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1. Crystal structure of a bacterial photoactivated adenylate cyclase determined by serial femtosecond and serial synchrotron crystallography

Author

Sofia M. Kapetanaki, Nicolas Coquelle, David von Stetten, Martin Byrdin, Ronald Rios-Santacruz, Richard Bean, Johan Bielecki, Mohamed Boudjelida, Zsuzsana Fekete, Geoffrey W. Grime, Huijong Han, Caitlin Hatton, Sravya Kantamneni, Konstantin Kharitonov, Chan Kim, Marco Kloos, Faisal H. M. Koua, Iñaki de Diego Martinez, Diogo Melo, Lukas Rane, Adam Round, Ekaterina Round, Abhisakh Sarma, Robin Schubert, Joachim Schulz, Marcin Sikorski, Mohammad Vakili, Joana Valerio, Jovana Vitas, Raphael de Wijn, Agnieszka Wrona, Ninon Zala, Arwen Pearson, Katerina Dörner, Giorgio Schirò, Elspeth F. Garman, András Lukács, and Martin Weik

Subjects
bacterial adenylate cyclase, bluf domain, ac domain, serial femtosecond crystallography, xfels, synchrotrons, room-temperature x-ray crystallography, cryo-macromolecular crystallography, protein structure, light-activated enzymes, oscillatoria acuminata, Crystallography, QD901-999
Abstract

OaPAC is a recently discovered blue-light-using flavin adenosine dinucleotide (BLUF) photoactivated adenylate cyclase from the cyanobacterium Oscillatoria acuminata that uses adenosine triphosphate and translates the light signal into the production of cyclic adenosine monophosphate. Here, we report crystal structures of the enzyme in the absence of its natural substrate determined from room-temperature serial crystallography data collected at both an X-ray free-electron laser and a synchrotron, and we compare these structures with cryo-macromolecular crystallography structures obtained at a synchrotron by us and others. These results reveal slight differences in the structure of the enzyme due to data collection at different temperatures and X-ray sources. We further investigate the effect of the Y6W mutation in the BLUF domain, a mutation which results in a rearrangement of the hydrogen-bond network around the flavin and a notable rotation of the side chain of the critical Gln48 residue. These studies pave the way for picosecond–millisecond time-resolved serial crystallography experiments at X-ray free-electron lasers and synchrotrons in order to determine the early structural intermediates and correlate them with the well studied picosecond–millisecond spectroscopic intermediates.

Published
2024
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2. Form factor determination of biological molecules with X-ray free electron laser small-angle scattering (XFEL-SAS)

Author

Clement E. Blanchet, Adam Round, Haydyn D. T. Mertens, Kartik Ayyer, Melissa Graewert, Salah Awel, Daniel Franke, Katerina Dörner, Saša Bajt, Richard Bean, Tânia F. Custódio, Raphael de Wijn, E. Juncheng, Alessandra Henkel, Andrey Gruzinov, Cy M. Jeffries, Yoonhee Kim, Henry Kirkwood, Marco Kloos, Juraj Knoška, Jayanath Koliyadu, Romain Letrun, Christian Löw, Jana Makroczyova, Abhishek Mall, Rob Meijers, Gisel Esperanza Pena Murillo, Dominik Oberthür, Ekaterina Round, Carolin Seuring, Marcin Sikorski, Patrik Vagovic, Joana Valerio, Tamme Wollweber, Yulong Zhuang, Joachim Schulz, Heinrich Haas, Henry N. Chapman, Adrian P. Mancuso, and Dmitri Svergun

Subjects
Biology (General), QH301-705.5
Abstract

Abstract Free-electron lasers (FEL) are revolutionizing X-ray-based structural biology methods. While protein crystallography is already routinely performed at FELs, Small Angle X-ray Scattering (SAXS) studies of biological macromolecules are not as prevalent. SAXS allows the study of the shape and overall structure of proteins and nucleic acids in solution, in a quasi-native environment. In solution, chemical and biophysical parameters that have an influence on the structure and dynamics of molecules can be varied and their effect on conformational changes can be monitored in time-resolved XFEL and SAXS experiments. We report here the collection of scattering form factors of proteins in solution using FEL X-rays. The form factors correspond to the scattering signal of the protein ensemble alone; the scattering contributions from the solvent and the instrument are separately measured and accurately subtracted. The experiment was done using a liquid jet for sample delivery. These results pave the way for time-resolved studies and measurements from dilute samples, capitalizing on the intense and short FEL X-ray pulses.

Published
2023
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3. Structure-based insights into evolution of rhodopsins

Author

Dmitrii Zabelskii, Natalia Dmitrieva, Oleksandr Volkov, Vitaly Shevchenko, Kirill Kovalev, Taras Balandin, Dmytro Soloviov, Roman Astashkin, Egor Zinovev, Alexey Alekseev, Ekaterina Round, Vitaly Polovinkin, Igor Chizhov, Andrey Rogachev, Ivan Okhrimenko, Valentin Borshchevskiy, Vladimir Chupin, Georg Büldt, Natalia Yutin, Ernst Bamberg, Eugene Koonin, and Valentin Gordeliy

Subjects
Biology (General), QH301-705.5
Abstract

Zabelskii et al. present a structural and functional analysis of the lightdriven proton pump LR (Mac) from the fungus Leptosphaeria maculans. Their findings indicate that the archaeal ancestry of eukaryotic type 1 rhodopsins, and that the archaeal host of the proto-mitochondrial endosymbiont was capable of light-driven proton pumping.

Published
2021
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4. An Approach to Heterologous Expression of Membrane Proteins. The Case of Bacteriorhodopsin.

Author

Dmitry Bratanov, Taras Balandin, Ekaterina Round, Vitaly Shevchenko, Ivan Gushchin, Vitaly Polovinkin, Valentin Borshchevskiy, and Valentin Gordeliy

Subjects
Medicine, Science
Abstract

Heterologous 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.

Published
2015
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5. Structural and functional investigation of flavin binding center of the NqrC subunit of sodium-translocating NADH:quinone oxidoreductase from Vibrio harveyi.

Author

Valentin Borshchevskiy, Ekaterina Round, Yulia Bertsova, Vitaly Polovinkin, Ivan Gushchin, Andrii Ishchenko, Kirill Kovalev, Alexey Mishin, Galina Kachalova, Alexander Popov, Alexander Bogachev, and Valentin Gordeliy

Subjects
Medicine, Science
Abstract

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.

Published
2015
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6. Crystal structure of Escherichia coli-expressed Haloarcula marismortui bacteriorhodopsin I in the trimeric form.

Author

Vitaly Shevchenko, Ivan Gushchin, Vitaly Polovinkin, Ekaterina Round, Valentin Borshchevskiy, Petr Utrobin, Alexander Popov, Taras Balandin, Georg Büldt, and Valentin Gordeliy

Subjects
Medicine, Science
Abstract

Bacteriorhodopsins 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.

