14 results on '"Steffen Brünle"'
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2. Serial millisecond crystallography for routine room-temperature structure determination at synchrotrons
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Tobias Weinert, Natacha Olieric, Robert Cheng, Steffen Brünle, Daniel James, Dmitry Ozerov, Dardan Gashi, Laura Vera, May Marsh, Kathrin Jaeger, Florian Dworkowski, Ezequiel Panepucci, Shibom Basu, Petr Skopintsev, Andrew S. Doré, Tian Geng, Robert M. Cooke, Mengning Liang, Andrea E. Prota, Valerie Panneels, Przemyslaw Nogly, Ulrich Ermler, Gebhard Schertler, Michael Hennig, Michel O. Steinmetz, Meitian Wang, and Jörg Standfuss
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Science - Abstract
Serial crystallography was developed for protein crystal data collection with X-ray free-electron lasers. Here the authors present several examples which show that serial crystallography using high-viscosity injectors can also be routinely employed for room-temperature data collection at synchrotrons.
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- 2017
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3. The Molybdenum Storage Protein
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Tonie Baars, Iram Aziz, Juliane Poppe, Steffen Brünle, Klaus Schneider, and Ulrich Ermler
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- 2023
4. Ultrafast structural changes direct the first molecular events of vision
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Thomas Gruhl, Tobias Weinert, Matthew Rodrigues, Christopher J Milne, Giorgia Ortolani, Karol Nass, Eriko Nango, Saumik Sen, Philip J M Johnson, Claudio Cirelli, Antonia Furrer, Sandra Mous, Petr Skopintsev, Daniel James, Florian Dworkowski, Petra Båth, Demet Kekilli, Dmitry Ozerov, Rie Tanaka, Hannah Glover, Camila Bacellar, Steffen Brünle, Cecilia M Casadei, Azeglio D Diethelm, Dardan Gashi, Guillaume Gotthard, Ramon Guixà-González, Yasumasa Joti, Victoria Kabanova, Gregor Knopp, Elena Lesca, Pikyee Ma, Isabelle Martiel, Jonas Mühle, Shigeki Owada, Filip Pamula, Daniel Sarabi, Oliver Tejero, Ching-Ju Tsai, Niranjan Varma, Anna Wach, Sébastien Boutet, Kensuke Tono, Przemyslaw Nogly, Xavier Deupi, So Iwata, Richard Neutze, Jörg Standfuss, Gebhard FX Schertler, and Valerie Panneels
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basis-sets ,Multidisciplinary ,software ,energy-storage ,dynamics ,Photobiology ,isomerization ,rhodopsin ,excited-state ,retinal chromophore ,counterion displacement ,Visual system ,crystallography ,X-ray crystallography - Abstract
Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs). A photon is absorbed by the 11-cis retinal chromophore of rhodopsin, which isomerizes within 200 femtoseconds to the all-trans conformation, thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains experimentally unclear. Here we use ultrafast time-resolved crystallography at room temperature to determine how an isomerized twisted all-trans retinal stores the photon energy that is required to initiate the protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at a 1-ps time delay after photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Notably, the very early structural motions in the protein side chains of rhodopsin appear in regions that are involved in later stages of the conserved class A GPCR activation mechanism. Our study sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation., 視覚に関わるタンパク質の超高速分子動画 --薄暗いところで光を感じる仕組み--. 京都大学プレスリリース. 2023-03-23.
