upon pH change. In Y236F, a signal at 1744 cm -1 was obtained for the step to pH 10.5, but the mutant is unstable and the amide I signature was different for each pH value studied.

In L329F, no signal could be identified except for some conformational changes in the amide I/II range. Both residues are located in the hydrophobic patch surrounding E325 (Figure 17B). Finally, the effect of mutant Y236F was tested. Again, the signature of E325 was not identified, and for each pH step, the amide I signature was different, as can be seen when comparing the step to pH 10 and to pH 10.5. It is noted that Y236 is hydrogen-bonded to H322 [43] . Mutations in the hydrophobic pocket of E325 perturb structure and the conformational flexibility of LacY.

CHAPTER IV

Perfusion-SEIRAS approach for the study of membrane transport proteins ___________________________________________________________________________ Mutation E325D exhibits a shift in the pKa from ∼10.5 to ∼8.3 (Figure 18), consistent with a change in the microenvironment of the COOH. Mutation E325D retains 15-20% of WT transport activity with reduced affinity [23][27] [32] . Mutant R302A is also defective with respect to affinity for galactoside [23] , but catalyzes transmembrane exchange [44] , and the mutation has little or no effect on the pKa of E325.

However, mutation R302K is particularly interesting, as it causes the pKa of E325 to decrease to pH 8.3 (Figure 19B), clearly an indication of interaction between the two side chains. The E325 signal at 1745 cm -1 can be observed at pH 8 and pH 8.5 in the difference spectra (Figure 19A). Ana Filipa Santos Seiça bridge [45] and cause very small, but reproducible, acid shifts in the E325 titration (Figure 20). Finally, mutations Y236F (helix VII) and L329F (helix X) yield unstable E325 pH titrations as mentioned above, and the spectra differ at each pH tested. Both of these side chains comprise part of the hydrophobic pocket in which E325 is situated. One of the problems for LacY turnover is deprotonation when the midpoint for both galactoside and H + binding is pH 10.5, which translates into a Kd for H + of ∼30 pM. As discussed, it is unlikely that either decreasing the cytosolic H + concentration or the pKa of E325 is a realistic possibility.

An increase in water accessibility to the hydrophobic site in which E325 is located is a possibility. However, as shown by three independent H/D exchange studies [46][47][48] , the backbone amide protons in LacY are largely accessible to water. Another possibility is that R302 and E325 may be in closer proximity in another conformation of LacY [49][50] , and the distance between the two residues is important in this regard [32] . Neither the R302K mutation nor the R302A mutation causes gross alteration in structure, as is also the case for other mutations in the vicinity of E325 that disrupt a salt bridge or cause a change in conformation, but do not affect the pKa of E325 Finally, we determine that mutation of the residues in the immediate neighborhood of E325 have little effect on the pKa of E325, while replacement of R302 with a positively charged Lys caused a two-pH unit acid shift, which led us to concluded that R302 is important for deprotonation of E325.

CHAPTER IV Perfusion-SEIRAS approach for the study of membrane transport proteins

___________________________________________________________________________

GENERAL

CONCLUSION General Conclusion

___________________________________________________________________________

In this thesis 2 different infrared markers bands in the protein were studied, using reaction induced vibrational spectroscopies, an analytical technique that studies proteins at a molecular level and is a valuable tool for the investigation of protein structure and protein reactions.

Firstly, before studying the infrared markers it was necessary to understand the influence of the gold nanostructure morphology and protein immobilization procedure on the enhancement factor of protein signals in infrared. Two different immobilization approaches were used to characterize different silicon crystals after the deposition of metal nanoparticles for different deposition times.

The first approach for protein immobilization was the formation of a negatively charged 11-MUA SAM on a gold film, taking advantage of the sulfur group, which forms a strong covalent bond with the gold surface. The second immobilization approach has the advantage of directing the orientation of the protein in the surface through the affinity of the genetically introduced His-tag for the Ni-NTA surface. Once the protein was immobilized in these two different immobilizations the enhancement factor of the amide I was obtained for the different silicon crystals.

The best deposition time was obtained for 40s for the silicon wafer and the small ATR crystal and 60s for the big ATR crystal for both immobilizations. The enhancement determined for the protein immobilized in a Ni-NTA surface was consisted with the data for the 11-MUA immobilization, confirming that the coverage of the surface contributed to the enhancement and that the enhancement obtained for different deposition times was sensitive to infrared measurement technique and the size of the crystal. The enhancement was also independent on the type of immobilization.