Published
2014
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7. Mix-and-extrude: high-viscosity sample injection towards time-resolved protein crystallography

Author

Mohammad Vakili, Huijong Han, Christina Schmidt, Agnieszka Wrona, Marco Kloos, Iñaki de Diego, Katerina Dörner, Tian Geng, Chan Kim, Faisal Koua, Diogo Melo, Mathieu Rappas, Adam Round, Ekaterina Round, Marcin Sikorski, Joana Valerio, Tiankun Zhou, Kristina Lorenzen, and Joachim Schulz

Abstract

Time-resolved crystallography enabled the visualization of protein molecular motion during reaction. While light is commonly used to initiate reactions in time-resolved crystallography, only a small number of proteins can in fact be activated by light. However, many biological reactions can be triggered by the interaction of proteins with ligands. The sample delivery method presented here uses a mix-and-extrude approach based on 3D printed microchannels in conjunction with a micronozzle to study the dynamics of samples in viscous media that can be triggered by diffusive mixing. The device design allows for mixing of ligands and protein crystals in a time window of 2 to 20 seconds. The device characterization using a model system (fluorescence quenching of iq-mEmerald proteins by copper ions) demonstrated that ligand and protein crystals, each within the lipidic cubic phase, can be mixed efficiently. The potential use of this approach for time-resolved membrane protein crystallography to support in the development of new drugs is also discussed.Synopsis3D printed mixing-HVE devices address time-resolved membrane protein crystallography challenges via compact dual-flow LCP injection.Abstract Figure

Published
2022
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8. The XBI BioLab for life science experiments at the European XFEL

Author

Domingo Meza, Philipp Heuser, Kristina Lorenzen, Petra Fromme, Joachim Schulz, Yasmin Gül, Christian Betzel, Huijong Han, Poul Nissen, Victor S. Lamzin, Janos Hajdu, Robin Schubert, Inari Kursula, Jozef Ulicný, Matthias Wilmanns, Jana Makroczyová, Martin Aepfelbacher, Elena Tereschenko, Ekaterina Round, Charlotte Uetrecht, and Imrich Barák

Subjects
0301 basic medicine, Sample purification, Engineering, Other Physics Topics, 02 engineering and technology, single-particle imaging (SPI), General Biochemistry, Genetics and Molecular Biology, free-electron lasers (XFELs), XBI Laboratory, 03 medical and health sciences, structural biology, User Facility, sample preparation and characterization, coherent diffractive imaging (CDI), business.industry, Atomic force microscopy, Wet laboratory, Annan fysik, 021001 nanoscience & nanotechnology, Research Papers, European XFEL (EuXFEL), time-resolved experiments, serial femtosecond crystallography (SFX), 030104 developmental biology, Systems engineering, Russian federation, 0210 nano-technology, business
Abstract

An overview of a unique life science facility at the European XFEL is provided, and its importance in sample preparation, characterization and analysis prior to measurements at the different European XFEL instruments is discussed. The capabilities that the facility can provide for the research community are highlighted, alongside examples of recent successful studies., The science of X-ray free-electron lasers (XFELs) critically depends on the performance of the X-ray laser and on the quality of the samples placed into the X-ray beam. The stability of biological samples is limited and key biomolecular transformations occur on short timescales. Experiments in biology require a support laboratory in the immediate vicinity of the beamlines. The XBI BioLab of the European XFEL (XBI denotes XFEL Biology Infrastructure) is an integrated user facility connected to the beamlines for supporting a wide range of biological experiments. The laboratory was financed and built by a collaboration between the European XFEL and the XBI User Consortium, whose members come from Finland, Germany, the Slovak Republic, Sweden and the USA, with observers from Denmark and the Russian Federation. Arranged around a central wet laboratory, the XBI BioLab provides facilities for sample preparation and scoring, laboratories for growing prokaryotic and eukaryotic cells, a Bio Safety Level 2 laboratory, sample purification and characterization facilities, a crystallization laboratory, an anaerobic laboratory, an aerosol laboratory, a vacuum laboratory for injector tests, and laboratories for optical microscopy, atomic force microscopy and electron microscopy. Here, an overview of the XBI facility is given and some of the results of the first user experiments are highlighted.

Published
2021
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9. True-atomic-resolution insights into the structure and functional role of linear chains and low-barrier hydrogen bonds in proteins

Author

Valentin Borshchevskiy, Kirill Kovalev, Ekaterina Round, Rouslan Efremov, Roman Astashkin, Gleb Bourenkov, Dmitry Bratanov, Taras Balandin, Igor Chizhov, Christian Baeken, Ivan Gushchin, Alexander Kuzmin, Alexey Alekseev, Andrey Rogachev, Dieter Willbold, Martin Engelhard, Ernst Bamberg, Georg Büldt, Valentin Gordeliy, Moscow Institute of Physics and Technology [Moscow] (MIPT), Institute of Complex Systems (ICS), Forschungszentrum Jülich GmbH | Centre de recherche de Juliers, Helmholtz-Gemeinschaft = Helmholtz Association-Helmholtz-Gemeinschaft = Helmholtz Association, European X-ray Free Electron Laser GmbH, Structural Biology Brussels (SBB), Vrije Universiteit Brussel (VUB), Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), DESY, European Mol Biol Lab, D-22607 Hamburg, Germany, Institute of Biological Information Processing [Jülich] (IBI-7), Institute for Biophysical Chemistry, Hannover Medical School [Hannover] (MHH), Jülich Center for Structural Biology [Jülich] (JuStruct), Research Center for Molecular Mechanisms of Aging and Age-related Diseases, Moscow, Institut für Physikalische Biologie [Düsseldorfd], Heinrich Heine Universität Düsseldorf = Heinrich Heine University [Düsseldorf], Max Planck Institute of Molecular Physiology, Max-Planck-Gesellschaft, and Department of Structural Biology, Max-Planck-Institute of Biophysics, Frankfurt, Germany

Subjects
[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Structural Biology, Proteins, MESH: Proteins, Hydrogen Bonding, MESH: Hydrogen Bonding, MESH: Protons, Protons, Molecular Biology
Abstract

International audience; Hydrogen bonds are fundamental to the structure and function of biological macromolecules and have been explored in detail. The chains of hydrogen bonds (CHBs) and low-barrier hydrogen bonds (LBHBs) were proposed to play essential roles in enzyme catalysis and proton transport. However, high-resolution structural data from CHBs and LBHBs is limited. The challenge is that their 'visualization' requires ultrahigh-resolution structures of the ground and functionally important intermediate states to identify proton translocation events and perform their structural assignment. Our true-atomic-resolution structures of the light-driven proton pump bacteriorhodopsin, a model in studies of proton transport, show that CHBs and LBHBs not only serve as proton pathways, but also are indispensable for long-range communications, signaling and proton storage in proteins. The complete picture of CHBs and LBHBs discloses their multifunctional roles in providing protein functions and presents a consistent picture of proton transport and storage resolving long-standing debates and controversies.