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- 2023
5. Release of a photopharmacological drug from its protein target captured by time-resolved serial crystallography
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Maximilian Wranik, Tobias Weinert, Chavdar Slavov, Tiziana Masini, Antonia Furrer, Natacha Gaillard, Dario Gioia, Marco Ferrarotti, Daniel James, Hannah Glover, Melissa Carrillo, Demet Kekilli, Robin Stipp, Petr Skopintsev, Steffen Brünle, Tobias Mühlethaler, John Beale, Dardan Gashi, Karol Nass, Dmitry Ozerov, Philip Johnson, Claudio Cirelli, Camila Bacellar, Markus Braun, Meitian Wang, Florian Dworkowski, Christopher Milne, Andrea Cavalli, Josef Wachtveitl, Michel Steinmetz, and Jörg Standfuss
- Abstract
The binding and release of ligands from their protein targets is central to fundamental biological processes as well as to drug discovery. Photopharmacology introduces chemical triggers that allow the changing of ligand affinities and thus biological activity by light. Insight into the molecular mechanisms of photopharmacology is largely missing because the relevant transitions during the light-triggered reaction cannot be resolved by conventional structural biology. Using time-resolved serial crystallography at a synchrotron and X-ray free-electron laser, we have captured the release of azo-combretastatin A4 and the resulting conformational changes in tubulin. Nine structural snapshots from 1 ns to 100 ms complemented by simulations show how cis-to-trans isomerization of the azobenzene bond leads to a switch in ligand affinity, opening of an exit channel, and collapse of the binding pocket upon ligand release. The resulting global backbone rearrangements are related to the action mechanism of tubulin-binding drugs against gout, cancer, and COVID-19.
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- 2022
6. Watching the release of a photopharmacological drug from tubulin using time-resolved serial crystallography
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Maximilian Wranik, Tobias Weinert, Chavdar Slavov, Tiziana Masini, Antonia Furrer, Natacha Gaillard, Dario Gioia, Marco Ferrarotti, Daniel James, Hannah Glover, Melissa Carrillo, Demet Kekilli, Robin Stipp, Petr Skopintsev, Steffen Brünle, Tobias Mühlethaler, John Beale, Dardan Gashi, Karol Nass, Dmitry Ozerov, Philip J. M. Johnson, Claudio Cirelli, Camila Bacellar, Markus Braun, Meitian Wang, Florian Dworkowski, Chris Milne, Andrea Cavalli, Josef Wachtveitl, Michel O. Steinmetz, and Jörg Standfuss
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Multidisciplinary ,General Physics and Astronomy ,General Chemistry ,General Biochemistry, Genetics and Molecular Biology - Abstract
The binding and release of ligands from their protein targets is central to fundamental biological processes as well as to drug discovery. Photopharmacology introduces chemical triggers that allow the changing of ligand affinities and thus biological activity by light. Insight into the molecular mechanisms of photopharmacology is largely missing because the relevant transitions during the light-triggered reaction cannot be resolved by conventional structural biology. Using time-resolved serial crystallography at a synchrotron and X-ray free-electron laser, we capture the release of the anti-cancer compound azo-combretastatin A4 and the resulting conformational changes in tubulin. Nine structural snapshots from 1 ns to 100 ms complemented by simulations show how cis-to-trans isomerization of the azobenzene bond leads to a switch in ligand affinity, opening of an exit channel, and collapse of the binding pocket upon ligand release. The resulting global backbone rearrangements are related to the action mechanism of microtubule-destabilizing drugs.
