L’efflux actif, qui permet aux bactéries d’exporter les antibiotiques vers le milieu extérieur est l’un des mécanismes majeurs de résistance aux antibiotiques. L’une des pompes d’efflux de Pseudomonas aeruginosa, MexA-MexB-OprM, est constituée de trois protéines : i) MexA, une protéine membranaire de fusion qui se trouve dans le périplasme ; ii) MexB qui se trouve dans la membrane interne et qui reconnaît l’antibiotique et initie son transport grâce à la force protomotrice et iii) OprM un canal qui se trouve dans la membrane externe. Durant ma thèse, j’ai mis au point un test fonctionnel pour MexA et MexB. Ce test est basé sur la coreconstitution de ces protéines avec la bactériorhodopsine, une protéine membranaire qui génère un gradient de proton après activation par la lumière. L’activité de MexB est suivie de manière indirecte via la mesure du pH. En mesurant le pH à l’intérieur des liposomes, on peut connaître l’activité de MexB puisque ce dernier utilise la force protomotrice pour transporter ses substrats. Une mesure fiable du pH peut être obtenue grâce à la pyranine dont la fluorescence varie avec le pH. Grâce à ce test, j’ai prouvé que MexB possède une activité basale qui ne dépend pas de la présence de substrat et que l’activité de MexB devient optimale quand cette dernière est reconstituée en présence de MexA. Dans un deuxième temps, j’ai mis au point un test fonctionnel pour la pompe d’efflux entière. Pour cela, je prépare deux types distincts de protéoliposomes. Dans le premier type de liposome, j’encapsule de la pyranine, (pour suivre l’activité de MexB) et un substrat de MexB qui est un agent intercalant de l’ARN. Ce substrat est faiblement fluorescent dans un environnement aqueux et fortement fluorescent lorsqu’il est intercalé dans l'ARN. MexB et MexA sont reconstitués dans ces liposomes. Dans le deuxième type de liposomes, je reconstitue OprM et j’encapsule de l’ARN. Ces deux types de liposomes sont alors mélangés. Lorsque la pompe s’assemble et qu’il y a un transport actif à travers cette dernière, deux phénomènes sont observés: la diminution de la fluorescence de la pyranine (car MexB fait entrer des protons dans le premier type de liposome pour transporter le substrat) et l’augmentation de la fluorescence du substrat car ce dernier s’intercale dans l’ARN se trouvant dans le deuxième type de liposome. En mélangeant les deux types de liposomes, j’obtiens une preuve de la reconstitution in vitro de la pompe entière et j’ai mis en évidence qu’OprM s’ouvre en présence de MexA et MexB et que sa présence augmente l’activité de MexB. / Among the various mechanisms developed by the bacteria to counter to the effect of antibiotics, active efflux is on the front line. In Pseudomonas aeruginosa, a Gram negative bacteria, efflux transporters are organized as multicomponent systems where MexB, the pump located in the inner membrane, works in conjunction with MexA, a periplasmic protein, and OprM, an outer membrane protein. MexB is a proton motive force-dependent pump with broad substrate specificity. During my PhD, I have designed an original activity assay for MexB and MexA. The pump is coreconstituted into proteoliposomes together with bacteriorhodopsin (BR), a light-activated proton pump. In this system, upon illumination with visible light, the photo-induced proton gradient created by the BR is shown to be coupled to the active transport of substrates through the pump. The activity of MexB is monitored indirectly. Since MexB uses the protomotive force to transport antibiotics, one can determine substrate transport though MexB by monitoring the pH inside the liposomes. For that purpose, pyranine, a fluorescent probe whose fluorescence yield increases with increasing pH, is encapsulated inside the liposomes. This test makes the investigation of the pump possible. In the absence of MexA, MexB has a basal activity which is not substrate-dependent. Once MexB is reconstituted together with MexA, its activity is specific and substrate-dependent. Then I worked on the reconstitution of the whole efflux pump. For this, I prepare two different kinds of liposomes: i) Liposomes with reconstituted MexA and MexB in which pyranine and a nucleic acid intercalating agent are encapsulated, ii) Liposomes with reconstituted OprM and encapsulated RNA. The activity of MexB is monitored thanks to the addition of EthB, a substrate of MexB, that is poorly fluorescent in aqueous medium and highly fluorescent when intercalated into RNA. Upon generation of a pH gradient, I observe two concomitant phenomena: the decrease of pyranine fluorescence, as MexB is using protons to transport the substrate, and the increase of the fluorescence of the RNA intercalating agent as a result of its interaction with RNA. I have successfully assembled the efflux pump and monitored transport through it from one liposome to the other. I have demonstrated that OprM needs to interact with MexA and MexB in order to open and that MexB activity is accelerated when the pump is assembled.