Published
2021
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10. Multiscale computation delivers organophosphorus reactivity and stereoselectivity to immunoglobulin scavengers

Author

Alexey A. Belogurov, Spyros D. Chatziefthimiou, Richard A. Lerner, Ekaterina Round, Yuliana A. Mokrushina, T. V. Bobik, Alexander G. Gabibov, Anastasia V. Stepanova, Andrey V. Golovin, G. Michael Blackburn, Gleb Bourenkov, Matthias Wilmanns, Alexander Zlobin, O. G. Shamborant, Ivan V. Smirnov, Alexey Stepanov, Kirill A. Konovalov, Arthur O. Zalevsky, Stanislav S. Terekhov, Sofiya O. Pipiya, Jia Xie, and Alexander A. Makarov

Subjects
0301 basic medicine, phosphoryl transfer, Stereochemistry, antibody bioscavengers, 01 natural sciences, Molecular mechanics, 03 medical and health sciences, Stereospecificity, stereospecificity, Nucleophile, Reactivity (chemistry), designed biocatalysts, Multidisciplinary, QM/MM computation, 010405 organic chemistry, Chemistry, Metadynamics, Biological Sciences, Transition state, 0104 chemical sciences, Biophysics and Computational Biology, 030104 developmental biology, Covalent bond, Stereoselectivity, ddc:500
Abstract

Proceedings of the National Academy of Sciences of the United States of America 117(37), 22841 - 22848 (2020). doi:10.1073/pnas.2010317117, Quantum mechanics/molecular mechanics (QM/MM) maturation of an immunoglobulin (Ig) powered by supercomputation delivers novel functionality to this catalytic template and facilitates artificial evolution of biocatalysts. We here employ density functional theory-based (DFT-b) tight binding and funnel metadynamics to advance our earlier QM/MM maturation of A17 Ig-paraoxonase (WTIgP) as a reactibody for organophosphorus toxins. It enables regulation of biocatalytic activity for tyrosine nucleophilic attack on phosphorus. The single amino acid substitution l-Leu47Lys results in 340-fold enhanced reactivity for paraoxon. The computed ground-state complex shows substrate-induced ionization of the nucleophilic l-Tyr37, now H-bonded to l-Lys47, resulting from repositioning of l-Lys47. Multiple antibody structural homologs, selected by phenylphosphonate covalent capture, show contrasting enantioselectivities for a P-chiral phenylphosphonate toxin. That is defined by crystallographic analysis of phenylphosphonylated reaction products for antibodies A5 and WTIgP. DFT-b analysis using QM regions based on these structures identifies transition states for the favored and disfavored reactions with surprising results. This stereoselection analysis is extended by funnel metadynamics to a range of WTIgP variants whose predicted stereoselectivity is endorsed by experimental analysis. The algorithms used here offer prospects for tailored design of highly evolved, genetically encoded organophosphorus scavengers and for broader functionalities of members of the Ig superfamily, including cell surface-exposed receptors., Published by National Acad. of Sciences, Washington, DC

Published
2020
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11. Nucleation and Growth of Membrane Protein Crystals In Meso—A Fluorescence Microscopy Study

Author

Vadim Cherezov, Andrey Bogorodskiy, Georg Büldt, Vitaly Polovinkin, Alexey Mishin, Fedor Frolov, Valentin Gordeliy, Valentin Borshchevskiy, Thomas Gensch, and Ekaterina Round

Subjects
Ostwald ripening, 0303 health sciences, Materials science, biology, Nucleation, Crystal growth, Bacteriorhodopsin, General Chemistry, 010402 general chemistry, Condensed Matter Physics, 01 natural sciences, 0104 chemical sciences, law.invention, Crystal, 03 medical and health sciences, Crystallography, symbols.namesake, law, Phase (matter), Fluorescence microscope, symbols, biology.protein, General Materials Science, Crystallization, 030304 developmental biology
Abstract

Since the introduction of in meso crystallization of membrane proteins in lipidic cubic phase (LCP) by Landau and Rosenbusch in 1996, numerous studies attempted to elucidate the mechanism of in meso crystal nucleation and growth. Here we present a fluorescence microscopy study of the crystallization process of the light-driven proton pump bacteriorhodopsin. The crystallization starts with formation of microcrystals, followed by growth of a dominating crystal at the expense of smaller ones and formation of a depletion zone around it. These observations suggest an Ostwald ripening mechanism of the in meso crystal growth. Analysis of the microcrystal spatial distribution suggests that microcrystal nucleation occurs predominately at the LCP domain boundaries.

Published
2015
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12. New Insights on Signal Propagation by Sensory Rhodopsin II/Transducer Complex

Author

Johann P. Klare, Petr Utrobin, Ivan Gushchin, Vitaly Polovinkin, Alina Remeeva, Valentin Borshchevskiy, Valentin Gordeliy, Martin Engelhard, Sergei Grudinin, Georg Büldt, Taras Balandin, Andrii Ishchenko, Ekaterina Round, Institute of Complex Systems (ICS), ICS-6: Structural Biochemistry ( ICS-6), Institute of Crystallography [Aachen], Rheinisch-Westfälische Technische Hochschule Aachen (RWTH), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Moscow Institute of Physics and Technology [Moscow] (MIPT), Algorithms for Modeling and Simulation of Nanosystems (NANO-D), Inria Grenoble - Rhône-Alpes, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Laboratoire Jean Kuntzmann (LJK ), Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Institut National de Recherche en Informatique et en Automatique (Inria)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Max Planck Institute of Molecular Physiology, Max-Planck-Gesellschaft, Department of Physics, University of Osnabrück, Osnabrück University, Rheinisch-Westfälische Technische Hochschule Aachen University (RWTH), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Universität Osnabrück - Osnabrück University

Subjects
Models, Molecular, 0301 basic medicine, Magnetic Resonance Spectroscopy, Protein Conformation, Archaeal Proteins, Static Electricity, Plasma protein binding, Biology, Article, HAMP domain, Structure-Activity Relationship, 03 medical and health sciences, Sensory Rhodopsins, Protein structure, Membrane proteins, Halobacteriaceae, Protein Interaction Domains and Motifs, Negative phototaxis, Binding Sites, Multidisciplinary, 030102 biochemistry & molecular biology, Nanocrystallography, Sensory rhodopsin II, biology.organism_classification, [INFO.INFO-MO]Computer Science [cs]/Modeling and Simulation, 3. Good health, 030104 developmental biology, Biochemistry, Domain (ring theory), Biophysics, ddc:000, Protein Multimerization, Protein Binding, Signal Transduction
Abstract

The complex of two membrane proteins, sensory rhodopsin II (NpSRII) with its cognate transducer (NpHtrII), mediates negative phototaxis in halobacteria N. pharaonis. Upon light activation NpSRII triggers a signal transduction chain homologous to the two-component system in eubacterial chemotaxis. Here we report on crystal structures of the ground and active M-state of the complex in the space group I212121. We demonstrate that the relative orientation of symmetrical parts of the dimer is parallel (“U”-shaped) contrary to the gusset-like (“V”-shaped) form of the previously reported structures of the NpSRII/NpHtrII complex in the space group P21212, although the structures of the monomers taken individually are nearly the same. Computer modeling of the HAMP domain in the obtained “V”- and “U”-shaped structures revealed that only the “U”-shaped conformation allows for tight interactions of the receptor with the HAMP domain. This is in line with existing data and supports biological relevance of the “U” shape in the ground state. We suggest that the “V”-shaped structure may correspond to the active state of the complex and transition from the “U” to the “V”-shape of the receptor-transducer complex can be involved in signal transduction from the receptor to the signaling domain of NpHtrII.