Once characterized the surface and the optimum conditions for a higher enhancement of a protein immobilized, the protein F1-ATPase from the respiratory chain was studied by using nitrile probes in order to understand in which conformation the epsilon subunit can be found in presence of ATP.

The introduction of the nitrile probes at variants εH56C and εY114C, located in the NTD and CTD, respectively, led to a complex spectral signature pointing to the presence of Ana Filipa Santos Seiça several cysteines which can be seen from the deconvolution spectra. When direct comparing the spectrum of the two studied variants, the SCN difference signal is found in a comparable spectral range and a similar shape in presence and absence of ATP.

The results obtained do not give us evidence for an extended or contracted conformation of epsilon subunit, when the protein switches between ATP synthesis and ATP hydrolysis. However, it is not possible to exclude that switching between both modes of catalysis, might be connected with the movement of other parts of the epsilon subunit.

Finally, the use of a small infrared probe allowed us to label the residues of the epsilon subunit with a higher labeling efficiency as compared with big fluorescent probes without perturbing the secondary structure.

The other membrane protein studied from the respiratory chain was E.coli Complex I, where we followed the movement of small and highly flexible nitrile infrared label attached to the amphipathic helix in the membrane arm.

The placement of nitrile labels to cysteine residues of complex I allowed to monitor minor movements of the amphipathic horizontal helix of NuoL in the membrane arm upon reduction by NADH. When subtracting the spectra before and after addition of NADH differences of 15 to 18 cm -1 for the signals of the label visualize experimentally a reorganization of distinct positions within the helix towards a more hydrophobic environment or an environment with weaker hydrogen bonds upon NADH addition. The most likely explanation for our observation is that the movement of the positions on the amphipathic helix is an indirect effect that results from a structural compensation of the movement of the membrane arm during reduction.

The other infrared marker studied was the ν(C=O) group of the transport proteins Staphylococcus Epidermidis GlcPSe and E. Coli Lactose Permease using SEIRAS approach combined with a perfusion cell in order to understand the functioning of the protein, which residues are crucial for a high pKa and if there is a residue responsible for the coupling mechanism of symport.

For the protein GlcPSe, our results revealed that Asp22 has a pKa of 8.5 ± 0.1. A neutral replacement of the negatively charged Asp22 led to positive charge displacements over the entire pH range, suggesting that the polarity change of the wild type reflects the protonation state of Asp22.

3 . 7 . 1 - 3 . 7 . 4 -

 371374 Infrared Absorption Spectroscopy………………...………...82 3.7 -RESULTS AND DISCUSSION………………...……………….…..………………..82 Cyanide Labelling -Cyanide Label of F 1 -ATPase Residues……………………..82 3.7.1.1 -Determination of the Label Efficiency of F 1 -ATPase………………...……………83 3.7.1.2 -Immobilization of F 1 -ATPase…………….…………………..………………...85 3.7.1.3 -Deconvolution of the amide I signal of F 1 -ATPase and Influence of ATP………………………………………………………………………………………....86 3.7.2 -FTIR spectra of the introduced IR label in εH56C and εY114C……....………..88 3.7.3 -Labeled Cysteines……....………………....……....………………....……....……..92 3.7.3.1 -Interpretation of the Observed Shifts of the Labeled Cysteines……....……………92 Cyanide Label of Complex I Residues……....………………................................93 3.7.4.1 -Deconvolutionof the amide I band of Complex I Residues After Labelling……............................................................................................................……...93 3.7.4.2 -Cyanide Labelling of the Residues K551C L and Y590C L ……....……………….....95 3.7.4.3 -Interpretation of the νSCN Shifts in the Residues K551C L and Y590C L ………………………………………………………………………….….-MECHANISM OF THE TRANSPORTERS……………………………...………….115 4.1.1 -Carrier or Channel Proteins……………………………………..……………….115 4.1.2 -Symport and Antiport………………………………………………..…………...116

Figure 3 :

 3 Figure 3: Conformational states of GlcPSe (PDB 1PV7) and LacY (PDB: 1PW4), from the major facilitator superfamily. N domains are colored wheat and the C domains gray.