Sodium/proton exchangers are ubiquitous secondary active transporters that can be found in all kingdoms of life. These proteins facilitate the transport of protons in exchange for sodium ions to help regulate internal pH, sodium levels, and cell volume. Na+/H+ exchangers belong to the SLC9 family and are involved in many physiological processes including cell proliferation, cell migration and vesicle trafficking. Dysfunction of these proteins has been linked to physiological disorders, such as hypertension, heart failure, epilepsy and diabetes. The goal of my thesis is to establish the molecular basis of ion exchange in Na+/H+ exchangers. By establishing how they bind and catalyse the movement of ions across the membrane, we hope we can better understand their role in human physiology. In my thesis, I will first present an overview of Na+/H+ exchangers and their molecular mechanism of ion translocation as was currently understood by structural and functional studies when I started my PhD studies. I will outline our important contributions to this field, which were to (i) obtain the first atomic structures of the same Na+/H+ exchanger (NapA) in two major alternating conformations, (ii) show how a transmembrane embedded lysine residue is essential for carrying out electrogenic transport, and (iii) isolate and recorde the first kinetic data of a mammalian Na+/H+ exchanger (NHA2) in an isolated liposome reconstitution system.

Récemment, le système de sécrétion de type VI (SST6) a été identifié comme un nouvel acteur clé dans la compétition inter-bactérienne parmi le large arsenal dont dispose les bactéries. L’une des particularités du SST6 est de cibler à la fois des cellules eucaryotes et procaryotes. Le T6SS est un complexe protéique formé par l’assemblage de deux ‘sous-complexes’. Le premier sert à l’ancrage de la machinerie au sein de l’enveloppe bactérienne et le second agit comme une arbalète moléculaire. Le mécanisme d’action du SST6 est très similaire à celui d’autres machineries contractiles telles que celui des bactériophages : la contraction d’un fourreau propulse une flèche, composée d’un tube avec une aiguille à son extrémité, directement dans la cellule cible afin de délivrer les différentes toxines. Mon projet de thèse consiste à comprendre quelles sont la structure et la biogénèse des deux différents complexes et de comprendre comment ils sont assemblés. Nous utilisons comme modèle la bactérie pathogène à Gram négatif Escherichia coli entéroagrégative. J’ai pu démontrer que le complexe membranaire est assemblé en premier, avec l’adressage de la lipoprotéine de membrane externe TssJ, puis le recrutement séquentiel de TssM et TssL, deux protéines de membrane interne. Le complexe membranaire recrute ensuite une plateforme d’assemblage, appelée ‘baseplate’. Nous avons identifié et caractérisé les composants de cette ‘baseplate’ qui sert de plateforme d’assemblage pour le recrutement du reste de la machinerie (fourreau et flèche). Enfin, nous avons identifié et déterminé le rôle de la protéine TssA, une protéine qui coordonne la polymérisation du fourreau et de la flèche. / Among the broad weaponry of bacteria, the recently identified type VI secretion system (T6SS) emerges as one of the key player in bacterial competition. T6SS is a versatile machinery that targets both eukaryotic and prokaryotic cells. This molecular weapon assembles two evolutionarily different sub-assemblies. One complex anchors the machinery to the cell envelope while the second acts as a molecular crossbow. The mechanism of action of the T6SS is similar to other known contractile machineries such as bacteriophages: the contraction of a sheath propels an arrow, constituted of a tail tube capped by a cell-puncturing device, directly into the prey cell to deliver effector toxins. My Ph.D project was to provide mechanistic details on the structure and biogenesis of the two T6SS sub-complexes and to understand how they are connected, using entero-aggregative Escherichia coli as model bacterium. I have demonstrated that the membrane complex is assembled first and starts with the positioning of the outer membrane TssJ lipoprotein and proceeds inward, from the outer to the inner membrane, through the sequential recruitment of the TssM and TssL subunits. After assembly, the membrane complex recruits an assembly platform called the baseplate. We identified and characterized the components of this baseplate, which serves as assembly platform for the tail. We further demonstrated that the functional and physical interaction between the T6SS membrane complex and the baseplate is mediated by multiple contacts. Finally, we identified and deciphered the role of TssA, a protein that coordinates the polymerizations of the tail tube and sheath.