Published
2017
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13. Biological user support at the European XFEL

Author

Joachim Schulz, Huijong Han, Robin Schubert, Kristina Lorenzen, Domingo Meza, and Ekaterina Round

Subjects
Inorganic Chemistry, Structural Biology, General Materials Science, Physical and Theoretical Chemistry, Condensed Matter Physics, Biochemistry
Published
2019
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14. Integral Membrane Proteins Can Be Crystallized Directly from Nanodiscs

Author

Pavel Kuzmichev, Mikhail Nikolaev, Valentin Gordeliy, Valentin Borshchevskiy, Vitaly Polovinkin, Alexander I. Kuklin, Ivan Gushchin, Adam Round, Georg Büldt, F. Bernhard, Dieter Willbold, Ekaterina Round, Taras Balandin, and Vitaly Shevchenko

Subjects
0301 basic medicine, biology, Chemistry, Mesophase, Bacteriorhodopsin, General Chemistry, Condensed Matter Physics, law.invention, Folding (chemistry), 03 medical and health sciences, Crystallography, 030104 developmental biology, Structural biology, Membrane protein, law, Phase (matter), ddc:540, biology.protein, General Materials Science, Crystallization, Integral membrane protein
Abstract

Membrane-like nanodiscs (ND) have become an important tool for the cell-free expression, solubilization, folding, and in vitro structural and functional studies of membrane proteins (MPs). Direct crystallization of MPs embedded in NDs would be of high importance for structural biology. However, despite considerable efforts we have been as yet unable to obtain crystals suitable for X-ray crystallography. In the present work, we show that an ND-trapped MP can be transferred into the cubic phase and crystallized in meso. Bacteriorhodopsin (BR) reconstituted into nanodiscs was mixed with a lipidic mesophase and crystallization was induced by adding a precipitant. The resulting crystals diffract beyond 1.8 A. The structure of BR was solved at 1.9 A and found to be indistinguishable from previous structures obtained with the protein solubilized in detergent. We suggest the proposed protocol of in meso crystallization to be generally applicable to ND-trapped MPs.

Published
2017
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15. Highly Sensitive Coherent Anti-Stokes Raman Scattering Imaging of Protein Crystals

Author

Egor Zinovev, Valentin Gordeliy, Nelya V. Doroshkevich, Sergey N. Shashkov, Ekaterina Round, A. V. Vlasov, Kahramon Z. Mamatkulov, and Grigory Arzumanyan

Subjects
0301 basic medicine, Free electron model, Chemistry, business.industry, General Chemistry, Laser, Biochemistry, Catalysis, Synchrotron, Highly sensitive, law.invention, Micrometre, 03 medical and health sciences, symbols.namesake, 030104 developmental biology, Colloid and Surface Chemistry, Optics, Structural biology, law, symbols, business, Protein crystallization, Raman scattering
Abstract

Serial crystallography at last generation X-ray synchrotron sources and free electron lasers enabled data collection with micrometer and even submicrometer size crystals, which have resulted in amazing progress in structural biology. However, imaging of small crystals, which although is highly demanded, remains a challenge, especially in the case of membrane protein crystals. Here we describe a new extremely sensitive method of the imaging of protein crystals that is based on coherent anti-Stokes Raman scattering.

Published
2016

16. Mechanism of transmembrane signaling by sensor histidine kinases

Author

Ivan, Gushchin, Igor, Melnikov, Vitaly, Polovinkin, Andrii, Ishchenko, Anastasia, Yuzhakova, Pavel, Buslaev, Gleb, Bourenkov, Sergei, Grudinin, Ekaterina, Round, Taras, Balandin, Valentin, Borshchevskiy, Dieter, Willbold, Gordon, Leonard, Georg, Büldt, Alexander, Popov, and Valentin, Gordeliy

Subjects
Models, Molecular, Protein Domains, Escherichia coli Proteins, Escherichia coli, Membrane Proteins, Crystallization, Phosphoproteins, Signal Transduction
Abstract

One of the major and essential classes of transmembrane (TM) receptors, present in all domains of life, is sensor histidine kinases, parts of two-component signaling systems (TCSs). The structural mechanisms of TM signaling by these sensors are poorly understood. We present crystal structures of the periplasmic sensor domain, the TM domain, and the cytoplasmic HAMP domain of the

Published
2016

17. Active State of Sensory Rhodopsin II: Structural Determinants for Signal Transfer and Proton Pumping

Author

Martin Engelhard, Anastasia Reshetnyak, Valentin Gordeliy, Andrii Ishchenko, Sergei Grudinin, Valentin Borshchevskiy, Ekaterina Round, Ivan Gushchin, Georg Büldt, Moscow Institute of Physics and Technology [Moscow] (MIPT), Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institute of Complex Systems (ICS), Forschungszentrum Jülich GmbH | Centre de recherche de Juliers, Helmholtz-Gemeinschaft = Helmholtz Association-Helmholtz-Gemeinschaft = Helmholtz Association, Institute of Crystallography [Aachen], Rheinisch-Westfälische Technische Hochschule Aachen University (RWTH), Algorithms for Modeling and Simulation of Nanosystems (NANO-D), Inria Grenoble - Rhône-Alpes, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria)-Laboratoire Jean Kuntzmann (LJK), Université Pierre Mendès France - Grenoble 2 (UPMF)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS)-Université Pierre Mendès France - Grenoble 2 (UPMF)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS), Max Planck Institute of Molecular Physiology, Max-Planck-Gesellschaft, This work was supported by the program 'Chaires d'excellence' edition 2008 of ANR France, by a CEA(IBS)-HGF(FZJ) STC 5.1 specific agreement, and by a Marie Curie grant for the training and career development of researchers (FP7-PEOPLE- 2007-1-1-ITN, project SBMPs). This work was performed in the framework of Russian State contract numbers 02.740.11.0299, 02.740.11.5010, 974 (in the framework of activity 1.2.2), and P211 (in the framework of activity 1.3.2) of the Federal Target Program 'Scientific and academic research cadres of innovative Russia' for the years 2009- 2013., European Project: 211800,EC:FP7:PEOPLE,FP7-PEOPLE-2007-1-1-ITN,SBMPS(2008), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Rheinisch-Westfälische Technische Hochschule Aachen (RWTH), and Centre National de la Recherche Scientifique (CNRS)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Université Joseph Fourier - Grenoble 1 (UJF)-Université Pierre Mendès France - Grenoble 2 (UPMF)-Centre National de la Recherche Scientifique (CNRS)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Université Joseph Fourier - Grenoble 1 (UJF)-Université Pierre Mendès France - Grenoble 2 (UPMF)