[ 10 ] 9 [ 14 ] 4 [ 16 ]

 10914416 . Mitchell P. Chemiosmotic coupling in oxidative and photosynthetic phosphorylation. Biochimica et Biophysica Acta (BBA)-Bioenergetics. 2011; 1807(12) : 1507-1538. doi: 10.1016/j.bbabio.2011.09.018 [11]. Kriegel SB, Uchida T, Osawa M, Friedrich T, Hellwig P. Biomimetic environment to study E. coli complex I through surface-enhanced IR absorption spectroscopy. Biochemistry 2014; 53: 6340-6347. doi: 10.1021/bi500955a [12]. Steimie S, Schnick C, Burger E., Nuber F et al. Cysteine scanning reveals minor local rearrangements of the horizontal helix of respiratory complex I. Molecular microbiology. 2015; 98(1): 151-161. doi: 10.1111/mmi.13112 [13]. Jonckheere AI, Smeitink J.M, Rodenburg RJT. Mitochondrial ATP synthase: architecture, function and pathology. J Inherit Metab Dis. 2012; 35(2): 211-225. doi: 10.1007/s10545-011-9382-. Shah NB, Hutcheon ML, Haarer BK, Duncan TM. F1-ATPase of Escherichia coli the ϵ-inhibited state forms after atp hydrolysis, is distinct from the adp-inhibited state, and responds dynamically to catalytic site ligands. Journal of Biological Chemistry. 2013 ; 288(13), 9383-9395. doi: 10.1074/jbc.M113.451583 [15]. Laget PP, Smith JB (1979). Inhibitory properties of endogenous subunit ε in the E. coli F1 ATPase. Arch. Biochem. Biophys. 1979; 197(1): 83-89. doi: 10.1016/0003-9861(79)90222-. West IC. General principles of membrane transport. Biomembranes: A Multi-Volume Treatise. 1995; (1): 225-243. doi: 10.1016/S1874-5342(06)80059-0 [17]. Grytsyk N, Sugihara J, Kaback HR, Hellwig P. PKa of Glu325 in LacY. PNAS. 2017; 114(7): 1530-1535. doi: 10.1073/pnas.1621431114 [18]. Iancu CV, Zamoon J, Woo SB, Aleshin A, Choe JY. Crystal structure of a glucose/H+ symporter and its mechanism of action. PNAS. 2013; 110(44): 17862-17867. doi: 10.1073/pnas.1311485110 [19]. Yan, N. Structural advances for the major facilitator superfamily (MFS) transporters. Trends in Biochemical Sciences. 2013; 38(3): 151-159. doi: 10.1016/j.tibs.2013.01.003

Figure 1 :

 1 Figure 1: Electromagnetic spectrum…………...………...….…………………......…..17

Figure 2 : 19 Figure 3 : 22 Figure 4 : 26 Figure 5 :

 2193224265 Figure2: Electronic vibrational energy states in Raman Spectra[7] ………….…….........19 Figure 3: Normal mode vibrations of water [4] ……………………….............…......…...22 Figure 4: Schematic representation of the transmission cell………………………..…..26 Figure 5: Schematic representation of the ATR principle [19] ………....……...................27

  …..........................................................28

Figure 4 :

 4 Figure 4: Schematic representation of the transmission cell

Figure 5 :

 5 Figure 5: Schematic representation of the ATR principle[19]

Figure 2 : 39 Figure 3 : 40 Figure 4 : 41 Figure 5 .Figure 6 . 44 Figure 7 . 47 Figure 8 .Figure 9 :

 239340441564474789 Figure 2: Schematic image of a protein immobilized on a self-assembled monolayer gold surface………………………………………………………………………...………...39 Figure 3: Schematic image of the protein Cyt C immobilized on an 11-MUA selfassembled monolayer gold surface………………………………...…...….…………....40 Figure 4: Schematic image of a protein immobilized on a Ni-NTA SAM surface in a gold layer…………………………………………………………………………...………..41 Figure 5. Schematic representation of a perfusion cell……………………....…………43 Figure 6. Scheme of a reaction-induced infrared difference spectroscopy [28] …........….44 Figure 7. Different silicon crystal used for the immobilization of proteins and the respective infrared technique A) ATR-perfusion cell, B) ATR-electrochemical cell and C) Transmission mode………………………………………………………………….47 Figure 8. Spectra of the formation of an 11-mercaptoundecanoic acid (MUA) selfassembled monolayer on a gold surface followed by attenuated total reflection (A) (reference spectrum: gold layer with evaporated ethanol) and transmission (B) (reference spectrum: gold layer).……...………………………………………..……………..……48 Figure 9: Absorbance spectra of Amide I band dry (A) and in solution (B and C) of cytochrome c protein immobilized in a 11-MUA surface in function of the time deposition of gold on different crystals A) silicon wafer for transmission 1cm diameter (reference spectrum gold layer) B) small ATR crystal 3mm diameter (reference spectrum 11-MUA monolayer) and C) big ATR crystal 2cl diameter (reference spectrum gold layer). The enhancement factor was obtained from the intensity of the amide I band of the protein immobilized on a gold surface and the protein on the silicon crystal.…………………50

