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

Abstract P-40: The Shape and Size of the Recombinant Virus-like Particles Were Checked by Means of Electron Microscope

Background: Nucleocapsid protein of hepatitis-B virus (HBcAg) recombinantly synthesized in prokaryotic and eukaryotic cells is known to be capable to self-assemble into highly immunogenic stable viral-like particles (VLP) of icosahedral shape with a characteristic size of 32 nm (Schödel et al., 1994; Murray and Shiau, 1999). The VLP formation is tolerant to the insertion of some artificial epitopes to N- and C-termini of HBcAg monomer and also into major insertion region (MIR), forming a spike on the surface of VLP (Tordjeman et al., 1993, Peyret et al., 2015). Methods: We have investigated the possibility of heterologous expression of de novo designed gene coding the first 148 amino acid residues of HBcAg (Pumpens and Grens, 1999). The gene was specially designed to be suitable for the insertions of genes coding fluorescent proteins, which are desired for the studies of VLP distribution in tissues by confocal microscopy. Gene was optimized for overexpression in E. coli producer strains and special attention was taken to obtain a simple purification scheme, which reliably reduces the amount of pyrogens in purified VLP. The MIPT scientific platform of electron microscopy equipped with the transmission electron microscope Tecnai Polara G2 (Thermo Scientific (FEI)) was used. Carbon-coated (Lacey Carbon and 10 nm thin carbon) copper 200 mesh grids were treated with glow-discharge and coated with VLP suspension in deionized water. The samples were stained with uranyl acetate solution, air-dried, and inspected at the accelerating voltage of 300 kV. Results: The 32 nm size of heterologously synthesized VLP was successfully proved, and spherical shape was seen using negative contrasting

Role of Mitochondrial Protein Import in Age-Related Neurodegenerative and Cardiovascular Diseases

Mitochondria play a critical role in providing energy, maintaining cellular metabolism, and regulating cell survival and death. To carry out these crucial functions, mitochondria employ more than 1500 proteins, distributed between two membranes and two aqueous compartments. An extensive network of dedicated proteins is engaged in importing and sorting these nuclear-encoded proteins into their designated mitochondrial compartments. Defects in this fundamental system are related to a variety of pathologies, particularly engaging the most energy-demanding tissues. In this review, we summarize the state-of-the-art knowledge about the mitochondrial protein import machinery and describe the known interrelation of its failure with age-related neurodegenerative and cardiovascular diseases

Structural insights into thrombolytic activity of destabilase from medicinal leech

Destabilase from the medical leech Hirudo medicinalis belongs to the family of i-type lysozymes. It has two different enzymatic activities: microbial cell walls destruction (muramidase activity), and dissolution of the stabilized fibrin (isopeptidase activity). Both activities are known to be inhibited by sodium chloride at near physiological concentrations, but the structural basis remains unknown. Here we present two crystal structures of destabilase, including a 1.1 Å-resolution structure in complex with sodium ion. Our structures reveal the location of sodium ion between Glu34/Asp46 residues, which were previously recognized as a glycosidase active site. While sodium coordination with these amino acids may explain inhibition of the muramidase activity, its influence on previously suggested Ser49/Lys58 isopeptidase activity dyad is unclear. We revise the Ser49/Lys58 hypothesis and compare sequences of i-type lysozymes with confirmed destabilase activity. We suggest that the general base for the isopeptidase activity is His112 rather than Lys58. pKa calculations of these amino acids, assessed through the 1 μs molecular dynamics simulation, confirm the hypothesis. Our findings highlight the ambiguity of destabilase catalytic residues identification and build foundations for further research of structure–activity relationship of isopeptidase activity as well as structure-based protein design for potential anticoagulant drug development.</p

