A three-dimensional movie of structural changes in bacteriorhodopsin

Snapshots of bacteriorhodopsin Bacteriorhodopsin is a membrane protein that harvests the energy content from light to transport protons out of the cell against a transmembrane potential. Nango et al. used timeresolved serial femtosecond crystallography at an x-ray free electron laser to provide 13 structural snapshots of the conformational changes that occur in the nanoseconds to milliseconds after photoactivation. These changes begin at the active site, propagate toward the extracellular side of the protein, and mediate internal protonation exchanges that achieve proton transport. Science, this issue p. 1552 Time-resolved serial crystallography using an x-ray free electron laser reveals structural changes in bacteriorhodopsin. Bacteriorhodopsin (bR) is a light-driven proton pump and a model membrane transport protein. We used time-resolved serial femtosecond crystallography at an x-ray free electron laser to visualize conformational changes in bR from nanoseconds to milliseconds following photoactivation. An initially twisted retinal chromophore displaces a conserved tryptophan residue of transmembrane helix F on the cytoplasmic side of the protein while dislodging a key water molecule on the extracellular side. The resulting cascade of structural changes throughout the protein shows how motions are choreographed as bR transports protons uphill against a transmembrane concentration gradient.

Takashi Kameshima | Gebhard F. X. Schertler | Makina Yabashi | Takanori Nakane | Takaki Hatsui | Antoine Royant | Tomohiro Nishizawa | So Iwata | Kensuke Tono | Ana-Nicoleta Bondar | Osamu Nureki | Shigeki Owada | Rie Tanaka | Ayumi Yamashita | Yasumasa Joti | Richard Neutze | Eriko Nango | Daewoong Nam | O. Nureki | R. Neutze | Changyong Song | D. Nam | T. Hatsui | Y. Joti | T. Kameshima | K. Tono | M. Yabashi | S. Iwata | T. Nishizawa | M. Kubo | M. Sugahara | E. Nango | Tomoyuki Tanaka | J. Davidsson | P. Nogly | E. Mizohata | M. Fukuda | G. Schertler | A. Royant | A. Bondar | T. Nakane | T. Shimamura | M. Murata | D. Im | J. Standfuss | S. Matsuoka | Tetsunari Kimura | Jan Davidsson | Minoru Kubo | Cecilia Wickstrand | Tetsunari Kimura | Tomoyuki Tanaka | Changyong Song | T. Arima | Jun Kobayashi | Toshiaki Hosaka | Eiichi Mizohata | Przemyslaw Nogly | Michihiro Sugahara | Takashi Nomura | Tatsuro Shimamura | D. Im | Takaaki Fujiwara | Yasuaki Yamanaka | Byeonghyun Jeon | Kazumasa Oda | Masahiro Fukuda | Rebecka Andersson | Petra Båth | Robert Dods | Shigeru Matsuoka | Satoshi Kawatake | Michio Murata | Jörg Standfuss | S. Owada | R. Tanaka | Jun Kobayashi | P. Båth | Cecilia Wickstrand | J. Kobayashi | Satoshi Kawatake | T. Hosaka | Changyong Song | T. Arima | Ayumi Yamashita | Takashi Nomura | T. Fujiwara | Y. Yamanaka | Byeonghyun Jeon | K. Oda | R. Andersson | R. Dods | J. Kobayashi | S. Matsuoka | C. Song | Rie Tanaka | B. Jeon | Byeonghyun Jeon | Takaaki Fujiwara | Michio Murata

[1]  O. Jardetzky,et al.  Simple Allosteric Model for Membrane Pumps , 1966, Nature.

[2]  L. T. Eyck,et al.  Crystallographic fast Fourier transforms , 1973 .

[3]  W. L. Jorgensen,et al.  Comparison of simple potential functions for simulating liquid water , 1983 .

[4]  M. Karplus,et al.  CHARMM: A program for macromolecular energy, minimization, and dynamics calculations , 1983 .

[5]  M. Sheves,et al.  Controlling the pKa of the bacteriorhodopsin Schiff base by use of artificial retinal analogues. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[6]  R. Govindjee,et al.  REGENERATION OF BLUE AND PURPLE MEMBRANES FROM DEIONIZED BLEACHED MEMBRANES OF Halobacterium halobium , 1988 .

[7]  D. Oesterhelt,et al.  Time‐resolved X‐ray diffraction study of structural changes associated with the photocycle of bacteriorhodopsin. , 1991, The EMBO journal.

[8]  M. Engelhard,et al.  Asp85 is the only internal aspartic acid that gets protonated in the M intermediate and the purple‐to‐blue transition of bacteriorhodopsin A solid‐state13C CP‐MAS NMR investigation , 1992, FEBS letters.

[9]  Albert J. M. Duisenberg,et al.  Indexing in single‐crystal diffractometry with an obstinate list of reflections , 1992 .

[10]  Two light-transducing membrane proteins: bacteriorhodopsin and the mammalian rhodopsin. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[11]  J. Lanyi,et al.  Glutamic Acid 204 is the Terminal Proton Release Group at the Extracellular Surface of Bacteriorhodopsin (*) , 1995, The Journal of Biological Chemistry.