Figure 10 . 52 Figure 11 . 55 Ana Filipa Santos Seiça 2 -

 105211552 Figure 10. Spectra of the surface modification process followed by attenuated total reflection. A) Self-assembly of the TSP monolayer on the bare Au surface (reference spectrum: gold layer covered with DMSO), B) Cross-linking of ANTA with the TSP monolayer (reference spectrum: DTSP SAM in water), C) complexation of Ni 2+ by NTA (reference spectrum: TSP-NTA SAM covered with water) and D) absorption of GlcPSe on the Ni-NTA SAM in different depositions times of the gold surface (reference spectrum: Ni-NTA modified gold layer). 1) Enhancement of amide I band of GlcPSe in function of the time deposition of gold in the small silicon ATR crystal…………...…...52 Figure 11. Scanning electron microscopy image of a gold layer on a silicon wafer (1) and after a Ni-NTA immobilization (2) and simulated electric field intensity of random gold nanoparticles on a silicon substrate when excited with unpolarized light. Field intensity calculated around Amide I band 1660 cm -1 for different deposition time (a) 20 sec, (b) 40 sec and (c) 60 sec...….…………………………………………….……………...……..55

Figure 1 :

 1 Figure1: AFM image of electroless deposition of an Au film on the silicon crystal[15] .

Figure 11 ,

 11 increasing the time deposition of gold leads to different morphology of the gold particles before and after a Ni-NTA immobilization. SEM characterization and theoretical calculations show that the time deposition of the gold nanoparticles has a significant influence on the enhancement. Increasing the time from 20s Figure 11.1(a) to 40s Figure 11.1(b) leads to an increase of density, a decrease of the interparticle distances a much higher enhancement. During the deposition time, the thickness and the shape of the AuNPs differs before the immobilization. For 20 seconds (Figure 11.1.a) of deposition time the AuNPs are smaller Ana Filipa Santos Seiça in size, with less density as compared to 40 sec (Figure 11.1.b). For the 60s see Figure 11.1c an uncontrolled deposition of the nanostructures over the surface has occurred, which in turn results in agglomeration or multilayer formation of nanostructures with narrow spacing between the particles.Similar morphologies are observed after the immobilization in Figure11.2 (a-c). This indicates that the accessibility of gold nanoparticles on the surface was not affected by introduction of the linkers confirming that the AuNPs are therefore still available for functionalization because the (-SH) bond forms a strong covalent interaction between the gold surface on one side and the biofunctional molecule on the other.The best deposition time seems to be for the 40s as observed from the results obtained for the different immobilizations, as for the theoretical results calculated for the silicon wafer before and after the Ni-NTA immobilization. The crystal has sufficient coverage of the nanostructures on the surface and hence, a high density of biofunctional molecules immobilized onto the surface. With longer deposition times, the inner surface area decreases due to the increasing connection between islands, and consequently the enhancement decreases.A series of factors are crucial for obtaining the best enhancement in SEIRAS such as sample density, surface structure or crystal structure. These parameters along with different time deposition of the gold are important factors to consider in order to obtain an optimum enhancement.

  Figure1A) Overview of E. coli F1FO -ATPase (PDB 5t4o), subunits α in blue, β in red, γ in orange, ε in violet, c in green, a in grey, b in yellow, and δ in pink[14] and B) The binding-change mechanism of the F1 complex. Three catalytic sites in three different conformations: loose, open, and tight. Substrate (ADP + Pi) initially binds to the open site and is converted to ATP at the tight site. (1) Rotation of the γ subunit causes a conformational change, resulting in a change in the formation of the sites and ATP is released from the enzyme. (2) Substrate again binds to the open site, and another ATP is

Figure 2 .Figure 3 . 74 Figure 4 .Figure 5 .

 237445 Figure 2. Comparison of the secondary structure of epsilon subunit in E.coli F1-ATPase (A) in an extended conformation and isolated epsilon (B) in a compacted conformation (PDB 3OAA) [27] ………...……………………………………………………………...73 Figure 3. Overall architecture of Complex I. The membrane domain is from E.coli Complex I (PDB: 3RKO), and the peripheral domain is from T. Thermophiles Complex I (PDB 3M9S)………….……………………………………………………..……….....74 Figure 4. Reaction scheme of a chemical conjugation between maleimide and sulfhydryl group on the protein (image taken from thermofisher site)………….......................……77 Figure 5. Cysteine cyanylation scheme[START_REF] Fafarman | Site-Specific Conversion of Cysteine Thiols into Thiocyanate Creates an IR Probe for Electric Field in Proteins[END_REF] ……………………...………….…………...77

Figure 7 .