Raman Scattering:From Structural Biology to Medical Applications

This is a review of relevant Raman spectroscopy (RS) techniques and their use in structural biology, biophysics, cells, and tissues imaging towards development of various medical diagnostic tools, drug design, and other medical applications. Classical and contemporary structural studies of different water-soluble and membrane proteins, DNA, RNA, and their interactions and behavior in different systems were analyzed in terms of applicability of RS techniques and their complementarity to other corresponding methods. We show that RS is a powerful method that links the fundamental structural biology and its medical applications in cancer, cardiovascular, neurodegenerative, atherosclerotic, and other diseases. In particular, the key roles of RS in modern technologies of structure-based drug design are the detection and imaging of membrane protein microcrystals with the help of coherent anti-Stokes Raman scattering (CARS), which would help to further the development of protein structural crystallography and would result in a number of novel high-resolution structures of membrane proteins&mdash;drug targets; and, structural studies of photoactive membrane proteins (rhodopsins, photoreceptors, etc.) for the development of new optogenetic tools. Physical background and biomedical applications of spontaneous, stimulated, resonant, and surface- and tip-enhanced RS are also discussed. All of these techniques have been extensively developed during recent several decades. A number of interesting applications of CARS, resonant, and surface-enhanced Raman spectroscopy methods are also discussed

Abstract P-4: Robust Method for Background Subtraction in Serial X-ray Diffraction Data

Background: Membrane receptors play an important role in signal transduction across the cell membrane in all living organisms. Their structural studies have been enabled by multiple technological breakthroughs in their heterologous expression, stabilization, crystallization, and crystallographic data collection as well as in cryogenic electron microscopy (cryoEM). During the last decade, serial femtosecond crystallography (SFX) using X-ray free electron lasers (XFELs) has enabled structure determination of previously inaccessible proteins, including several G-protein-coupled receptors (GPCR), that produce only micrometer-sized crystals, thus paving the way towards understanding their activation mechanism and rational drug discovery. In addition to experimental difficulties, membrane protein structure determination is also often accompanied by data processing challenges. In particular, the lipidic cubic phase that serves as a carrier for membrane protein microcrystals, as well as various XFEL beam-shaping devices may generate substantial background scattering that could complicate the structure factor extraction from the diffraction images. Methods: In this work, we tested an adaptation of the denoising algorithm via matrix decomposition to XFEL-SFX data. We benchmarked its performance using high-background data from PAL-XFEL and established its applicability to serial crystallography image denoising, as well as compared it to the CrystFEL-based image denoising algorithm. Results: We find that, although the decomposition-based image denoising does not outperform CrystFEL median subtraction, it performs better than the integration without any additional subtraction. We find the non-negative matrix factorization performing better than more traditional singular-value decomposition methods, both in terms of visual interpretability and final data quality. Conclusion: We hope that this work will draw attention to background subtraction methods in structural biology, and will pave the way towards processing of most challenging datasets in structural biology, in particularly, those collected from membrane proteins