The expression of polysaccharide capsules is common in bacteria and associated with virulence in some pathogenic strains. Strains of the Gram-negative bacterium Escherichia coli express a structurally diverse range of capsular polysaccharides. E. coli strains expressing group 2 capsules are associated with a number of extra-intestinal infections, including sepsis, urinary tract infections, and neonatal meningitis. Group 2 capsular polysaccharides are synthesised on the cytoplasmic face of the inner membrane. Evidence from previous work suggests that export of polysaccharides across the Gram-negative membranes involves four transport proteins which interact to form a continuous membrane-spanning translocation complex (the KpsMTED translocon). Polysaccharide translocation across the inner membrane requires the ABC transporter KpsMT, in which KpsM is the integral inner membrane component and KpsT is the ATPase. Transport across the periplasmic space and outer membrane involves the integral inner membrane protein KpsE and the outer membrane protein KpsD, respectively. This thesis addressed some of the key areas in the study of group 2 polysaccharide transport by employing the K5 capsule as a model system. Using biochemical and molecular genetics approaches, the study focused on establishing functional and structural characteristics of the translocon members and analysing protein-protein interactions within the complex. This study demonstrated that KpsE can self-associate as dimers, tetramers and possibly higher order oligomers in the absence of other capsule gene products and the K5 substrate. A mutagenesis study of KpsE revealed that the periplasmic, membrane-associated C-terminus is essential for correct protein function. Work presented here confirmed previous data, which suggested a direct interaction between KpsE and KpsM, by alternative methods, and demonstrated that the C-terminal domain of KpsE is required for this interaction. Further experiments suggested that KpsE and KpsM can both form higher order oligomers when interacting as a complex. The C-terminus of KpsE is not required for an interaction between KpsE and KpsD, and the two proteins are thus more likely to interact via their respective periplasmic domains. Generation of a theoretical model of the secondary structure and topology of KpsD predicted that KpsD is made primarily of β-sheets with some interspersed α-helices, including a larger coiled coil region. The theoretical topology model proposed an N-terminal transmembrane domain made of eight membrane-spanning regions, and a large periplasmic domain. Substituted-cysteine accessibility method and myc-epitope insertion analysis were both assessed for their suitability for topology analysis of KpsD. Myc-epitope insertion was identified as the recommended approach for future topology study. Myc-epitope tagging of the periplasmic C-terminus of KpsD revealed that a native C-terminus is essential for correct KpsD function.In conclusion, this thesis contributes to the model of group 2 polysaccharide export in E. coli, and, more generally, provides clues about the transport of high-molecular weight molecules across Gram-negative membranes. It is hoped that a thorough understanding of polysaccharide transport might reveal therapeutic targets to block capsule export in pathogenic E. coli in the future.