 7 Figure 7. Absorbance spectrum of potassium thiocyanate (KSCN) in methanol (2066 cm -1 ), aqueous buffer pH 7.0 (2064 cm -1 ), ethanol (2061 cm -1 ), DMSO (2057 cm -1 ) and in THF (2044 cm -1 )…………………………………………………………………..…83

Figure 8 .Figure 9 .Figure 10 .Figure 11 .Figure 12 .Figure 13 .Figure 14 :Figure 15 .Figure 16 .Figure 17 .Figure 18 .

 89101112131415161718 Figure 8. Infrared (A-C) and fluorescence label (D) of the protein F1-ATPase followed by UV-Visible spectroscopy with the corresponding scheme of the label in each step. A) The protein is incubated for 30 min with DTT. B) Formation of the mixed proteinthionitrobenzoic acid disulfide (PS-TNB) after sephadex removal of excess of TNB. C) Addition of potassium cyanide and formation of the protein-thiocyanate (PS-CN). D) ATTO 594 a fluorescent dye was covalently coupled to the ε subunit……………..……84

Figure 4 :

 4 Figure 4: Reaction scheme of a chemical conjugation between maleimide and sulfhydryl group on the protein (image taken from thermofisher site).

Figure 6 :

 6 Figure6: Reaction scheme of the coupled enzyme test in ATP hydrolysis[START_REF] Fischer | Comparison of ΔpH-and Δφ-driven ATP synthesis catalyzed by the H+-ATPases from Escherichia coli or chloroplasts reconstituted into liposomes[END_REF] .

Figure 15 :

 15 Figure 15: Absorbance spectrum of the residue K551C L of Complex I from E.coli labeled immobilized in a Ni-NTA SAM.

3 -

 3 Interpretation of the νSCN Shifts in the Residues K551CL and Y590CL

Figure 1 :Figure 2 :Figure 3 :Figure 4 :Figure 5 :Figure 6 :Figure 7 :Figure 8 :Figure 9 :

 123456789 Figure 1: Scheme of Channel and Carrier Proteins (image taken from "Scheme facilitated diffusion in cell membrane by Mariana Ruiz Villareal, public domain).………............116 Figure 2: Mode of Transport for the Uniporter Transport Protein GLUT1 [9] ……...…117 Figure 3: Structure of the Inward-Facing Conformation of GlcPSe (A) and the H + -Binding Site (B) (PDB: 4LDS) [18] ……………….……………………...………………...…….119 Figure 4: Mechanism of Glucose/H + Symport of GlcPSe. S, Carbohydrate Substrate (glucose) of the Transporter; H + , proton [18 ……………………........….……….……...120 Figure 5: Representation of LacY in an inward-open conformation (PDB 1PV7). Cytoplasmic View of the E325 Environment and of the Positions of the Mutations (PDB ID code 2V8N) [29] ……...……………….…………………………………..…..……..121 Figure 6: Perfusion-Induced FTIR Difference Spectra of GlcPSe WT (a) and GlcPSe D22A (b) Obtained from the Sample Equilibrated at pH 5.5 without Sugar Subtracted from the sample Equilibrated at pH 8.5 respectively (black line) and reverse (red line)……………...………………………………………………………………..…...125 Figure 7: pH-induced difference Spectra of the GlcPSe WT (A), GlcPSe D22A (B), Equilibrated at pH 5.5 was Subtracted from the Sample Equilibrated at 7.5 (a), 8.5 (b) and 9.5 (c) in Absence of Glucose……………………...…………..………………..……..126 Figure 8: Deconvolution of Amide I of Initial Purified Samples of GlcPSe WT (A), GlcPSe D22A (B) prepared in 50mM KPi/0.05% DDM pH 7.5 on the Ni-NTA Surface………………………………………………………………………………...128 Figure 9: pH-dependent Change of the GlcPSe WT Equilibrated at pH 5.5 Subtracted from the Sample Equilibrated at 7.5 (d), 8.5 (e) and 9.5 (f) in the Presence of Glucose………………………………………………………………………………..130

Figure 10 :Figure 11 :Figure 12 :Figure 13 :Figure 14 :Figure 15 :Figure 16 :Figure 17 :

 1011121314151617 Figure 10: Titration Curves for the ν(C=O) Vibrational Mode of Asp22 at 1750 cm -1 of GlcPSe WT with (filled red circle) and without (full black circle) Glucose and GlcPSe D22A (full dark-blue circle)…………………………..………………………….……131 Figure 11: Structure of the Residues Located in the H + Binding site (PDB: 4LDS) of the Protein GlcPSe…………………...……………………...……………………..….…...132

