Structural and functional investigation of flavin binding center of the NqrC subunit of sodium-translocating NADH:Quinone oxidoreductase from Vibrio harveyi

Na+-translocating NADH:quinone oxidoreductase (NQR) is a redox-driven sodium pump operating in the respiratory chain of various bacteria, including pathogenic species. The enzyme has a unique set of redox active prosthetic groups, which includes two covalently bound flavin mononucleotide (FMN) residues attached to threonine residues in subunits NqrB and NqrC. The reason of FMN covalent bonding in the subunits has not been established yet. In the current work, binding of free FMN to the apo-form of NqrC from Vibrio harveyi was studied showing very low affinity of NqrC to FMN in the absence of its covalent bonding. To study structural aspects of flavin binding in NqrC, its holo-form was crystallized and its 3D structure was solved at 1.56 Å resolution. It was found that the isoalloxazine moiety of the FMN residue is buried in a hydrophobic cavity and that its pyrimidine ring is squeezed between hydrophobic amino acid residues while its benzene ring is extended from the protein surroundings. This structure of the flavin-binding pocket appears to provide flexibility of the benzene ring, which can help the FMN residue to take the bended conformation and thus to stabilize the one-electron reduced form of the prosthetic group. These properties may also lead to relatively weak noncovalent binding of the flavin. This fact along with periplasmic location of the FMN-binding domains in the vast majority of NqrC-like proteins may explain the necessity of the covalent bonding of this prosthetic group to prevent its loss to the external medium

THz streak camera performance for single-shot characterization of XUV pulses with complex temporal structures

The THz-field-driven streak camera has proven to be a powerful diagnostic-technique that enables the shot-to-shot characterization of the duration and the arrival time jitter of free electron laser (FEL) pulses. Here we investigate the performance of three computational approaches capable to determine the duration of FEL pulses with complex temporal structures from single-shot measurements of up to three simultaneously recorded spectra. We use numerically simulated FEL pulses in order to validate the accuracy of the pulse length retrieval in average as well as in a single-shot mode. We discuss requirements for the THz field strength in order to achieve reliable results and compare our numerical study with the analysis of experimental data that were obtained at the FEL in Hamburg - FLASH. © 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreemen

Application of amphipols for surface-enhanced Raman spectroscopy and X-ray crystallography studies of membrane proteins