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- 2022
7. Dynamics and mechanism of a light-driven chloride pump
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Sandra Mous, Guillaume Gotthard, David Ehrenberg, Saumik Sen, Tobias Weinert, Philip J. M. Johnson, Daniel James, Karol Nass, Antonia Furrer, Demet Kekilli, Pikyee Ma, Steffen Brünle, Cecilia Maria Casadei, Isabelle Martiel, Florian Dworkowski, Dardan Gashi, Petr Skopintsev, Maximilian Wranik, Gregor Knopp, Ezequiel Panepucci, Valerie Panneels, Claudio Cirelli, Dmitry Ozerov, Gebhard F. X. Schertler, Meitian Wang, Chris Milne, Joerg Standfuss, Igor Schapiro, Joachim Heberle, and Przemyslaw Nogly
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Multidisciplinary - Abstract
Chloride transport by microbial rhodopsins is an essential process for which molecular details such as the mechanisms that convert light energy to drive ion pumping and ensure the unidirectionality of the transport have remained elusive. We combined time-resolved serial crystallography with time-resolved spectroscopy and multiscale simulations to elucidate the molecular mechanism of a chloride-pumping rhodopsin and the structural dynamics throughout the transport cycle. We traced transient anion-binding sites, obtained evidence for how light energy is used in the pumping mechanism, and identified steric and electrostatic molecular gates ensuring unidirectional transport. An interaction with the π-electron system of the retinal supports transient chloride ion binding across a major bottleneck in the transport pathway. These results allow us to propose key mechanistic features enabling finely controlled chloride transport across the cell membrane in this light-powered chloride ion pump. ISSN:0036-8075 ISSN:1095-9203
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- 2022
8. Molybdate pumping into the molybdenum storage protein via an ATP-powered piercing mechanism
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Ulrich Ermler, Juliane Poppe, Deryck J. Mills, Steffen Brünle, Martin Lorenz Eisinger, Janet Vonck, and Julian David Langer
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0301 basic medicine ,Pyrophosphatase ,Multidisciplinary ,biology ,chemistry.chemical_element ,Biological Sciences ,Molybdate ,010402 general chemistry ,biology.organism_classification ,01 natural sciences ,0104 chemical sciences ,03 medical and health sciences ,chemistry.chemical_compound ,Crystallography ,030104 developmental biology ,Protein structure ,chemistry ,Azotobacter vinelandii ,ATP hydrolysis ,Molybdenum ,Side chain ,UMP kinase - Abstract
The molybdenum storage protein (MoSto) deposits large amounts of molybdenum as polyoxomolybdate clusters in a heterohexameric (αβ) 3 cage-like protein complex under ATP consumption. Here, we suggest a unique mechanism for the ATP-powered molybdate pumping process based on X-ray crystallography, cryoelectron microscopy, hydrogen-deuterium exchange mass spectrometry, and mutational studies of MoSto from Azotobacter vinelandii . First, we show that molybdate, ATP, and Mg 2+ consecutively bind into the open ATP-binding groove of the β-subunit, which thereafter becomes tightly locked by fixing the previously disordered N-terminal arm of the α-subunit over the β-ATP. Next, we propose a nucleophilic attack of molybdate onto the γ-phosphate of β-ATP, analogous to the similar reaction of the structurally related UMP kinase. The formed instable phosphoric-molybdic anhydride becomes immediately hydrolyzed and, according to the current data, the released and accelerated molybdate is pressed through the cage wall, presumably by turning aside the Metβ149 side chain. A structural comparison between MoSto and UMP kinase provides valuable insight into how an enzyme is converted into a molecular machine during evolution. The postulated direct conversion of chemical energy into kinetic energy via an activating molybdate kinase and an exothermic pyrophosphatase reaction to overcome a proteinous barrier represents a novelty in ATP-fueled biochemistry, because normally, ATP hydrolysis initiates large-scale conformational changes to drive a distant process.