O desenvolvimento de uma vacina contra malária causada por P. falciparum é prejudicado pelo alto nível de polimorfismo apresentado pelos antígenos desse parasito. O dimorfismo alélico é um padrão no qual os alelos observados de um gene se encontram divididos em duas famílias. A proteína dimórfica MSP-6 se associa à proteína MSP-1 (também dimórfica) na superfície do merozoíto. Genes de msp-6 de 21 isolados obtidos de pacientes do Brasil, mais 2 isolados da Tanzânia, África, foram seqüenciados para estudo da diversidade nucleotídica e distribuição geográfica dos alelos. As duas famílias possuem distribuição global. Não foi verificada associação entre o dimorfismo de MSP-1 e MSP-6. O gene ortólogo de msp-6 em P. reichenowi, grupo irmão de P. falciparum, foi seqüenciado para estudos evolutivos. Os alelos dimórficos de MSP-6 aparentam ter surgido de uma população ancestral polimórfica, tendo sido mantidos no presente por seleção balanceada. O alto grau de conservação encontrado dentro de cada família alélica torna MSP-6 um potencial alvo de uma vacina contra a malária. / The development of a vaccine against malaria caused by Plasmodium falciparum has been hampered by the high level of antigen polymorphism exhibited by this parasite. Allelic dimorphism is a pattern in which every observed allele of a gene is clearly grouped into one of two families. The dimorphic protein MSP-6 forms a complex with MSP-1 (also dimorphic) on merozoite surface. The msp-6 genes were sequenced in isolates obtained from 21 patients from Brazil, plus 2 isolates from Tanzania, Africa, to study nucleotide diversity and geographic distribution of alleles. Both families are globally distributed. Moreover, no association was observed between the MSP-1 and MSP-6 allelic types. Orthologous gene of msp-6 in P. reichenowi, chimpanzee parasite and sister group of P. falciparum, was sequenced for evolutionary studies. Dimorphic alleles of MSP-6 seem to have originated from an ancestral polymorphic population and are maintained by balancing selection. The high degree of conservation observed within each allelic family makes MSP-6 an promising target for vaccine development.

Cilia are conserved organelles that extend from the surface of most eukaryotic cells. During development cilia play key roles in force generation and perception of the extracellular environment. Ciliary defects cause a broad class of human diseases called ciliopathies characterized by pleiotropic symptoms including cystic kidneys, retinal degeneration, cardiac malformations and skeletal deformations. Perception of the environment relies on specific proteins being localized to the ciliary membrane compartment. The mechanism for sorting and trafficking membrane proteins to the cilium is poorly understood. To address this question, I developed a fluorescence-based pulse-chase assay to measure the transport kinetics of ciliary membrane proteins. This assay was used to determine the importance of candidate proteins to the delivery of fibrocystin, polycystin-2, and smoothened to cilia. Using this assay, I found that ciliary delivery of fibrocystin and polycystin-2 requires IFT20, GMAP210 and the exocyst while smoothened delivery is largely independent of these proteins. In addition, I determined that polycystin-2, but not smoothened or fibrocystin require the biogenesis of lysosome related organelles complex-1 (BLOC-1) for ciliary delivery. Consistent with a requirement for BLOC-1 in ciliary transport of polycystin-2, BLOC-1 mutant mice have cystic kidney disease. BLOC-1 functions in endosomal sorting and I find that disrupting the recycling endosome also reduced ciliary polycystin-2 and causes its accumulation in the recycling endosome. This is the first demonstration of a role for BLOC-1 in ciliary biogenesis and highlights the complexity of trafficking pathways to the cilium.

Les ATPases-P4 sont des transporteurs membranaires couplant l'hydrolyse de l'ATP au transport de lipides dans les membranes cellulaires eucaryotes. Avec leurs partenaires, les protéines CDC50, les ATPases-P4 transportent les phospholipides, en particulier la phosphatidylsérine (PS) et la phosphatidyléthanolamine (PE), du feuillet exoplasmique au feuillet cytosolique des membranes, assurant ainsi le maintien de l'asymétrie membranaire.Drs2p est l'une des cinq ATPases-P4 de la levure Saccharomyces cerevisiae. Elle est localisée dans les membranes du trans-Golgi (TGN), et elle a comme partenaire la protéine Cdc50p, qui est nécessaire à l'adressage correct et probablement au transport catalysé par Drs2p. Drs2p est principalement responsable du transport de la phosphatidylsérine (PS) dans les membranes du TGN et son activité est essentielle pour le maintien de la PS dans le feuillet cytosolique de ces membranes. En raison du rôle crucial de la PS dans de nombreuses voies de signalisation, aussi bien à l’extérieur (au cours de l’apoptose par exemple) qu’à l’intérieur de la cellule (par le recrutement de protéines impliquées dans des processus