Les amphipoles (APols) sont devenus des outils importants pour la stabilisation, le repliement, et les études structurales et fonctionnelles in vitro des protéines membranaires (MPs). Les MPs sont les unités fonctionnelles des biomembranes et représentent environ un tiers des protéines qui sont codées par le génome. La première partie de mon travail est dédiée à la cristallisation de MPs piégée par des APol. La cristallisation directe de protéines solubilisées en APol sera d'une grande importance pour la biologie structurale. Cependant, malgré des efforts considérables, il n'est pas certain que les complexes MP/APol peuvent être utilisés pour former des cristaux bien ordonnés utilisables en cristallographie des rayons X. Le premier objectif de cette thèse est de montrer que les MPs piégées par des APol peuvent être cristallisées in meso. Pour faire cela, nous avons utilisé des bicouches amphiliques interconnectées qui sont ajustables pour certaines MPs. Cette méthode a été récemment développée dans notre laboratoire. Nous avons utilisé la bactériorhodopsin (BR) piégée avec APol A8-35 comme système modèle pour nos études cristallographiques. Le premier cristal obtenu diffractait à 3 Å, alors qu'une nouvelle méthode de cristallisation en nanovolume, exploitant des précipitants secs, améliore les pics de diffraction aux rayons X observés jusqu'a 2 Å. La structure de BR a été résolue à 2 Å et s'est révélée identique aux autre structures obtenues précédemment à partir de protéine solubilisée en détergents. Nous suggérons que le protocole proposé, de cristallisation in meso, est applicable aux MPs solubilisées avec des APols.La deuxième partie est liée aux applications des APols pour les études de MPs à l'aide de spectroscopie Raman exaltée de surface (SERS). La spectroscopie SERS a énormément évolué depuis sa découverte en 1970. C'est un outil analytique puissant pour sélectionner les molécules qui adsorbent sur des nanoparticules et des nanostructures à base de métaux nobles, possiblement au niveau de la molécule unique. Malheureusement les études de MPs sont loin de l'application courante du SERS à cause de la difficulté résultante de la nature amphiphilique des MPs. La capacité des APols à piéger les MPs et de les garder solubles, stables et fonctionnelles ouvre la voie pour des applications extrêmement intéressantes des études SERS, éventuellement au niveau de la molécule unique. De plus, le deuxième objectif de ce travail de thèse était de démontrer la faisabilité de l'utilisation de SERS avec des MPs piégées par des APols. Le même modèle (BR/A8-35) a été utilisé pour les études cristallographiques et pour les agrégats de NP d'argent. Cette tâche a été réalisée a un niveau suffisant pour commencer des études de MPs avec la méthode SERS.Le premier chapitre de cette thèse commence avec des informations générales à propos de l'importance des études de MPs et les problèmes inhérents à leur manipulation. Plus loin dans le chapitre, un bref résumé des APols, de leurs propriétés et leurs applications est présenté. La majeure partie de l'introduction est dédiée aux points importants des différentes approches de cristallisation de MPs et de spectroscopie Raman, en particulier SERS spectroscopie, et leurs applications aux protéines. La fin de la partie “Introduction” présente les conclusions à propos des applications des APols pour les études de cristallographie aux rayons X et pour les études de spectroscopie SERS sur les MPs, définissant les objectifs principaux pour ce travail. Le chapitre “Materials and methods” consiste en une description détaillée des matériels et des protocoles utilisés dans cette étude. Le résultat des études de cristallisation et de SERS et leurs interprétations sont présentés comme deux différentes parties dans le dernier chapitre “Results and discussions”. Le chapitre “Conclusions and perspectives est présent dans chaque partie.Amphipols (APols) have become important tools for the stabilization, folding, and in vitro structural and functional studies of membrane proteins (MPs). MPs are the main functional units of biomembranes and represent roughly one-third of the proteins encoded in the genome. The first part of my work was dedicated to crystallization of a MP trapped by APol. Direct crystallization of MPs solubilized in APols would be of high importance for structural biology. However, despite considerable efforts, it is still not clear whether MP/APol complexes can be used to form well-ordered crystals suitable for X-ray crystallography. The first major goal of this PhD thesis work was to show that APol-trapped MP can be crystallized in meso. To perform it we utilized special, flexibly adjustable for a certain MP, interconnected amphiphilic bilayers (IAB) approach which has been recently developed in our laboratory. We used bacteriorhodopsin (BR) trapped with APol A8-35 as a model system for our crystallization studies. The first obtained crystals diffracted to 3 Å, while a new developed type of high throughput nanovolume crystallization, exploiting dry precipitants, shifted the observed X ray diffraction peaks beyond 2 Å. The structure of BR was solved to 2 Å and found to be indistinguishable from previous structures obtained with a detergent-solubilized protein. We suggest that the proposed protocol of in meso crystallization is generally applicable to APol-trapped MPs.The second, to a certain extent, complementary part of the present work was related to application of APols to the surface-enhanced Raman scattering (SERS) studies of MPs. SERS spectroscopy has been developed dramatically since its discovery in the 1970s. It is a powerful analytical tool for selective sensing of molecules adsorbed onto noble metal nanoparticles (NPs) and nanostructures, including at the single molecule (SM) level. Unfortunately, MPs studies are far away from the main stream of SERS applications due to the great handling difficulties resulting from the amphiphilic nature of MPs. The ability of APols to trap MPs and keep them soluble, stable and functional opens the way for highly interesting applications of SERS studies, possibly at the SM level. Thus, the second goal of this PhD thesis work was to prove our concept of feasibility of SERS with MPs trapped by APols. Using the same as in the crystallization studies model BR/A8-35 complexes and silver NP aggregates, the task was fulfilled to a degree enough to start with the SERS studies of MPs.The first chapter of the PhD thesis begins with general information about the importance of MP studies and the problems with their handling. Further in this chapter, a brief overview of APols, their properties and applications is presented. The largest part of the “Introduction” is dedicated to main points of different MP crystallization approaches and Raman spectroscopy, in particular SERS spectroscopy, and their applications to proteins. The end of the “Introduction” part presents the conclusions about APol application for X-ray crystallography and SERS spectroscopy studies of MPs, setting the main goals for the present work. The “Materials and methods” chapter consists of detailed description of the materials and protocols used in this study. The results of crystallization and SERS studies and their interpretations are presented as two different parts in the last “Results and discussions” chapter. The “Conclusions and perspectives” sections accompany each of these parts