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- 2019
9. The Molybdenum Storage Protein: A soluble<scp>ATP</scp>hydrolysis‐dependent molybdate pump
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Steffen Brünle, Ulrich Ermler, Katharina Wiesemann, Klaus Schneider, Ron Hail, and Juliane Poppe
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Models, Molecular ,0301 basic medicine ,Protein Conformation ,Protein subunit ,Protein Data Bank (RCSB PDB) ,Molybdate ,Crystallography, X-Ray ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Adenosine Triphosphate ,Bacterial Proteins ,ATP hydrolysis ,Metalloproteins ,Molecular Biology ,Molybdenum ,Azotobacter vinelandii ,Binding Sites ,biology ,Nitrogenase ,Cell Biology ,Phosphate ,biology.organism_classification ,Crystallography ,030104 developmental biology ,chemistry ,Polyoxometalate ,Protein Binding - Abstract
A continuous FeMo cofactor supply for nitrogenase maturation is ensured in Azotobacter vinelandii by developing a cage-like molybdenum storage protein (MoSto) capable to store ca. 120 molybdate molecules ( MoO 4 2 - ) as discrete polyoxometalate (POM) clusters. To gain mechanistic insight into this process, MoSto was characterized by Mo and ATP/ADP content, structural, and kinetic analysis. We defined three functionally relevant states specified by the presence of both ATP/ADP and POM clusters (MoStofunct ), of only ATP/ADP (MoStobasal ) and of neither ATP/ADP nor POM clusters (MoStozero ), respectively. POM clusters are only produced when ATP is hydrolyzed to ADP and phosphate. Vmax was ca. 13 μmolphosphate ·min-1 ·mg-1 and Km for molybdate and ATP/Mg2+ in the low micromolar range. ATP hydrolysis presumably proceeds at subunit α, inferred from a highly occupied α-ATP/Mg2+ and a weaker occupied β-ATP/no Mg2+ -binding site found in the MoStofunct structure. Several findings indicate that POM cluster storage is separated into a rapid ATP hydrolysis-dependent molybdate transport across the protein cage wall and a slow molybdate assembly induced by combined auto-catalytic and protein-driven processes. The cage interior, the location of the POM cluster depot, is locked in all three states and thus not rapidly accessible for molybdate from the outside. Based on Vmax , the entire Mo storage process should be completed in less than 10 s but requires, according to the molybdate content analysis, ca. 15 min. Long-time incubation of MoStobasal with nonphysiological high molybdate amounts implicates an equilibrium in and outside the cage and POM cluster self-formation without ATP hydrolysis. DATABASES: The crystal structures MoSto in the MoSto-F6, MoSto-F7, MoStobasal , MoStozero , and MoSto-F1vitro states were deposited to PDB under the accession numbers PDB 6GU5, 6GUJ, 6GWB, 6GWV, and 6GX4.
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- 2018
10. The Crystal Structure of RosB: Insights into the Reaction Mechanism of the First Member of a Family of Flavodoxin-like Enzymes
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Valentino Konjik, Amanda Vanselow, Ulrich Ermler, Matthias Mack, Steffen Brünle, Ulrike Demmer, and Roger Sandhoff
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Models, Molecular ,0301 basic medicine ,Protein Conformation ,Flavodoxin ,Stereochemistry ,Riboflavin ,030106 microbiology ,Substituent ,Flavoprotein ,Crystallography, X-Ray ,Mass Spectrometry ,Catalysis ,Cofactor ,03 medical and health sciences ,chemistry.chemical_compound ,Bacterial Proteins ,Biosynthesis ,Transaminases ,biology ,ATP synthase ,Substrate (chemistry) ,Active site ,General Chemistry ,030104 developmental biology ,chemistry ,Biocatalysis ,biology.protein - Abstract
8-demethyl-8-aminoriboflavin-5′-phosphate (AFP) synthase (RosB) catalyzes the key reaction of roseoflavin biosynthesis by forming AFP from riboflavin-5′-phosphate (RP) and glutamate via the intermediates 8-demethyl-8-formylriboflavin-5′-phosphate (OHC-RP) and 8-demethyl-8-carboxylriboflavin-5′-phosphate (HO2C-RP). To understand this reaction in which a methyl substituent of an aromatic ring is replaced by an amine we structurally characterized RosB in complex with OHC-RP (2.0 A) and AFP (1.7 A). RosB is composed of four flavodoxin-like subunits which have been upgraded with specific extensions and a unique C-terminal arm. It appears that RosB has evolved from an electron- or hydride-transferring flavoprotein to a sophisticated multi-step enzyme which uses RP as a substrate (and not as a cofactor). Structure-based active site analysis was complemented by mutational and isotope-based mass-spectrometric data to propose an enzymatic mechanism on an atomic basis.