cellulaires essentiels), il est important de comprendre le mécanisme par lequel l’asymétrie de la PS est établie.Afin de progresser dans la compréhension du mécanisme moléculaire du transport de lipides, nous avons mis au point une procédure qui nous a permis de co-exprimer Drs2p et Cdc50p dans Saccharomyces cerevisiae. La purification de Drs2p par chromatographie d'affinité sur résine streptavidine a permis d'obtenir une fraction purifiée contenant très majoritairement Drs2p et Cdc50p, à raison de 1-2 mg/L de culture. Les deux protéines sont sous forme de complexe avec une stœchiométrie d'association de 1:1. Le complexe purifié est fonctionnel, et présente une activité d’hydrolyse de l’ATP stimulée par son substrat, la PS. Cette stimulation n’est cependant possible qu’en présence de PI4P, un phosphoinositide impliqué dans la régulation du trafic membranaire.De par leur rôle crucial dans le maintien de l'asymétrie membranaire, les ATPases-P4 ne peuvent qu'être régulées. Comme de nombreuses ATPases de type P sont soumises à une auto-régulation de leur activité, nous avons examiné la possibilité d’une telle auto-régulation dans le cas des ATPases P4. Pour ce faire, une approche par mutagenèse dirigée et protéolyse ménagée associée à l’identification par spectrométrie de masse des peptides ont été effectuées. La protéolyse ménagée du complexe purifié Drs2p/Cdc50p montre une activité ATPasique dépendante au PI4P de 30-50 fois plus importante. La protéolyse par la thrombine engendre un Drs2p dépourvu d'une partie N-terminale (R104) et d'une partie C-terminale (R1290) qui reste toujours associé à Cdc50p. Ce résultat montre qu'une coupure appropriée au niveau des extrémités terminales de Drs2p peut augmenter de façon significative, en présence du PI4P, l'activité ATPasique du complexe, nous amenant ainsi à identifier un rôle auto-inhibiteur des extrémités N- et/ou C-terminales de Drs2p.Ce travail ouvre des perspectives quant à la caractérisation structurale et fonctionnelle du mécanisme de transport de lipides par le complexe. Par ailleurs, il laisse entrevoir la possibilité d’étudier les bases moléculaires des pathologies associées aux mutations de certaines ATPases P4 humaines. / Maintenance of phospholipid asymmetry in eukaryotic cell membranes is essential for cellular integrity and function. P4-ATPases, from the P-type ATPases family, are energy-dependent transporters, together with their CDC50 accessory subunits couple ATP hydrolysis to lipid transport from the exoplasmic to cytoplasmic leaflet to maintain membrane asymmetry.Drs2p is one of these P4-ATPases in the yeast Saccharomyces cerevisiae. Drs2p is localised in trans-Golgi (TGN) membranes in association with its binding partner Cdc50p, which contributes to the correct addressing of Drs2p and probably in the catalyzed transport by Drs2p. Drs2p transport principally phosphatidylserine (PS) in TGN membranes. The PS is important for a several signalling pathways, for example, in apoptosis and recruitment of the proteins implied in various essential cellular process, so, it's very important to understand the mechanism that establishes this asymmetry.To gain in comprehension of molecular mechanism of lipid transport, robust protocols for expression and purification are required. In this work, we present a procedure for high-yield co-expression of Drs2p and Cdc50p. The purification of Drs2p and Cdc50p is achieved in a single step by affinity chromatography on streptavidin beads, yielding, 1-2 mg purified Drs2p/Cdc50p per liter of culture. This procedure allows purification of the complex Drs2p/Cdc50p with stoichiometry to 1:1. Our complex is functional, overal ATP hydrolysis by the complex is dependent of PS, favourite substrate of Drs2p. This hydrolyze is critically dependent on the presence of PI4P, a phosphoinositide involved in regulation of membrane trafficking.Like many P-type ATPases auto-regulate their activity, we examined the possibility that P4-ATPases are auto-regulated. In this work, we use directed mutagenesis and limited proteolysis associated with mass spectrometry for identify peptides. We show that limited proteolysis of a purified complex Drs2p/Cdc50p resulted in up to a 30-50 fold increase of it ATPase activity, which however remained dependent on PI4P. Using thrombin as the protease, Cdc50p remained intact and in complex with Drs2p, which was cleaved at two positions, namely after R104 and after R1290. Our results therefore reveal that trimming off appropriate regions of the terminal extensions of Drs2p can increase its ATPase activity in the presence of PI4P by an enormous factor, thereby identifying a role of N and/or C-terminal extensions in auto-inhibition of Drs2p.Our results open perspectives on the structural and the functional characterization of the lipid transport mechanism by the complex Drs2p/Cdc50p. Furthermore, our procedures open up the possibility of studying the molecular bases of the pathologies associated with the mutations of human P4-ATPases.