[12]  J. Rosenbusch,et al.  Lipidic cubic phases: a novel concept for the crystallization of membrane proteins. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[13]  B. Roux,et al.  Structure and dynamics of a proton wire: a theoretical study of H+ translocation along the single-file water chain in the gramicidin A channel. , 1996, Biophysical journal.

[14]  G. A. Jeffrey,et al.  An Introduction to Hydrogen Bonding , 1997 .

[15]  A. Vagin,et al.  MOLREP: an Automated Program for Molecular Replacement , 1997 .

[16]  H Luecke,et al.  Proton transfer pathways in bacteriorhodopsin at 2.3 angstrom resolution. , 1998, Science.

[17]  Sándor Suhai,et al.  Self-consistent-charge density-functional tight-binding method for simulations of complex materials properties , 1998 .

[18]  Alexander D. MacKerell,et al.  All-atom empirical potential for molecular modeling and dynamics studies of proteins. , 1998, The journal of physical chemistry. B.

[19]  Robert H. Blessing,et al.  Difference structure‐factor normalization for heavy‐atom or anomalous‐scattering substructure determinations , 1999 .

[20]  Karl Edman,et al.  High-resolution X-ray structure of an early intermediate in the bacteriorhodopsin photocycle , 1999, Nature.

[21]  H Luecke,et al.  Structural changes in bacteriorhodopsin during ion transport at 2 angstrom resolution. , 1999, Science.

[22]  D. Oesterhelt,et al.  Closing in on bacteriorhodopsin: progress in understanding the molecule. , 1999, Annual review of biophysics and biomolecular structure.

[23]  P. Ormos,et al.  Structural alterations for proton translocation in the M state of wild-type bacteriorhodopsin , 2000, Nature.

[24]  B. Schobert,et al.  Coupling photoisomerization of retinal to directional transport in bacteriorhodopsin. , 2000, Journal of molecular biology.

[25]  Richard Henderson,et al.  Molecular mechanism of vectorial proton translocation by bacteriorhodopsin , 2000, Nature.

[26]  E. Pebay-Peyroula,et al.  Helix deformation is coupled to vectorial proton transport in the photocycle of bacteriorhodopsin , 2000, Nature.

[27]  J. Hajdu,et al.  Potential for biomolecular imaging with femtosecond X-ray pulses , 2000, Nature.

[28]  Stefan Zaefferer,et al.  New developments of computer-aided crystallographic analysis in transmission electron microscopy , 2000 .

[29]  Efthimios Kaxiras,et al.  A QM/MM Implementation of the Self-Consistent Charge Density Functional Tight Binding (SCC-DFTB) Method , 2001 .

[30]  R B Rose,et al.  Structure of an early intermediate in the M-state phase of the bacteriorhodopsin photocycle. , 2001, Biophysical journal.

[31]  J. Herzfeld,et al.  Tight Asp-85--Thr-89 association during the pump switch of bacteriorhodopsin. , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[32]  Judith Herzfeld,et al.  Interaction of internal water molecules with the schiff base in the L intermediate of the bacteriorhodopsin photocycle. , 2002, Biochemistry.

[33]  V. Hornak,et al.  Crystallographic structure of the K intermediate of bacteriorhodopsin: conservation of free energy after photoisomerization of the retinal. , 2002, Journal of molecular biology.

[34]  M. Murakami,et al.  Specific damage induced by X-ray radiation and structural changes in the primary photoreaction of bacteriorhodopsin. , 2002, Journal of molecular biology.

[35]  Karl Edman,et al.  Bacteriorhodopsin: a high-resolution structural view of vectorial proton transport. , 2002, Biochimica et biophysica acta.

[36]  Randy J Read,et al.  Electronic Reprint Biological Crystallography Phenix: Building New Software for Automated Crystallographic Structure Determination Biological Crystallography Phenix: Building New Software for Automated Crystallographic Structure Determination , 2022 .

[37]  Klaus Schulten,et al.  Structural changes during the formation of early intermediates in the bacteriorhodopsin photocycle. , 2002, Biophysical journal.

[38]  R. Henderson,et al.  Proton translocation by bacteriorhodopsin in the absence of substantial conformational changes. , 2002, Journal of molecular biology.

[39]  Marius Schmidt,et al.  Application of singular value decomposition to the analysis of time-resolved macromolecular x-ray data. , 2003, Biophysical journal.

[40]  R. Gennis,et al.  Water-mediated hydrogen-bonded network on the cytoplasmic side of the Schiff base of the L photointermediate of bacteriorhodopsin. , 2003, Biochemistry.

[41]  R. Gennis,et al.  Water molecule rearrangements around Leu93 and Trp182 in the formation of the L intermediate in bacteriorhodopsin's photocycle. , 2003, Biochemistry.

[42]  P. Nollert,et al.  Lipidic cubic phases as matrices for membrane protein crystallization. , 2004, Methods.