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- 2016
11. Die Kristallstruktur von RosB: Einblicke in den Reaktionsmechanismus des ersten Mitglieds einer flavodoxinähnlichen Enzymfamilie
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Amanda Vanselow, Steffen Brünle, Ulrike Demmer, Matthias Mack, Valentino Konjik, Ulrich Ermler, and Roger Sandhoff
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0301 basic medicine ,Streptomyces davawensis ,03 medical and health sciences ,030104 developmental biology ,Roseoflavin ,Stereochemistry ,Chemistry ,General Medicine - Published
- 2016
12. The molybdenum storage protein - A bionanolab for creating experimentally alterable polyoxomolybdate clusters
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Steffen Brünle, Ulrich Ermler, Juliane Poppe, Ron Hail, and Ulrike Demmer
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chemistry.chemical_element ,Structural difference ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Inorganic Chemistry ,Metal ,Bacterial Proteins ,Cluster (physics) ,Storage protein ,Production system ,chemistry.chemical_classification ,Molybdenum ,Azotobacter vinelandii ,biology ,010405 organic chemistry ,biology.organism_classification ,0104 chemical sciences ,Crystallography ,chemistry ,Covalent bond ,visual_art ,visual_art.visual_art_medium ,Protein Binding - Abstract
Various N2-fixing bacteria contain a cage-like molybdenum storage protein (MoSto) that deposits more than 100 Mo as discrete polyoxomolybdate (POMo) clusters. To explore the relationship between modifiable cage properties/preparation conditions on one side and the types of POMo clusters formed on the other we established a recombinant production system for MoSto of Azotobacter vinelandii and prepared site-specifically mutated, “in vivo-like and in vitro” POMo cluster-loaded and POMo cluster-free MoSto. Seven representative X-ray structures revealed highly different POMo clusters inside architecturally rather related MoSto cages. The only significant structural difference includes a small polypeptide segment, the β-linker, which protrudes differently far into the cage interior. The β-linker is positioned outwards in in vivo-like structures of MoSto (treated with ATP and Na2MoO4 during preparation) and inwards in in vitro structures (obtained after loading the purified POMo-cluster free MoSto with ATP and Na2MoO4). Non-covalent Mo8, Mo6–7 and Mo8–14 clusters are exclusively present in in vivo-like structures. Instead, in vitro structures contain a new well-defined Mo5-7 cluster II. The digit(s) behind Mo defines the (variable) number of metal atoms in the respective POMo clusters. In comparison to the native MoSto structures the Lα131H variant is characterized by a new non-covalent Mo3 cluster and a ca. 5 A shifted Mo5–7 cluster II, by which the covalent Mo8 cluster becomes structurally modified. Altogether, the unique bionanolab in the MoSto cage is able to create a large variety of POMo clusters by expansions, fusions and positional/orientational variations of a few discrete polynuclear Mo-O building blocks.