abstract: Coronaviruses are the causative agents of SARS, MERS and the ongoing COVID-19 pandemic. Coronavirus envelope proteins have received increasing attention as drug targets, due to their multiple functional roles during the infection cycle. The murine coronavirus mouse hepatitis virus strain A59, a hepatic and neuronal tropic coronavirus, is considered a prototype of the betacoronaviruses. The envelope protein of the mouse hepatitis virus (MHV-E) was extensively screened with various membrane mimetics by solution state nuclear magnetic resonance spectroscopy to find a suitable mimetic, which allowed for assignment of ~97% of the backbone atoms in the transmembrane region. Following resonance assignments, the binding site of the ion channel inhibitor hexamethylene amiloride (HMA) was mapped to MHV-E using chemical shift perturbations in both amide and aromatic transverse relaxation optimized spectroscopy (TROSY) spectra, which indicated the inhibitor binding site is located at the N-terminal opening of the channel, in accord with one of the proposed HMA binding sites in the envelope protein from the related SARS (severe acute respiratory syndrome) betacoronavirus. Structure calculation of residues M1-K38 of MHV-E, encompassing the transmembrane region, is currently in progress using dihedral angle restraints obtained from isotropic chemical shifts and distance restraints obtained from manually assigned NOE cross-peaks, with the ultimate aim of generating a model of the MHV-E viroporin bound to the inhibitor HMA. This work outlines the first NMR studies on MHV-E, which have provided a foundation for structure based drug design and probing interactions, and the methods can be extended, with suitable modifications, to other coronavirus envelope proteins. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2020

Intramembrane proteases from the rhomboid-like superfamily are enzymes widely distributed and conserved in all domains of life. They participate in many important processes such as membrane protein quality control or mitochondrial dynamics. Their activity is also linked with diseases like Parkinson's disease or cancer. This makes them potential therapeutic targets. In this work we tried to elucidate in more detail the mechanism of action of the main model intramembrane protease, GlpG from E. coli. We also focused on the mechanism of eukaryotic rhomboid RHBDL2, one of the four mammalian rhomboids, function of which is poorly understood. To acquire more detailed information about substrate-enzyme interaction, we synthesized a series of novel peptidyl-chloromethylketone inhibitors derived from natural rhomboid substrate TatA from P. stuartii. Crystal structure of the complex of GlpG with these inhibitors revealed four substrate binding subsites (S1 to S4) of the enzyme and explained its observed substrate specificity structurally. This study showed that substrate cleavage rate can be dramatically modified by changing the substrate sequence in positions P1 to P5. This helped us develop fluorogenic transmembrane peptide substrates for rhomboid proteases, which are usable in detergent and liposomes, and...

Understanding the conformational and dynamic changes of biomacromolecular complexes in different states, such as the membrane protein photoreceptor-transducer complex NpSRII/NpHtrII, is a key step to gaining insight into the functional mechanism of these important classes of protein complexes, since ~30 % of the human proteome are membrane proteins, yet they are largely underrepresented in terms of structural information with <1 % of all structures in the protein data bank. Hence for the development of methods suitable to study the conformation and dynamics of such complexes there is a strong demand and a vast potential field of applications. Here we combined method development at the interface between biomolecular simulations and model-based analysis of EPR- and fluorescence spectroscopic data with application studies using state-of-the-art spectroscopic techniques in conjunction with site-directed spin- or fluorescence labeling. In an initial benchmark study on the rigid globular protein complex Rpo4/7, we compared experimental inter fluorescence label distances or spin label distance distributions to a variety of predicted inter label distances based on molecular dynamics simulations, Monte Carlo sampling and a discrete rotamer library analysis. We found that while for the molecular dynamics simulations with explicit solvent considerable sampling challenges have to be overcome to reproduce the experimentally observed inter label distance distributions, the Monte Carlo sampling performed well when compared to the experimental data and was computationally less demanding. Significantly more efficient and equally accurate for our examples was the so-called rotamer library analysis available for the spin labels since it relies on a pre-calculated set of rotational isomers. In general, predictions for the mean distances were in agreement within the error margins while distribution shapes were more challenging to reproduce. Overall this study shows a positive evaluation for the assessed tools and the developed simulation protocols as well as their potential applications. Using the combination of EPR and fluorescence spectroscopy for distance