Figure 18 :Figure 19 :Figure 20 :

 181920 Figure 18: Titration Curves for the ν(C = O) Vibrational Mode of Glu325 at 1745 cm -1 : pseudo-WT with (red) and LacYww/E325D (blue)………………….……..…………142

  IR Dichroic Values of GlcPSe WT prepared in 50mM KPi/0.05% DDM pH 7.5, 8.5 and 5.5 and pD 7.5 prepared in 50mM KPi/0.05% DDM adsorbed on the Ni-NTA modified gold surface………………….………………………………………………129 CHAPTER IV Perfusion-SEIRAS approach for the study of membrane transport proteins ___________________________________________________________________________ 115 Ana Filipa Santos Seiça

4. 4 - 4 . 4 . 1 -

 4441 RESULTS AND DISCUSSION Perfusion-Induced IR Spectroscopy of GLCPSe 4.4.1.1 -Assignment of the Signal for Residue Asp22

Figure 7 :

 7 Figure 7: pH-induced difference spectra of the GlcPSe WT (A), GlcPSe D22A (B), equilibrated at pH 5.5 was subtracted from the sample equilibrated at 7.5 (a), 8.5 (b) and 9.5 (c) in absence of glucose

Figure 8 :

 8 Figure 8: Deconvolution of amide I of initial purified samples of GlcPSe WT (A), GlcPSe D22A (B) prepared in 50mM KPi/0.05% DDM pH 7.5 on the Ni-NTA surface.

Figure 9 :

 9 Figure 9: pH-dependent change of the GlcPSe WT equilibrated at pH 5.5 subtracted from the sample equilibrated at 7.5 (d), 8.5 (e) and 9.5 (f) in the presence of glucose.

4 -

 4 Determination of the pKa of the Residue Asp22

Figure 10 :

 10 Figure 10: Titration curves for the ν(C=O) vibrational mode of D22 at 1750 cm -1 of GlcPSe WT with (filled red circle) and without (full black circle) glucose and GlcPSe D22A (full dark-blue circle).

  approach for the study of membrane transport proteins ___________________________________________________________________________ 4.4.1.5 -Perfusion-Induced Conformational Changes in a Monolayer of GLCPSe I105S, A167E and A167E-I105G Mutations of residues I105S, A167E and the double mutation I105G-A167E (Figure 11), were studied for different pH values and the pKa of those residues were determined in order to understand if a single or a double mutation could affect the pKa of D22 in presence or absence of glucose.

Figure 11 :

 11 Figure 11: Structure of the residues located in the H + binding site (PDB: 4LDS) of the protein GlcPSe

  structural change of the protein upon substitution of the amino acid alanine for a glutamate.

Figure 12 :

 12 Figure 12: Deconvolution of amide I of initial purified samples of GlcPSe I105S (A), GlcPSe A167E (B) and GlcPSe A167E-I105G (C) prepared in 50mM KPi/0.05% DDM pH 7.5 on the Ni-NTA surface.

Figure 13 : 6 -

 136 Figure 13: pH and ligand-induced difference spectra of GlcPSe I105S (A), GlcPSe A167E (B) and GlcPSe A167E-I105G (C) equilibrated at pH 5.5 was subtracted from the sample equilibrated at 7.5 (a), 8.5 (b) and 9.5 in absence of glucose and equilibrated at pH 5.5 subtracted from the sample equilibrated at 7.5 (d), 8.5 (e) and 9.5 (f) in presence of glucose

Figure 14 :

 14 Figure 14: Titration curves for the ν(C=O) vibrational mode of Asp22 at 1750 cm -1 of GlcPSe A167E with (full red circle) and without (full green circle) glucose / GlcPSe I105S with (full black circle) and without (full blue circle) glucose

  approach for the study of membrane transport proteins ___________________________________________________________________________ 137

Figure 15 :

 15 Figure 15: Transient currents recorded at the SSM after a substrate concentration jump at symmetrical pH as indicated (a). pH dependence in GlcPSe I105G-A167E mutant at symmetrical pH as indicated. Transient currents were induced by 30 mM D-glucose at different pH values, as indicated. Mean and SD of the respective charge translocations (peak integrals) from at least three datasets. The apparent pKa is indicated (b) (data provided by Dr. Madej from Rogensburg University).

4. 4 . 2 . 2 -

 422 Effect of Mutant Y236F, L329F and F243W on Glu325Three additional mutants were studied and show strongly perturbed spectra (Figure.17A).