Subjects
Models, Molecular, Protein Folding, Archaeal Proteins, Natronobacterium, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Crystallography, X-Ray, Models, Biological, Signal, 03 medical and health sciences, Structural Biology, Sensory Rhodopsins, membrane protein, Molecular Biology, 030304 developmental biology, G protein-coupled receptor, 0303 health sciences, Halobacteriaceae, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biology, Chemistry, 030302 biochemistry & molecular biology, Sensory rhodopsin II, Proton Pumps, Carotenoids, Transmembrane protein, Protein Structure, Tertiary, Transport protein, Crystallography, active state, Rhodopsin, Biophysics, biology.protein, Bacterial rhodopsins, Signal transduction, Crystallization, signaling, Signal Transduction, bacterial rhodopsins
Abstract

International audience; The molecular mechanism of transmembrane signal transduction is still a pertinent question in cellular biology. Generally, a receptor can transfer an external signal via its cytoplasmic surface as found for GPCRs like rhodopsin or via the membrane domain like it is utilized by sensory rhodopsin II (SRII) in complex with its transducer HtrII. In the absence of HtrII SRII functions as a proton pump. Here, we report on the crystal structure of the active state of SRII from Natronomonas pharaonis (NpSRII). The problem of a dramatic loss of the diffraction quality upon loading of the active state was overcome by growing better crystals and reducing the occupancy of the state. The determined conformational changes at the region comprising helices F and G are similar to those observed for the NpSRII-transducer complex but they are much more pronounced. Meaning of these differences for proton pumping ability and understanding of the signal transduction by NpSRII is discussed.

Published
2011
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18. Structural and Functional Investigation of Flavin Binding Center of the NqrC Subunit of Sodium-Translocating NADH:Quinone Oxidoreductase from Vibrio harveyi

Author

Galina S. Kachalova, Alexander Popov, Ekaterina Round, Kirill Kovalev, Vitaly Polovinkin, Alexey Mishin, Valentin Gordeliy, Alexander V. Bogachev, Ivan Gushchin, Valentin Borshchevskiy, Andrii Ishchenko, Yulia V. Bertsova, Moscow Institute of Physics and Technology [Moscow] (MIPT), Institute of Complex Systems (ICS), Forschungszentrum Jülich GmbH | Centre de recherche de Juliers, Helmholtz-Gemeinschaft = Helmholtz Association-Helmholtz-Gemeinschaft = Helmholtz Association, Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University (MSU), Moscow Inst Phys & Technol, Lab Adv Studies Membrane Prot, Dolgoprudnyi, Russia, A.N. Bach Institute of Biochemistry, Russian Academy of Sciences [Moscow] (RAS), European Synchrotron Radiation Facility (ESRF), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Forschungszentrum Jülich GmbH, Institut de biologie structurale [1992-2019] (IBS - UMR 5075 [1992-2019]), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)

Subjects
animal structures, Stereochemistry, Flavin Mononucleotide, [SDV]Life Sciences [q-bio], Recombinant Fusion Proteins, Molecular Sequence Data, Respiratory chain, lcsh:Medicine, Flavin mononucleotide, Flavin group, Calorimetry, Molecular Dynamics Simulation, Quinone oxidoreductase, Crystallography, X-Ray, Cofactor, chemistry.chemical_compound, Protein structure, Quinone Reductases, Bacterial Proteins, Oxidoreductase, Amino Acid Sequence, lcsh:Science, Vibrio, chemistry.chemical_classification, Multidisciplinary, Binding Sites, biology, lcsh:R, Protein Structure, Tertiary, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Protein Subunits, chemistry, Biochemistry, biology.protein, lcsh:Q, ddc:500, Sequence Alignment, Research Article, Protein Binding
Abstract

International audience; 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 har-veyi 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 moi-ety 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 conforma-tion 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.

Published
2015
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19. Crystal structure of a light-driven sodium pump

Author

Ivan Gushchin, Alexey Alekseev, Georg Büldt, Alexander Popov, Christian Bamann, Dieter Willbold, Vitaly Shevchenko, Ekaterina Round, Thomas Gensch, Kirill Kovalev, Christoph Fahlke, Valentin Gordeliy, Vitaly Polovinkin, Taras Balandin, Valentin Borshchevskiy, and Ernst Bamberg

Subjects
Models, Molecular, Rhodopsin, Ion Transport, biology, Sodium, Sodium-Potassium-Exchanging ATPase, Potassium, Rational design, chemistry.chemical_element, Crystal structure, Crystallography, X-Ray, Protein Structure, Tertiary, Crystallography, chemistry.chemical_compound, Monomer, chemistry, Structural Biology, biology.protein, Molecular Biology, Flavobacteriaceae, Ion transporter
Abstract

Recently, the first known light-driven sodium pumps, from the microbial rhodopsin family, were discovered. We have solved the structure of one of them, Krokinobacter eikastus rhodopsin 2 (KR2), in the monomeric blue state and in two pentameric red states, at resolutions of 1.45 A and 2.2 and 2.8 A, respectively. The structures reveal the ion-translocation pathway and show that the sodium ion is bound outside the protein at the oligomerization interface, that the ion-release cavity is capped by a unique N-terminal α-helix and that the ion-uptake cavity is unexpectedly large and open to the surface. Obstruction of the cavity with the mutation G263F imparts KR2 with the ability to pump potassium. These results pave the way for the understanding and rational design of cation pumps with new specific properties valuable for optogenetics.

Published
2015

20. MeshAndCollect: an automated multi-crystal data-collection workflow for synchrotron macromolecular crystallography beamlines

Author

Alexander Popov, Gleb Bourenkov, Andrew A. McCarthy, Ulrich Zander, Christoph Mueller-Dieckmann, Gordon A. Leonard, Daniele de Sanctis, Ekaterina Round, Olof Svensson, Valentin Gordeliy, European Synchrotron Radiation Facility (ESRF), DESY, European Mol Biol Lab, D-22607 Hamburg, Germany, European Molecular Biology Laboratory [Grenoble] (EMBL), Unit for Virus Host-Cell Interactions [Grenoble] (UVHCI), Université Joseph Fourier - Grenoble 1 (UJF)-European Molecular Biology Laboratory [Grenoble] (EMBL)-Centre National de la Recherche Scientifique (CNRS), Moscow Inst Phys & Technol, Lab Adv Studies Membrane Proteins, Dolgoprudnyi 141700, Russia, Res Ctr Juelich, ICS, ICS Mol Biophys 6, D-52425 Julich, Germany, Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Centre National de la Recherche Scientifique (CNRS)-European Molecular Biology Laboratory [Grenoble] (EMBL)-Université Joseph Fourier - Grenoble 1 (UJF), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), European Molecular Biology Laboratory [Hamburg] (EMBL), and Institut de biologie structurale [1992-2019] (IBS - UMR 5075 [1992-2019])