[43]  Fei Long,et al.  REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use. , 2004, Acta crystallographica. Section D, Biological crystallography.

[44]  Karl Edman,et al.  Deformation of Helix C in the Low Temperature L-intermediate of Bacteriorhodopsin* , 2004, Journal of Biological Chemistry.

[45]  T. Kouyama,et al.  Crystal structure of the L intermediate of bacteriorhodopsin: evidence for vertical translocation of a water molecule during the proton pumping cycle. , 2004, Journal of molecular biology.

[46]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[47]  Alexander D. MacKerell,et al.  Extending the treatment of backbone energetics in protein force fields: Limitations of gas‐phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations , 2004, J. Comput. Chem..

[48]  Q. Cui,et al.  A critical evaluation of different QM/MM frontier treatments with SCC-DFTB as the QM method. , 2005, The journal of physical chemistry. B.

[49]  Jeremy C. Smith,et al.  Tuning of retinal twisting in bacteriorhodopsin controls the directionality of the early photocycle steps. , 2005, The journal of physical chemistry. B.

[50]  B. Schobert,et al.  Structural changes in the L photointermediate of bacteriorhodopsin. , 2007, Journal of molecular biology.

[51]  Jeremy C. Smith,et al.  Key role of active-site water molecules in bacteriorhodopsin proton-transfer reactions. , 2008, The journal of physical chemistry. B.

[52]  Michael Wulff,et al.  Structural dynamics of light-driven proton pumps. , 2009, Structure.

[53]  E. Round,et al.  X-ray-radiation-induced changes in bacteriorhodopsin structure. , 2011, Journal of molecular biology.

[54]  Randy J. Read,et al.  Overview of the CCP4 suite and current developments , 2011, Acta crystallographica. Section D, Biological crystallography.

[55]  Philip R. Evans,et al.  An introduction to data reduction: space-group determination, scaling and intensity statistics , 2011, Acta crystallographica. Section D, Biological crystallography.

[56]  Michael Gaus,et al.  DFTB3: Extension of the self-consistent-charge density-functional tight-binding method (SCC-DFTB). , 2011, Journal of chemical theory and computation.

[57]  Anton Barty,et al.  CrystFEL: a software suite for snapshot serial crystallography , 2012 .

[58]  Hirotada Ohashi,et al.  Beamline, experimental stations and photon beam diagnostics for the hard x-ray free electron laser of SACLA , 2013 .

[59]  M. Elstner,et al.  Parametrization and Benchmark of DFTB3 for Organic Molecules. , 2013, Journal of chemical theory and computation.

[60]  Anton Barty,et al.  Crystallographic data processing for free-electron laser sources , 2013, Acta crystallographica. Section D, Biological crystallography.

[61]  M. Facciotti,et al.  Deprotonation of D96 in bacteriorhodopsin opens the proton uptake pathway. , 2013, Structure.

[62]  Garth J. Williams,et al.  Time-resolved serial crystallography captures high-resolution intermediates of photoactive yellow protein , 2014, Science.

[63]  Kunio Hirata,et al.  Determination of damage-free crystal structure of an X-ray–sensitive protein using an XFEL , 2014, Nature Methods.

[64]  Takashi Kameshima,et al.  Development of an X-ray pixel detector with multi-port charge-coupled device for X-ray free-electron laser experiments. , 2014, The Review of scientific instruments.

[65]  Anton Barty,et al.  Cheetah: software for high-throughput reduction and analysis of serial femtosecond X-ray diffraction data , 2014, Journal of applied crystallography.

[66]  Anton Barty,et al.  Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography , 2014, Nature Communications.

[67]  E. Round,et al.  Low-dose X-ray radiation induces structural alterations in proteins. , 2014, Acta crystallographica. Section D, Biological crystallography.

[68]  R. Neutze,et al.  Bacteriorhodopsin: Would the real structural intermediates please stand up? , 2015, Biochimica et biophysica acta.

[69]  Sébastien Boutet,et al.  Direct observation of ultrafast collective motions in CO myoglobin upon ligand dissociation , 2015, Science.

[70]  Yoshiki Tanaka,et al.  Diverse application platform for hard X-ray diffraction in SACLA (DAPHNIS): application to serial protein crystallography using an X-ray free-electron laser , 2015, Journal of synchrotron radiation.

[71]  Anton Barty,et al.  Indications of radiation damage in ferredoxin microcrystals using high-intensity X-FEL beams. , 2014, Journal of synchrotron radiation.

[72]  Makina Yabashi,et al.  Data processing pipeline for serial femtosecond crystallography at SACLA , 2016, Journal of applied crystallography.

[73]  Garth J. Williams,et al.  Lipidic cubic phase injector is a viable crystal delivery system for time-resolved serial crystallography , 2016, Nature Communications.

[74]  Anton Barty,et al.  Femtosecond structural dynamics drives the trans/cis isomerization in photoactive yellow protein , 2016, Science.

[75]  Garth J. Williams,et al.  Lipidic cubic phase injector is a viable crystal delivery system for time-resolved serial crystallography , 2016, Nature Communications.