Figure 18 :

 18 Figure 18: Titration curves for the ν(C=O) vibrational mode of Glu325 at 1745 cm -1 : pseudo-WT with (red) and LacYww/E325D (blue) (part of the results were performed by Dr. Grytsyk).

  approach for the study of membrane transport proteins ___________________________________________________________________________ 144

Figure 20 :

 20 Figure 20: Titration curves for the ν(C=O) vibrational mode of Glu325 at 1745 cm -1 LacYww/D240A (orange), LacYww/K319L (cyan), and LacYww/H322Q (gray). Part of the results were performed by Dr. Grytsyk.

  approach for the study of membrane transport proteins ___________________________________________________________________________ 145 Ana Filipa Santos Seiça The data obtained here indicate that R302 interacts with E325. While the R302A mutation does not significantly change the microenvironment of E325, introduction of a Lys induces a downshift of approximately two pH units and a broader COOH vibrational mode characteristic of a residue with significant rotational freedom.

Conformation H2O (cm -1 ) D2O (cm -1 )

	use of D2O, however, this can affect the native secondary structure of the protein. Current
	Fourier transform infrared spectrometers allow for accurate subtraction of the water band,
	α-helix	1650-1657	1642-1660
	Antiparallel β-sheet	1612-1640	1613-1625
		1670-1690	1672-1694
	Parallel β-sheet	1626-1640	1615-1638
	Turn	1655-1675	1653-1691
	Unordered	1642-1657	1639-1654
	Ana Filipa Santos Seiça

Spectral region/ cm -1 Refractive index Depth of penetration at 45° Hardness/ kg/mm 2 pH Range ZnSe

		20,000 -500	2.43	1.66	120	5-9
	Ge	5,000 -600	4.01	0.65	780	1-14
	Si	10,000 -100	3.42	0.81	1150	1-12
	Diamond 45,000 -10	2.40	1.66	5700	1-14

Table 1 .

 1 ATP hydrolysis of F1-εY114C, before and after label with the fluorescence probe ATTO 594 in presence or absence of ATP at 37°C. The concentration of F1 during the measurement was 3, 10 and 20nM. …………………………………..

.……….…..…....79Table 2 :

 2 Activation factor calculated from the ratio of ATP hydrolysis with and without 0.03% (v / v) LDAO……………………………………………………...…….….……80

	Ana Filipa Santos Seiça

III Cyanide label of proteins from the respiratory chain ___________________________________________________________________________ 80

			CHAPTER
	3 nM	97.86	462.24	99.57	414.69	92.18	362.39
	10 nM	95.94	455.00	92.56	395.99	87.42	347.73
	20 nM	91.89	372.42	81.26	293.07	78.64	298.39

cm -1 ) Abs 5 x 10 -2 CHAPTER IV Perfusion-SEIRAS approach for the study of membrane transport proteins ___________________________________________________________________________ 129

				1654					1654
	A	5 x 10 -2 Abs	70%		B		62%
											1631
											28%
						1629
						20%
			1680						1679
			6%						5%				1613
		1691					1614	1689					3%
		1%					3%	1%
		1700	1680	1660	1640	1620	1600	1700	1680	1660	1640	1620	1600
			Wavenumber (cm -1 )			Wavenumber (

SEIRAS approach for the study of membrane transport proteins ___________________________________________________________________________ 130

	CHAPTER IV
	wild type pD 7.5 pH 5.5 pH 7.5 pH 8.5 Perfusion-Ana Filipa Santos Seiça A parallel A perpendicular RATR (amide I, α-helix) 0.022 0.016 1.38 0.016 0.008 2.00 0.016 0.013 1.23 0.013 0.008 1.63	angle ~ 90 ~ 52 ~ 90 ~ 72

Acknowledgements

To Dr. Elise Glattard from the Laboratory of To IDEX Program Doctoral International and University of Strasbourg for helping and providing the funding for the PhD.

To Dr. Zahia Boubegtiten for her friendship, support, advice and for her kindness.

Ana Filipa Santos Seiça How can deprotonation of LacY occur at physiological pH with a pKa of 10.5? One possibility is a structural change that exposes protonated Glu325 to a more aqueous local environment. Another is to bring Arg302 (helix IX) close to Glu325 (helix X), although the two residues are 6-7 Å apart with the hydroxyl group of Tyr236 between them in the current structure. Thus, Arg302 and Glu325 may be in closer proximity in another conformation of LacY. It was suggested that positively charged Arg302 may be important with respect to deprotonation of Glu325 [32] , and further evidence supporting this possibility has been presented [25] .

