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Retinal isomerization in bacteriorhodopsin captured by a femtosecond x-ray laser

Journal article
Authors P. Nogly
T. Weinert
D. James
S. Carbajo
D. Ozerov
A. Furrer
D. Gashi
V. Borin
P. Skopintsev
K. Jaeger
K. Nass
Petra Båth
Robert Bosman
J. Koglin
M. Seaberg
T. Lane
D. Kekilli
S. Brunle
T. Tanaka
W. T. Wu
C. Milne
T. White
A. Barty
U. Weierstall
V. Panneels
E. Nango
S. Iwata
M. Hunter
I. Schapiro
G. Schertler
Richard Neutze
J. Standfuss
Published in Science
Volume 361
Issue 6398
Pages 145-+
ISSN 0036-8075
Publication year 2018
Published at Department of Chemistry and Molecular Biology
Pages 145-+
Language en
Keywords resolved serial crystallography, primary photochemical event, photoactive yellow protein, excited-state dynamics, coherent-light, source, free-electron lasers, structural-changes, schiff-base, photoisomerization, spectroscopy, Science & Technology - Other Topics
Subject categories Spectroscopy


INTRODUCTION Retinal is a light-sensitive protein ligand that is used by all domains of life to process the information and energy content of light. Retinal-binding proteins are integral membrane proteins that drive vital biological processes, including light sensing for spatial orientation and circadian clock adjustment, as well as maintaining electrochemical gradients through ion transport. They also form the basis for optogenetic manipulation of neural cells. How the protein environment guides retinal isomerization on a subpicosecond time scale toward a single high-yield product is a fundamental outstanding question in photobiology. RATIONALE Light-induced isomerization of retinal is among the fastest reactions known in biology. It has been widely studied by spectroscopic techniques to probe the evolution of spectral intermediates over time. Using x-ray free-electron lasers (XFELs), it is now possible to observe ultrafast photochemical reactions and their induced molecular motions within proteins on scales of femtoseconds to milliseconds with near-atomic structural resolution. In this work, we used XFEL radiation to study the structural dynamics of retinal isomerization in the light-driven proton-pump bacteriorhodopsin (bR). The principal mechanism of isomerization in this prototypical retinal-binding protein has direct relevance for all other members of this important family of membrane proteins, and it provides insight into how protein environments catalyze photochemical reactions in general. RESULTS We collected high-resolution x-ray diffraction data from bR microcrystals injected across the femtosecond x-ray pulses of the Linac Coherent Light Source after excitation of the retinal chromophore by an optical laser pulse. X-ray diffraction images were sorted into temporal subgroups with a precision of about 200 fs. A series of 18 overlapping difference Fourier electron density maps reveal structural changes over the first picosecond of retinal photoexcitation. Complementary data for time delays of 10 ps and 8.33 ms allow us to resolve the later stages of the reaction. In combination with refined crystallographic structures at pump-probe delays corresponding to where the spectroscopically characterized I, J, K, and M intermediates form in solution, our time-resolved structural data reveal the trajectory of retinal isomerization and provide atomic details at key points along the reaction. The aspartic acid residues of the retinal counterion and functional water molecules in close proximity to the retinal Schiff base respond collectively to the formation and decay of the excited state. This collective motion sets the stage for retinal isomerization, which proceeds via a twisted retinal configuration. Quantum mechanics/molecular mechanics simulations provide theoretical support for this structural evolution. CONCLUSION Our observations reveal how, concomitant with the formation of the earliest excited state, the retinal-binding pocket opens up in close proximity to the isomerizing bond. We propose that ultrafast charge transfer along retinal is a driving force for collective motions that contribute to the stereoselectivity and efficiency of retinal isomerization within a protein scaffold. Vibrational quake-like motions extending from retinal to the protein may also be a mechanism through which excess energy is released in a nonradiative fashion.

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