Obtaining high-quality X-ray data of bacteriorhodopsin functional states

La synthèse de l'adénosine triphosphate (ATP) est un événement clé dans la bioénergétique cellulaire. ATP synthesis est possible quand un gradient de potentiel électrochimique de protons est présent sur les membranes des cellules ou des organelles. Ce gradient est produit par les réactions d'oxydoréduction ou les réactions photochimiques qui sont contrôlées par l'enzyme. Bactériorhodopsine (bR) est la protéine la plus simple et la plus étudiée qui convertit l'énergie lumineuse en potentiel électrochimique. bR est un protéine transmembranaire de Halobacterium salinarum. bR absorbe des photons de lumière et transmet un proton à partir du cytoplasme vers l'espace extracellulaire. Grâce à sa disponibilité en relativement grandes quantités, la procédure de purification facile et stable, bR reste un des protéines membranaire les plus étudiés au cours des 40 dernières années.Pour comprendre le mécanisme moléculaire de la bR fonctionnement il faut connaître les changements structurels, provoqués par l'absorption de photon, qui accompagnent le cycle de travail des protéines et poussent à transporter le proton. Cela implique l'obtention des structures cristallographiques de bR état fonctionnel avec une résolution atomique. Selon cette approche, il est important d'avoir les cristaux protéiques très ordonnés et les méthodes de fixage des molécules de protéines dans les états intermédiaires. Les méthodes de fixage dans des conditions cryogéniques ont été développées précédemment. Les cristaux de la qualité désirée peuvent être obtenus par la cristallisation in meso où lipide mésophase bicontinue est utilisé pour la cristallisation des protéines membranaires.Le mécanisme de la cristallisation in meso est actuellement étudié pauvrement. Cette situation limite grandement son application potentielle pour des protéines membranaires. Malgré ses limites l’approche in meso a récemment permis d'obtenir les structures de base ainsi que les structures intermédiaires des états de bR. Cependant, différents groupes de scientifiques ont publié de différents structures cristallographiques des mêmes états intermédiaires. Les mécanismes de protons transport proposés par des auteurs différents sont contradictoires. Les raisons de l'absence de consensus dans les structures intermédiaires restent floues. Les raisons possibles discutées dans la littérature sont: la qualité insuffisante de la diffraction des cristaux protéiques, twinning merohedral et détérioration des cristaux par l'irradiation de rayonnement X, ainsi que la génération de nouvelles protéines états provoqués par rayons X.L'objectif de l'étude était de trier les raisons de contradictions dans le domaine de l'analyse cristallographique de bR états fonctionnels et de trouver des moyens de surmonter les problèmes connexes. Ceci implique plusieurs sous-objectifs distincts: l'étude de twinning merohedral de bR cristaux; étude des changements dans la structure bR induit par les 'irradiation de rayonnement X; étude des changements structurels dans bR par les petites doses de radiations. Un autre objectif de ce travail était d'étudier un rôle de molécules de la matrice de in meso cristallisation dans la stabilisation des cristaux de protéines membranaires.The synthesis of adenosine triphosphate (ATP) is a key event in the cell bioenergetics. ATP synthesis is only possible when a proton electrochemical potential gradient is present on the membranes of cell or organelle. This gradient is produced by enzyme-controlled redox or photochemical reactions. Bacteriorhodopsin (bR) is the simplest and most studied protein that converts light energy into electrochemical potential. Being transmembrane protein of Halobacterium salinarum it absorbs light photon and transfers a proton from the cytoplasmic to the extracellular space. Due to its availability of relatively large quantities, easy purification procedure and protein stability bR remains one of the most extensively studied membrane proteins during the past 40 years.Current state of investigated problems. To understand the molecular mechanism of bR functioning is necessary to know the structural changes caused by light absorption which accompany the protein working cycle and lead to the directional transport of the proton. It implies obtaining of X-ray structures of bR functional states with atomic resolution. Following this approach it is important to have highly ordered three-dimensional protein crystals on the one hand and effective methods of trapping protein molecules in intermediate states on the other one. Trapping procedures for bR intermediate states under cryogenic conditions have been developed previously. Crystals of the desired quality can be obtained by in meso crystallization where lipid bicontinuous mesophase is used for the crystallization of membrane proteins. The mechanism of in meso crystallization is currently poorly investigated. This situation greatly limits its potential applicability for membrane proteins. Despite its limitations in meso approach have recently made possible to obtain the ground and some intermediate states structures of bR. However, different scientific groups have published different X-ray models of the same bR intermediate states. The proposed by different authors mechanisms of proton transport are contradictory. The reasons for the lack of the consensus in intermediate structures remain unclear. The possible reasons for this contradiction which have been discussed in literature are: insufficient quality of diffraction data, merohedral twinning and radiation damage of protein crystals, as well as the generation of new protein states caused by X-ray illumination.The aim of the study was to sort out the reasons for contradictions in the field of X-ray crystallographic analysis of bR functional states and to find ways to overcome related problems. This implies several separate subgoals: study of merohedral twinning of bR crystals; study of X-ray-radiation-induced changes in bR structure; study of low-dose radiation-induced structural changes in bR structure. An additional goal of the work was to study a role of molecules of the in meso crystallization matrix in the stabilization of membrane protein crystals