Look fast Organisms from bacteria to humans sense and react to light. Proteins that contain the light-sensitive molecule retinal couple absorption of light to conformational changes that produce a signal or move ions across a membrane. Nogly et al. used an x-ray laser to probe the earliest structural changes to the retinal chromophore within microcrystals of the ion pump bacteriorhodopsin (see the Perspective by Moffat). The excited-state retinal wiggles but is held in place so that only one double bond of retinal is capable of isomerizing. A water molecule adjacent to the proton-pumping Schiff base responds to changes in charge distribution in the chromophore even before the movement of atoms begins. Science, this issue p. eaat0094; see also p. 127 Ultrafast crystallography captures the response of the pigment of bacteriorhodopsin to absorption of light. 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. Time-resolved serial crystallography resolves ultrafast atomic motions of retinal and the surrounding protein following photoexcitation. Retinal evolves from an all-trans conformation in the ground state toward a twisted 13-cis retinal over the course of a few hundred femtoseconds. The complex counterion, formed by two aspartic acid residues (Asp) and a water molecule (Wat), responds to changes in the electronic structure of the chromophore on the same time scale as the formation of the excited state. Ultrafast isomerization of retinal is the primary step in photoresponsive biological functions including vision in humans and ion transport across bacterial membranes. We used an x-ray laser to study the subpicosecond structural dynamics of retinal isomerization in the light-driven proton pump bacteriorhodopsin. A series of structural snapshots with near-atomic spatial resolution and temporal resolution in the femtosecond regime show how the excited all-trans retinal samples conformational states within the protein binding pocket before passing through a twisted geometry and emerging in the 13-cis conformation. Our findings suggest ultrafast collective motions of aspartic acid residues and functional water molecules in the proximity of the retinal Schiff base as a key facet of this stereoselective and efficient photochemical reaction.

中文翻译：

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飞秒 X 射线激光捕获的细菌视紫红质的视网膜异构化

快速观察 从细菌到人类，有机体都能感知光并做出反应。含有光敏分子视网膜的蛋白质将光的吸收与产生信号或使离子跨膜移动的构象变化结合起来。诺格利等人。使用 X 射线激光探测离子泵细菌视紫红质微晶内视网膜发色团的最早结构变化（参见 Moffat 的观点）。激发态的视网膜会摆动但被固定在适当的位置，因此只有一个视网膜双键能够异构化。与质子泵浦希夫碱相邻的水分子甚至在原子运动开始之前就对发色团中电荷分布的变化做出响应。科学，这个问题 p。eaat0094; 另见第。127 超快晶体学捕获细菌视紫红质色素对光吸收的响应。介绍 视网膜是一种光敏蛋白质配体，生命的所有领域都使用它来处理光的信息和能量含量。视网膜结合蛋白是不可或缺的膜蛋白，可驱动重要的生物过程，包括用于空间定向和生物钟调整的光感应，以及通过离子传输保持电化学梯度。它们还构成了对神经细胞进行光遗传学操作的基础。蛋白质环境如何在亚皮秒时间尺度上引导视网膜异构化以产生单一的高产量产品是光生物学中一个基本的突出问题。基本原理 光诱导的视黄醛异构化是生物学中已知的最快反应之一。它已被光谱技术广泛研究，以探测光谱中间体随时间的演变。使用 X 射线自由电子激光器 (XFEL)，现在可以以接近原子的结构分辨率在飞秒到毫秒的范围内观察蛋白质内的超快光化学反应及其诱导的分子运动。在这项工作中，我们使用 XFEL 辐射来研究光驱动质子泵细菌视紫红质 (bR) 中视网膜异构化的结构动力学。这种原型视网膜结合蛋白中异构化的主要机制与这一重要膜蛋白家族的所有其他成员直接相关，它提供了对蛋白质环境如何催化光化学反应的深入了解。结果 我们从通过光学激光脉冲激发视网膜生色团后通过直线加速器相干光源的飞秒 X 射线脉冲注入的 bR 微晶收集了高分辨率 X 射线衍射数据。X 射线衍射图像以大约 200 fs 的精度被分类到时间子组中。一系列 18 个重叠的差分傅立叶电子密度图揭示了视网膜光激发第一皮秒内的结构变化。10 ps 和 8.33 ms 的时间延迟的补充数据使我们能够解决反应的后期阶段。结合泵-探针延迟处的精细晶体结构，对应于光谱表征的 I、J、K 和 M 中间体在溶液中的形成位置，我们的时间分辨结构数据揭示了视网膜异构化的轨迹，并提供了反应关键点的原子细节。靠近视网膜希夫碱的视网膜反离子和功能性水分子的天冬氨酸残基共同响应激发态的形成和衰变。这种集体运动为视网膜异构化奠定了基础，通过扭曲的视网膜构型进行。量子力学/分子力学模拟为这种结构演化提供了理论支持。结论我们的观察揭示了，随着最早激发态的形成，视网膜结合口袋是如何在异构化键附近打开的。我们建议沿视网膜的超快电荷转移是集体运动的驱动力，有助于蛋白质支架内视网膜异构化的立体选择性和效率。从视网膜延伸到蛋白质的类似振动的振动运动也可能是一种机制，通过这种机制，多余的能量以非辐射方式释放。时间分辨串行晶体学解决了光激发后视网膜和周围蛋白质的超快原子运动。在几百飞秒的过程中，视网膜从基态的全反式构象演变为扭曲的 13 顺式视网膜。由两个天冬氨酸残基 (Asp) 和一个水分子 (Wat) 形成的复合反离子，在与激发态形成相同的时间尺度上响应生色团电子结构的变化。视网膜的超快异构化是光响应生物功能的主要步骤，包括人类的视觉和跨细菌膜的离子传输。我们使用 X 射线激光来研究光驱动质子泵细菌视紫红质中视网膜异构化的亚皮秒结构动力学。一系列在飞秒范围内具有近原子空间分辨率和时间分辨率的结构快照显示了在通过扭曲的几何形状并以 13 顺式构象出现之前，兴奋的全反式视网膜样本在蛋白质结合袋内的构象状态。