Subjects
Halobacterium salinarum, Models, Molecular, Diffraction, Thermolysin, Analytical chemistry, Bacillus, Crystallography, X-Ray, Workflow, law.invention, Computational science, hierarchical cluster analysis, Set (abstract data type), macromolecular crystallography, Structural Biology, law, Proton transport, Animals, Cluster Analysis, Plant Proteins, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Proteins, General Medicine, multi-crystal data collection, Plants, Bombyx, Research Papers, Synchrotron, Hierarchical clustering, Data set, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Plant protein, Bacteriorhodopsins, Insect Proteins, synchrotron serial crystallography, Muramidase, Crystallization, Synchrotrons
Abstract

The fully automated collection and merging of partial data sets from a series of cryocooled crystals of biological macromolecules contained on the same support is presented, as are the results of test experiments carried out on various systems., Here, an automated procedure is described to identify the positions of many cryocooled crystals mounted on the same sample holder, to rapidly predict and rank their relative diffraction strengths and to collect partial X-ray diffraction data sets from as many of the crystals as desired. Subsequent hierarchical cluster analysis then allows the best combination of partial data sets, optimizing the quality of the final data set obtained. The results of applying the method developed to various systems and scenarios including the compilation of a complete data set from tiny crystals of the membrane protein bacterio­rhodopsin and the collection of data sets for successful structure determination using the single-wavelength anomalous dispersion technique are also presented.

Published
2015
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21. Erratum to: High-Resolution Structure of a Membrane Protein Transferred from Amphipol to a Lipidic Mesophase

Author

Ekaterina Round, Vitaly Polovinkin, Alexey Mishin, P. Utrobin, Alexander Popov, Taras Balandin, Dieter Willbold, Valentin Borshchevskiy, Valentin Gordeliy, Vladimir Chupin, Alexander I. Kuklin, M. Sintsov, P. Chervakov, J.-L. Popot, Ivan Gushchin, and Vitaly Shevchenko

Subjects
Crystallography, Membrane protein, Physiology, Chemistry, Biophysics, High resolution, Mesophase, Cell Biology, Human physiology
Published
2017
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22. Low-dose X-ray radiation induces structural alterations in proteins

Author

Ivan S. Erofeev, Dieter Willbold, Ivan Gushchin, Georg Büldt, Valentin Gordeliy, Andrii Ishchenko, Ekaterina Round, Valentin Borshchevskiy, Alexey Mishin, Martin Weik, Moscow Institute of Physics and Technology [Moscow] (MIPT), Institute of Complex Systems (ICS), Forschungszentrum Jülich GmbH | Centre de recherche de Juliers, Helmholtz-Gemeinschaft = Helmholtz Association-Helmholtz-Gemeinschaft = Helmholtz Association, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institute of Crystallography [Aachen], Rheinisch-Westfälische Technische Hochschule Aachen (RWTH), Heinrich Heine Universität Düsseldorf = Heinrich Heine University [Düsseldorf], Helmholtz-Gemeinschaft = Helmholtz Association, Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Rheinisch-Westfälische Technische Hochschule Aachen University (RWTH), and Thomas, Frank

Subjects
Models, Molecular, biology, Fourier Analysis, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Protein Conformation, X-Rays, X-ray, Active site, Proteins, Bacteriorhodopsin, Dose-Response Relationship, Radiation, General Medicine, Radiation, Crystallography, X-Ray, Transport protein, Crystallography, Protein structure, Membrane protein, Structural Biology, Bacteriorhodopsins, Catalytic Domain, biology.protein, Radiation damage
Abstract

X-ray-radiation-induced alterations to protein structures are still a severe problem in macromolecular crystallography. One way to avoid the influence of radiation damage is to reduce the X-ray dose absorbed by the crystal during data collection. However, here it is demonstrated using the example of the membrane protein bacteriorhodopsin (bR) that even a low dose of less than 0.06 MGy may induce structural alterations in proteins. This dose is about 500 times smaller than the experimental dose limit which should ideally not be exceeded per data set (i.e.30 MGy) and 20 times smaller than previously detected specific radiation damage at the bR active site. To date, it is the lowest dose at which radiation modification of a protein structure has been described. Complementary use was made of high-resolution X-ray crystallography and online microspectrophotometry to quantitatively study low-dose X-ray-induced changes. It is shown that structural changes of the protein correlate with the spectroscopically observed formation of the so-called bR orange species. Evidence is provided for structural modifications taking place at the protein active site that should be taken into account in crystallographic studies which aim to elucidate the molecular mechanisms of bR function.

Published
2014

23. Nanoparticle surface-enhanced Raman scattering of bacteriorhodopsin stabilized by amphipol A8-35

Author

Ekaterina Round, P. Utrobin, Taras Balandin, Valentin Gordeliy, Vitaly Polovinkin, Dieter Willbold, J.-L. Popot, Antoine Royant, Grigory Arzumanyan, Vladimir Chupin, O. Volkov, Valentin Borshchevskiy, D. von Stetten, Institute of Complex Systems (ICS), Forschungszentrum Jülich GmbH | Centre de recherche de Juliers, Helmholtz-Gemeinschaft = Helmholtz Association-Helmholtz-Gemeinschaft = Helmholtz Association, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Moscow Institute of Physics and Technology [Moscow] (MIPT), European Synchrotron Radiation Facility (ESRF), Heinrich Heine Universität Düsseldorf = Heinrich Heine University [Düsseldorf], Helmholtz-Gemeinschaft = Helmholtz Association, Physico-chimie moléculaire des membranes biologiques (PCMMB), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Forschungszentrum Jülich GmbH, Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Heinrich-Heine-Universität Düsseldorf [Düsseldorf], and Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)

Subjects
Silver, Physiology, Polymers, [SDV]Life Sciences [q-bio], Biophysics, Nanoparticle, Metal Nanoparticles, Nanotechnology, 02 engineering and technology, engineering.material, 010402 general chemistry, Photochemistry, Spectrum Analysis, Raman, 01 natural sciences, Silver nanoparticle, Colloid, symbols.namesake, Surface-Active Agents, Molecule, biology, Propylamines, Chemistry, Bacteriorhodopsin, Cell Biology, 021001 nanoscience & nanotechnology, 0104 chemical sciences, Solubility, Bacteriorhodopsins, symbols, biology.protein, engineering, Feasibility Studies, Noble metal, 0210 nano-technology, Raman spectroscopy, Hydrophobic and Hydrophilic Interactions, Raman scattering
Abstract

International audience; Surface-enhanced Raman spectroscopy (SERS) has developed dramatically since its discovery in the 1970s, because of its power as an analytical tool for selective sensing of molecules adsorbed onto noble metal nanoparticles (NPs) and nanostructures, including at the single-molecule (SM) level. Despite the high importance of membrane proteins (MPs), SERS application to MPs has not really been studied, due to the great handling difficulties resulting from the amphiphilic nature of MPs. The ability of amphipols (APols) to trap MPs and keep them soluble, stable, and functional opens up onto highly interesting applications for SERS studies, possibly at the SM level. This seems to be feasible since single APol-trapped MPs can fit into gaps between noble metal NPs, or in other gap-containing SERS substrates, whereby the enhancement of Raman scattering signal may be sufficient for SM sensitivity. The goal of the present study is to give a proof of concept of SERS with APol-stabilized MPs, using bacteriorhodopsin (BR) as a model. BR trapped by APol A8-35 remains functional even after partial drying at a low humidity. A dried mixture of silver Lee-Meisel colloid NPs and BR/A8-35 complexes give rise to SERS with an average enhancement factor in excess of 10(2). SERS spectra resemble non-SERS spectra of a dried sample of BR/APol complexes.