We have tested the effect of mutating Arg302 and other side chains in the vicinity of Glu325 in order to understand if the residues of the side chain that interact chemically with Glu325 and/or change the local environment affect the pKa.

-EXPERIMENTAL SECTION

-Construction of Mutants of LacY and GlcPSe

Construction of mutants, expression in E. coli, and purification of LacY were performed as described before [25][29] . All constructs contained a C-terminal His6 tag that was used for affinity purification with Talon resin. Purified proteins (10-15mg/mL) in 50 mM sodium phosphate [NaPi/0.02% DDM (pH 7.5)] were frozen in liquid nitrogen and stored at -80 °C until use. LacY and mutants were provided by Prof. H. Ronald Kaback (Department of Physiology, University of California, Los Angeles).

GlcPSe wild-type (WT) and mutants, D22A, I105S and A167E, were provided by Prof.

Jun-Yong Choe (The Chicago Medical School, Rosalind Franklin University of Medicine and Science, Chicago, IL). The GlcPSe gene was cloned into the pET-15b vector (Novagen) and was expressed in E. coli C41 cells. The protein was purified with Talon metal affinity resin (Clontech). The buffer of the purified protein was exchanged to 20 mM Tris (pH 7.5), 0.02% DDM, and the protein was brought to a concentration of 20 mg/mL by ultrafiltration using Amicon Ultra concentrator (Millipore). The double mutant A167E-I105S was provided by Dr. Gregor Madej (Institute of Biophysics and Physical Biochemistry, University of Regensburg, Germany).

CHAPTER IV

Perfusion-SEIRAS approach for the study of membrane transport proteins ___________________________________________________________________________ 123 Ana Filipa Santos Seiça

-Perfusion-Induced Difference Spectra

All measurements were recorded using a configuration allowing the simultaneous acquisition of Fourier transform infrared (FTIR) spectra in the ATR mode with perfusion of solutions with defined composition. The proteins were immobilized in a Ni-NTA surface [33] as described in Chapter II.

For the data presented here, the pump speed was kept constant at a flow rate of 0.2 mL•min -1 and the measurements were carried out at ∼7 °C with the solutions being kept on ice before use. Before each perfusion step, the input tube was carefully washed with water and buffer. The same procedure was repeated at least five times, and the difference spectra were averaged. Baseline correction was done whenever necessary.

-LACY

In order to monitor pH-induced difference spectra, one perfusion buffer with a pH value of 7.0 was used as a reference (25 mM KPi/100 mM KCl/0.01% dodecyl-β, Dmaltopyranoside) and a second perfusion solution with the same composition, but at different pH values ranging from 8.0 to 11.5, was employed.

The protein immobilized was equilibrated with the buffer solution, KPi at pH 7.0 for 20 min. Once the 20 min finished the spectrum was recorded as background, and the perfusion solution was changed to the second buffer with different pH (pH range: 8.0-11.5). After 20 min (pH 8.0-11.5) minus pH 7.0 difference spectra were recorded. The spectrum of the protein in the new state was recorded as background, and the solution was changed to pH 7.0. Finally, the pH 7.0 minus (pH 8.0-11.5) difference spectra were obtained. This procedure was repeated four times and the difference spectra were smoothed and averaged. A baseline correction was applied.

CHAPTER IV Perfusion-SEIRAS approach for the study of membrane transport proteins

___________________________________________________________________________

CHAPTER IV Perfusion-SEIRAS approach for the study of membrane transport proteins

___________________________________________________________________________

General Conclusion

___________________________________________________________________________

Ana Filipa Santos Seiça

The substitution of the residue Ile105 for a serine led to a change in the microenvironment of this residue, but the pKa of Asp22 corresponded to wild type values. Finally, an additional protonable site was introduced in analogy to the XylE protein, Ala167Glu, and studied together with the double mutation A167E-I105G, we found a strong perturbation of the protonation site of Asp22, indicating that the carboxyl group at position 22 is deprotonated at lower pH.

Relatively to the protein LacY, the residue Glu325 must be protonated for LacY to bind sugar effectively, while deprotonation is also essential for transport. Neutral replacement of Arg302 with Ala has little or no effect on the pKa of Glu325, while replacement with positively charged Lys causes a two-pH unit acid shift. Other mutations in the vicinity of the Glu325 showed little effect on the pKa, confirming that the residue Arg302 is important for deprotonation of Glu325.

APPENDIX I

Figure S1 (II)

Absorbance spectrum of the protein GlcPSe immobilized in a Ni-NTA surface