determination we studied the structural influence of RNA binding on Rpo4/7, and showed that the protein complex stays conformationally rigid and thereby serves as a guiding rail for the nascent RNA chain that leaves the RNA polymerase along the Rpo4/7 RNA binding interface. To enhance the interpretation of experimentally determined changes of conformation and dynamics in protein complexes and to discuss the observed changes in terms of structural information, we built models of the two transcription factors TFE and the Spt4/5 complex, as well as of Argonaute, a 713 amino acid four-domain protein nuclease from Methanocaldococcus jannaschii. These structural models not only allowed a more accurate planning of fluorescence or EPR labeling experiments, but also the models enabled the discussion of the experimental data in structural terms. Based on such an initial structure further computational analysis techniques may be applied to identify putative structural changes or dynamic modes. This was shown for the histidine transporter HisQMP2, where we combined normal mode analysis to model protein flexibility with the rotamer library analysis to screen for possible conformational changes in comparison to experimental inter spin distance data. The most prominent agreement with one mode led to a working hypothesis of a conformational change and provides the basis for validation in future experiments. Due to the inherent synergy effects, we applied a combined experimental and simulation approach for the EPR-based distance determination in the globular DNA-binding protein LexA to probe conformation and dynamics of the N-terminal DNA-binding domains with respect to the C-terminal domains within the LexA homodimer. While the C-terminal dimerization domains exhibit a well-defined conformation that proved to be independent of DNA-binding, large-scale changes in conformation and dynamics were detected for the N-terminal domains. They were only found in a defined conformation when bound to DNA while in its absence a large rotational freedom of the entire N-terminal domains contributed to the conformational ensemble. Combined with a biochemical characterization of the autocatalytic cleavage of LexA, our data explains how LexA induces the SOS response after DNA damage or under latent antibiotic stress. We further studied the membrane photoreceptor-transducer complex NpSRII/NpHtrII that governs the light-dependent swimming behavior in Natronomonas pharaonis by a two-component signaling system. This system comprises extraordinary features of sensitivity, signal amplification, integration and transducer cooperativity, yet the molecular details of these features are poorly understood, as is signal propagation itself. By combining time-resolved cw EPR spectroscopy of NpSRII/NpHtrII variants spin labeled in the HAMP1 domain with time-resolved optical absorbance spectroscopy to report on the receptor signaling state, we found a tight kinetic coupling of receptor and transducer during the relaxation back to the ground state and hence a prolonged activation period, that with ~500 - ~700 ms is sufficiently long to cause phosphorylation bursts of the cognate kinase CheA. This explains signal amplification already on the level of the NpSRII/NpHtrII dimers. We further determined the transient difference spectra from the time-resolved EPR data that show local differences in dynamics and steric restrictions upon light-activation. Comparing these experimentally observed differences to predictions confirms the assumed two-state structural model and shows this transition between the two states for a single HAMP domain in a light-dependent manner. Additionally, our approach integrates a dynamic view into the model, since the two states are shown to exhibit different local dynamics in a fashion described previously as a competing model for signaling by dynamic differences based on biochemical studies. Here we show unification of the two models into one congruent description encompassing a transition between the two previously suggested states by concerted structural and dynamic changes. In an independent analysis using all-atom and coarse grained molecular dynamics of the NpSRII/NpHtrII complex in the minimal unit that can exert kinase control, the trimer of receptor-transducer dimers, we revealed a distinct dynamical pattern encoded in the primary sequence of the coiled-coil heptad-repeats. Upon receptor activation, these segments alter their dynamics in a concerted fashion with regions such as HAMP1 and the adaptation region becoming more compact, while HAMP2 and the tip become more dynamic, leading to dynamic and to limited structural changes at the CheA-kinase binding sites. Together with an extensive validation against experimental data, these findings suggest the altered dynamics as the mechanism for signal propagation along the extended coiled-coil structure of NpHtrII. This working model, that explains the current body of experimental data, allows for further refinement by all-atom molecular dynamics and provides a basis to devise future experiments for validation. The presented studies outline the versatile methodology of combined experimental and simulation approaches to analyze the conformation and dynamics of biomacromolecules including membrane protein complexes.