Crystal Structure of a Proteolytic Fragment of the Sensor Histidine Kinase NarQ

Two-component signaling systems (TCSs) are a large and important class of sensory systems in bacteria, archaea, and some eukaryotes, yet their mechanism of action is still not fully understood from the structural point of view. Many TCS receptors are elongated flexible proteins with transmembrane (TM) regions, and are difficult to work with. Consequently, truncated fragments of the receptors are often used in structural studies. However, it is not fully clear whether the structures of the fragments correspond well to their native structures in the context of full-length proteins. Recently, we crystallized a fragment of Escherichia coli nitrate/nitrite sensor histidine kinase, NarQ, encompassing the sensor, TM, and HAMP domains. Here we report that a smaller proteolytic fragment consisting of the sensor and TM domains can also be crystallized using the in meso approach. The structure of the fragment is similar to the previously determined one, with minor differences in the vicinity of the truncation site. The results show that the crystallization of such sensor–TM fragments can be accomplished and can provide information on the packing of transmembrane helices, albeit limited, and that the proteolysis may or may not be a problem during crystallization

Sensor Histidine Kinase NarQ Activates via Helical Rotation, Diagonal Scissoring, and Eventually Piston-Like Shifts

Membrane-embedded sensor histidine kinases (HKs) and chemoreceptors are used ubiquitously by bacteria and archaea to percept the environment, and are often crucial for their survival and pathogenicity. The proteins can transmit the signal from the sensor domain to the catalytic kinase domain reliably over the span of several hundreds of angstroms, and regulate the activity of the cognate response regulator proteins, with which they form two-component signaling systems (TCSs). Several mechanisms of transmembrane signal transduction in TCS receptors have been proposed, dubbed (swinging) piston, helical rotation, and diagonal scissoring. Yet, despite decades of studies, there is no consensus on whether these mechanisms are common for all TCS receptors. Here, we extend our previous work on Escherichia coli nitrate/nitrite sensor kinase NarQ. We determined a crystallographic structure of the sensor-TM-HAMP fragment of the R50S mutant, which, unexpectedly, was found in a ligand-bound-like conformation, despite an inability to bind nitrate. Subsequently, we reanalyzed the structures of the ligand-free and ligand-bound NarQ and NarX sensor domains, and conducted extensive molecular dynamics simulations of ligand-free and ligand-bound wild type and mutated NarQ. Based on the data, we show that binding of nitrate to NarQ causes, first and foremost, helical rotation and diagonal scissoring of the α-helices at the core of the sensor domain. These conformational changes are accompanied by a subtle piston-like motion, which is amplified by a switch in the secondary structure of the linker between the sensor and TM domains. We conclude that helical rotation, diagonal scissoring, and piston are simply different degrees of freedom in coiled-coil proteins and are not mutually exclusive in NarQ, and likely in other nitrate sensors and TCS proteins as well

An Approach to Heterologous Expression of Membrane Proteins. The Case of Bacteriorhodopsin.

International audienceHeterologous overexpression of functional membrane proteins is a major bottleneck of structural biology. Bacteriorhodopsin from Halobium salinarum (bR) is a striking example of the difficulties in membrane protein overexpression. We suggest a general approach with a finite number of steps which allows one to localize the underlying problem of poor expression of a membrane protein using bR as an example. Our approach is based on constructing chimeric proteins comprising parts of a protein of interest and complementary parts of a homologous protein demonstrating advantageous expression. This complementary protein approach allowed us to increase bR expression by two orders of magnitude through the introduction of two silent mutations into bR coding DNA. For the first time the high quality crystals of bR expressed in E. Coli were obtained using the produced protein. The crystals obtained with in meso nanovolume crystallization diffracted to 1.67 Å

Crystal structure of Escherichia coli-expressed Haloarcula marismortui bacteriorhodopsin I in the trimeric form.