Published
2014

24. X-ray structure of a CDP-alcohol phosphatidyltransferase membrane enzyme and insights into its catalytic mechanism

Author

Przemyslaw Nogly, Andrii Ishchenko, Ana M. Esteves, Nuno Borges, Margarida Archer, Ekaterina Round, Isabel Moraes, Valentin Gordeliy, Alina Remeeva, Ivan Gushchin, Sergei Grudinin, Valentin Borshchevskiy, Pikyee Ma, Helena Santos, Instituto de Tecnologia Química e Biológica António Xavier (ITQB), Universidade Nova de Lisboa = NOVA University Lisbon (NOVA), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Moscow Institute of Physics and Technology [Moscow] (MIPT), Institute of Complex Systems (ICS), Forschungszentrum Jülich GmbH | Centre de recherche de Juliers, Helmholtz-Gemeinschaft = Helmholtz Association-Helmholtz-Gemeinschaft = Helmholtz Association, Institute of Crystallography [Aachen], Rheinisch-Westfälische Technische Hochschule Aachen (RWTH), Algorithms for Modeling and Simulation of Nanosystems (NANO-D), Inria Grenoble - Rhône-Alpes, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria)-Laboratoire Jean Kuntzmann (LJK), Centre National de la Recherche Scientifique (CNRS)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Université Joseph Fourier - Grenoble 1 (UJF)-Université Pierre Mendès France - Grenoble 2 (UPMF)-Centre National de la Recherche Scientifique (CNRS)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Université Joseph Fourier - Grenoble 1 (UJF)-Université Pierre Mendès France - Grenoble 2 (UPMF), Department of Life Sciences, Imperial College London, Membrane Protein Laboratory, Diamond Light Source, DIAMOND Light source, STFC Rutherford Appleton Laboratory (RAL), Science and Technology Facilities Council (STFC), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Rheinisch-Westfälische Technische Hochschule Aachen University (RWTH), and Université Pierre Mendès France - Grenoble 2 (UPMF)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS)-Université Pierre Mendès France - Grenoble 2 (UPMF)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, Stereochemistry, Protein-protein interactions, Archaeal Proteins, Molecular Sequence Data, General Physics and Astronomy, membrane proteins, Crystallography, X-Ray, Cytidine Diphosphate, Protein Structure, Secondary, General Biochemistry, Genetics and Molecular Biology, Conserved sequence, Cell membrane, Phosphotransferase, Catalytic Domain, medicine, Magnesium, Amino Acid Sequence, Integral membrane protein, Magnesium ion, Multidisciplinary, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Cell Membrane, Phosphotransferases, Active site, General Chemistry, Transmembrane protein, Protein Structure, Tertiary, medicine.anatomical_structure, Membrane protein, Biochemistry, Archaeoglobus fulgidus, Biocatalysis, biology.protein, lipids (amino acids, peptides, and proteins), ddc:500, Sequence Alignment
Abstract

International audience; Phospholipids have major roles in the structure and function of all cell membranes. Most integral membrane proteins from the large CDP-alcohol phosphatidyltransferase family are involved in phospholipid biosynthesis across the three domains of life. They share a conserved sequence pattern and catalyse the displacement of CMP from a CDP-alcohol by a second alcohol. Here we report the crystal structure of a bifunctional enzyme comprising a cytoplasmic nucleotidyltransferase domain (IPCT) fused with a membrane CDP-alcohol phosphotransferase domain (DIPPS) at 2.65 Å resolution. The bifunctional protein dimerizes through the DIPPS domains, each comprising six transmembrane α-helices. The active site cavity is hydrophilic and widely open to the cytoplasm with a magnesium ion surrounded by four highly conserved aspartate residues from helices TM2 and TM3. We show that magnesium is essential for the enzymatic activity and is involved in catalysis. Substrates docking is validated by mutagenesis studies, and a structure-based catalytic mechanism is proposed.

Published
2014
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25. High-resolution structure of a membrane protein transferred from amphipol to a lipidic mesophase

Author

V. Schevchenko, Alexander Popov, Ivan Gushchin, Alexey Mishin, Vitaly Polovinkin, Taras Balandin, Vladimir Chupin, M. Sintsov, P. Utrobin, Ekaterina Round, Dieter Willbold, P. Chervakov, J.-L. Popot, Valentin Gordeliy, Alexander I. Kuklin, and Valentin Borshchevskiy

Subjects
Physiology, Polymers, Protein Conformation, 030310 physiology, Lipid Bilayers, Biophysics, law.invention, 03 medical and health sciences, Surface-Active Agents, Protein structure, law, Solubility, Crystallization, 030304 developmental biology, 0303 health sciences, biology, Propylamines, Chemistry, Mesophase, Membrane Proteins, Bacteriorhodopsin, Cell Biology, Folding (chemistry), Solutions, Crystallography, Membrane protein, Structural biology, Bacteriorhodopsins, biology.protein, Hydrophobic and Hydrophilic Interactions
Abstract

Amphipols (APols) have become important tools for the stabilization, folding, and in vitro structural and functional studies of membrane proteins (MPs). 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 form well-ordered crystals suitable for X-ray crystallography. In the present work, we show that an APol-trapped MP can be crystallized in meso. Bacteriorhodopsin (BR) trapped by APol A8-35 was mixed with a lipidic mesophase, and crystallization was induced by adding a precipitant. The crystals diffract beyond 2 A. The structure of BR was solved to 2 A and found to be indistinguishable from previous structures obtained after transfer from detergent solutions. We suggest the proposed protocol of in meso crystallization to be generally applicable to APol-trapped MPs.