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- 2018
13. Serial millisecond crystallography for routine room-temperature structure determination at synchrotrons
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Gebhard F. X. Schertler, May Marsh, Steffen Brünle, Dmitry Ozerov, Andrew S. Doré, Mengning Liang, Robert M. Cooke, Przemyslaw Nogly, Florian S. N. Dworkowski, Michel O. Steinmetz, Ulrich Ermler, T. Geng, Ezequiel Panepucci, Laura Vera, Natacha Olieric, Valerie Panneels, Andrea E. Prota, Dardan Gashi, Meitian Wang, Petr Skopintsev, Jörg Standfuss, Kathrin Jaeger, Michael Hennig, Tobias Weinert, Robert K. Y. Cheng, Shibom Basu, and Daniel James
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0301 basic medicine ,Millisecond ,Multidisciplinary ,Materials science ,Serial communication ,Science ,Resolution (electron density) ,General Physics and Astronomy ,macromolecular substances ,General Chemistry ,Article ,General Biochemistry, Genetics and Molecular Biology ,Synchrotron ,law.invention ,Crystal ,03 medical and health sciences ,Crystallography ,030104 developmental biology ,Beamline ,law ,lcsh:Q ,Molecular replacement ,lcsh:Science ,Protein crystallization - Abstract
Historically, room-temperature structure determination was succeeded by cryo-crystallography to mitigate radiation damage. Here, we demonstrate that serial millisecond crystallography at a synchrotron beamline equipped with high-viscosity injector and high frame-rate detector allows typical crystallographic experiments to be performed at room-temperature. Using a crystal scanning approach, we determine the high-resolution structure of the radiation sensitive molybdenum storage protein, demonstrate soaking of the drug colchicine into tubulin and native sulfur phasing of the human G protein-coupled adenosine receptor. Serial crystallographic data for molecular replacement already converges in 1,000–10,000 diffraction patterns, which we collected in 3 to maximally 82 minutes. Compared with serial data we collected at a free-electron laser, the synchrotron data are of slightly lower resolution, however fewer diffraction patterns are needed for de novo phasing. Overall, the data we collected by room-temperature serial crystallography are of comparable quality to cryo-crystallographic data and can be routinely collected at synchrotrons., Nature Communications, 8, ISSN:2041-1723
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- 2017
14. Femtosecond-to-millisecond structural changes in a light-driven sodium pump
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Dardan Gashi, Xavier Deupi, Isabelle Martiel, Joachim Heberle, Jörg Standfuss, Gebhard F. X. Schertler, Karol Nass, Daniel James, Demet Kekilli, Antonia Furrer, Christopher J. Milne, D. Ehrenberg, Claudio Cirelli, Valerie Panneels, Gregor Knopp, Tobias Weinert, Christopher Arrell, Przemyslaw Nogly, Philip J. M. Johnson, S. Mous, Thomas Gruhl, Steffen Brünle, Roger Benoit, Dmitry Ozerov, Rajiv K. Kar, Igor Schapiro, Florian S. N. Dworkowski, Petr Skopintsev, and M. Wranik
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0301 basic medicine ,Time Factors ,Sodium ,Static Electricity ,chemistry.chemical_element ,Electrons ,010402 general chemistry ,01 natural sciences ,03 medical and health sciences ,Isomerism ,Rhodopsins, Microbial ,Schiff Bases ,Ion transporter ,Membrane potential ,Millisecond ,Binding Sites ,Crystallography ,Ion Transport ,Multidisciplinary ,biology ,Chemistry ,Lasers ,Spectrum Analysis ,0104 chemical sciences ,Microsecond ,030104 developmental biology ,Rhodopsin ,Femtosecond ,Retinaldehyde ,biology.protein ,Biophysics ,Quantum Theory ,Protons ,Sodium-Potassium-Exchanging ATPase ,Flavobacteriaceae ,Cation transport - Abstract
Light-driven sodium pumps actively transport small cations across cellular membranes1. These pumps are used by microorganisms to convert light into membrane potential and have become useful optogenetic tools with applications in neuroscience. Although the resting state structures of the prototypical sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) have been solved2,3, it is unclear how structural alterations over time allow sodium to be translocated against a concentration gradient. Here, using the Swiss X-ray Free Electron Laser4, we have collected serial crystallographic data at ten pump–probe delays from femtoseconds to milliseconds. High-resolution structural snapshots throughout the KR2 photocycle show how retinal isomerization is completed on the femtosecond timescale and changes the local structure of the binding pocket in the early nanoseconds. Subsequent rearrangements and deprotonation of the retinal Schiff base open an electrostatic gate in microseconds. Structural and spectroscopic data, in combination with quantum chemical calculations, indicate that a sodium ion binds transiently close to the retinal within one millisecond. In the last structural intermediate, at 20 milliseconds after activation, we identified a potential second sodium-binding site close to the extracellular exit. These results provide direct molecular insight into the dynamics of active cation transport across biological membranes. ISSN:0028-0836 ISSN:1476-4687
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