International audienceBacteriorhodopsins are a large family of seven-helical transmembrane proteins that function as light-driven proton pumps. Here, we present the crystal structure of a new member of the family, Haloarcula marismortui bacteriorhodopsin I (HmBRI) D94N mutant, at the resolution of 2.5 Å. While the HmBRI retinal-binding pocket and proton donor site are similar to those of other archaeal proton pumps, its proton release region is extended and contains additional water molecules. The protein's fold is reinforced by three novel inter-helical hydrogen bonds, two of which result from double substitutions relative to Halobacterium salinarum bacteriorhodopsin and other similar proteins. Despite the expression in Escherichia coli and consequent absence of native lipids, the protein assembles as a trimer in crystals. The unique extended loop between the helices D and E of HmBRI makes contacts with the adjacent protomer and appears to stabilize the interface. Many lipidic hydrophobic tail groups are discernible in the membrane region, and their positions are similar to those of archaeal isoprenoid lipids in the crystals of other proton pumps, isolated from native or native-like sources. All these features might explain the HmBRI properties and establish the protein as a novel model for the microbial rhodopsin proton pumping studies

Demonstration of electron diffraction from membrane protein crystals grown in a lipidic mesophase after lamella preparation by focused ion beam milling at cryogenic temperatures

Electron crystallography of sub-micrometre-sized 3D protein crystals has emerged recently as a valuable field of structural biology. In meso crystallization methods, utilizing lipidic mesophases, particularly lipidic cubic phases (LCPs), can produce high-quality 3D crystals of membrane proteins (MPs). A major step towards realizing 3D electron crystallography of MP crystals, grown in meso, is to demonstrate electron diffraction from such crystals. The first task is to remove the viscous and sticky lipidic matrix that surrounds the crystals without damaging the crystals. Additionally, the crystals have to be thin enough to let electrons traverse them without significant multiple scattering. In the present work, the concept that focused ion beam milling at cryogenic temperatures (cryo-FIB milling) can be used to remove excess host lipidic mesophase matrix is experimentally verified, and then the crystals are thinned to a thickness suitable for electron diffraction. In this study, bacteriorhodopsin (BR) crystals grown in a lipidic cubic mesophase of monoolein were used as a model system. LCP from a part of a hexagon-shaped plate-like BR crystal (∼10 µm in thickness and ∼70 µm in the longest dimension), which was flash-frozen in liquid nitro­gen, was milled away with a gallium FIB under cryogenic conditions, and a part of the crystal itself was thinned into a ∼210 nm-thick lamella with the ion beam. The frozen sample was then transferred into an electron cryo-microscope, and a nanovolume of ∼1400 × 1400 × 210 nm of the BR lamella was exposed to 200 kV electrons at a fluence of ∼0.06 e Å−2. The resulting electron diffraction peaks were detected beyond 2.7 Å resolution (with an average peak height to background ratio of >2) by a CMOS-based Ceta 16M camera. The results demonstrate that cryo-FIB milling produces high-quality lamellae from crystals grown in lipidic mesophases and pave the way for 3D electron crystallography on crystals grown or embedded in highly viscous media

Characterization of bR expressed in <i>E</i>. <i>coli</i>.

<p>A. Elution profile of the wild type bR on Sephacryl S200HR. The initial run is shown in red and the repeat run (after storage) in blue. The peak at 68.68 ml in the first run corresponds to the aggregated colorless protein. The second peak at 85.54 ml is the functional target protein. The fractions corresponding to the second peak were colored and those in the grey box were pooled. The repeat run after 5 days does not show any significant amount of aggregates. B. UV-Vis absorption spectra of the samples of the wild type bR and D85N and D96N mutants normalized by absorbance at 280 nm. Proteins were solubilized in DDM and purified using non-denaturing protocol. The spectrum of the wild type bR exhibited the retinal absorption peak at 555.5±1.0 nm with a peak ratio A₂₈₀/A_λret of 1.5 which corresponds to the highest purity of bR.</p