Published
2014

26. Structural insights into the proton pumping by unusual proteorhodopsin from nonmarine bacteria

Author

Alexander Popov, Pavel Kuzmichev, Dmitry A. Dolgikh, Pavel Chervakov, Ivan Gushchin, Andrii Ishchenko, Alexander S. Arseniev, Ekaterina Round, Valentin Gordeliy, Lada E. Petrovskaya, Valentin Borshchevskiy, Mikhail P. Kirpichnikov, V. A. Chupin, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Thomas, Frank, Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)

Subjects
Rhodopsin, Proton, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Protein Structure, Secondary, Crystallography, X-Ray, Protein Structure, Secondary, 03 medical and health sciences, MESH: Protein Structure, Tertiary, Structure-Activity Relationship, MESH: Structure-Activity Relationship, Bacterial Proteins, Proton transport, Rhodopsins, Microbial, Side chain, MESH: Bacterial Proteins, Bacillaceae, MESH: Rhodopsins, Microbial, 030304 developmental biology, 0303 health sciences, Multidisciplinary, Proteorhodopsin, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Retinylidene protein, MESH: Rhodopsin, 030302 biochemistry & molecular biology, MESH: Bacillaceae, Bacteriorhodopsin, Biological Sciences, MESH: Crystallography, X-Ray, Protein Structure, Tertiary, Crystallography, Helix, biology.protein
Abstract

Light-driven proton pumps are present in many organisms. Here, we present a high-resolution structure of a proteorhodopsin from a permafrost bacterium, Exiguobacterium sibiricum rhodopsin (ESR). Contrary to the proton pumps of known structure, ESR possesses three unique features. First, ESR's proton donor is a lysine side chain that is situated very close to the bulk solvent. Second, the α-helical structure in the middle of the helix F is replaced by 3 10 - and π-helix–like elements that are stabilized by the Trp-154 and Asn-224 side chains. This feature is characteristic for the proteorhodopsin family of proteins. Third, the proton release region is connected to the bulk solvent by a chain of water molecules already in the ground state. Despite these peculiarities, the positions of water molecule and amino acid side chains in the immediate Schiff base vicinity are very well conserved. These features make ESR a very unusual proton pump. The presented structure sheds light on the large family of proteorhodopsins, for which structural information was not available previously.

Published
2013

27. Ground state structure of D75N mutant of sensory rhodopsin II in complex with its cognate transducer

Author

Johann P. Klare, Taras Balandin, Valentin Gordeliy, Martin Engelhard, Andrii Ishchenko, Ekaterina Round, Valentin Borshchevskiy, Alina Remeeva, Sergei Grudinin, Ivan Gushchin, Georg Büldt, Institute of Complex Systems (ICS), Forschungszentrum Jülich GmbH | Centre de recherche de Juliers, Helmholtz-Gemeinschaft = Helmholtz Association-Helmholtz-Gemeinschaft = Helmholtz Association, Institute of Crystallography [Aachen], Rheinisch-Westfälische Technische Hochschule Aachen University (RWTH), Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Moscow Institute of Physics and Technology [Moscow] (MIPT), Algorithms for Modeling and Simulation of Nanosystems (NANO-D), Inria Grenoble - Rhône-Alpes, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria)-Laboratoire Jean Kuntzmann (LJK), Université Pierre Mendès France - Grenoble 2 (UPMF)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS)-Université Pierre Mendès France - Grenoble 2 (UPMF)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS), Fachbereich Physik [Osnabrück], Universität Osnabrück - Osnabrück University, Max Planck Institute of Molecular Physiology, Max-Planck-Gesellschaft, Rheinisch-Westfälische Technische Hochschule Aachen (RWTH), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Université Joseph Fourier - Grenoble 1 (UJF)-Université Pierre Mendès France - Grenoble 2 (UPMF)-Centre National de la Recherche Scientifique (CNRS)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Université Joseph Fourier - Grenoble 1 (UJF)-Université Pierre Mendès France - Grenoble 2 (UPMF), and Universitat Osnabruck

Subjects
Models, Molecular, Light, Archaeal Proteins, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Mutant, Biophysics, Biology, Crystallography, X-Ray, medicine.disease_cause, 03 medical and health sciences, 0302 clinical medicine, Rhodopsins, Microbial, Aspartic acid, medicine, Sensory Rhodopsins, Radiology, Nuclear Medicine and imaging, membrane protein, x-ray crystallography, 030304 developmental biology, Negative phototaxis, 0303 health sciences, Mutation, Halobacteriaceae, Radiation, Radiological and Ultrasound Technology, Intracellular Signaling Peptides and Proteins, Sensory rhodopsin II, Hydrogen Bonding, Chemotaxis, Carotenoids, Two-component regulatory system, Biochemistry, Multiprotein Complexes, retinal protein, Signal transduction, Halorhodopsins, 030217 neurology & neurosurgery, signal transduction
Abstract

International audience; The complex of sensory rhodopsin II (NpSRII) with its cognate transducer (NpHtrII) mediates negative phototaxis in halobacteria Natronomonas pharaonis. Upon light activation NpSRII triggers, by means of NpHtrII, a signal transduction chain homologous to the two component system in eubacterial chemotaxis. Here we report on the crystal structure of the ground state of the mutant NpSRII-D75N/NpHtrII complex in the space group I212121. Mutations of this aspartic acid in light-driven proton pumps dramatically modify or/and inhibit protein functions. However, in vivo studies show that the similar D75N mutation retains functionality of the NpSRII/NpHtrII complex. The structure provides the molecular basis for the explanation of the unexpected observation that the wild and the mutant complexes display identical physiological response on light excitation.

Published
2013
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28. X-ray-radiation-induced changes in bacteriorhodopsin structure

Author

Georg Büldt, Valentin Gordeliy, Alexandr N. Popov, Valentin Borshchevskiy, and Ekaterina Round

Subjects
Models, Molecular, structural changes, Protein Conformation, methods [Crystallography, X-Ray], membrane proteins, 010402 general chemistry, Crystallography, X-Ray, 01 natural sciences, chemistry [Bacteriorhodopsins], 03 medical and health sciences, Structural Biology, Proton transport, ddc:570, Molecule, crystallography, Molecular Biology, 030304 developmental biology, Electrochemical potential, intermediate states, 0303 health sciences, biology, Fourier Analysis, Chemistry, Active site, Bacteriorhodopsin, X-ray protein, 0104 chemical sciences, Crystallography, Membrane, radiation damage, Bacteriorhodopsins, biology.protein, Absorption (chemistry), Isomerization
Abstract

Bacteriorhodopsin (bR) provides light-driven vectorial proton transport across a cell membrane. Creation of electrochemical potential at the membrane is a universal step in energy transformation in a cell. Published atomic crystallographic models of early intermediate states of bR show a significant difference between them, and conclusions about pumping mechanisms have been contradictory. Here, we present a quantitative high-resolution crystallographic study of conformational changes in bR induced by X-ray absorption. It is shown that X-ray doses that are usually accumulated during data collection for intermediate-state studies are sufficient to significantly alter the structure of the protein. X-ray-induced changes occur primarily in the active site of bR. Structural modeling showed that X-ray absorption triggers retinal isomerization accompanied by the disappearance of electron densities corresponding to the water molecule W402 bound to the Schiff base. It is demonstrated that these and other X-ray-induced changes may mimic functional conformational changes of bR leading to misinterpretation of the earlier obtained X-ray crystallographic structures of photointermediates.

Published
2011
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