Dynamics of membrane proteins unraveled by time-resolved serial crystallography

摘要 膜蛋白对人类健康极其重要，30%的人类蛋白质组由以下组成： 膜蛋白，它们是重要细胞过程中的关键参与者，控制和介导 细胞之间的相互作用，调节进出细胞的运输，也是生物能源的主要参与者 转化包括呼吸;目前所有药物的40%靶向膜蛋白。然而 在原子水平上对它们的结构和功能的了解很少，因为不到800个独特的结构 膜蛋白的测定。此外，这些结构中的大多数仅显示静态 它们是膜蛋白的图像，而它们在细胞中的功能是高度动态的。这项建议旨在 开发新的方法来确定“工作中”的膜蛋白的“分子电影”，特别是， 确定细胞色素氧化酶催化循环的动力学，并研究其构象 β-肾上腺素能G蛋白偶联受体的动力学。该提案旨在开发和使用一种 这是专注于时间分辨飞秒（fs）X射线晶体学与XFEL的方法的组合。FS 晶体学，由我们的团队在以前的资金周期中开创，已经彻底改变了X射线 结晶学它克服了辐射损伤，使生物大分子的结构分析成为可能， 室温下在生理条件下基于用于结构测定的新的系列方法， 从一股完全水合的 蛋白质的纳米/微晶，与来自自由电子激光器的fs X射线脉冲相互作用。新发展 在晶体生长和特性分析方面，以及在开发新的注入器技术和数据方面， 评估方法，已经取得了进展，在非常快的速度的基础上，从这个结果SFX的新方法 该项目已导致70篇出版物，其中29篇在高影响力期刊上发表，并获得了专利（已接受， 临时申请）。这R 01更新是基于以前的工作的成功，但探索 通过将主要重点从概念验证转移到其改进和应用， 两个重要的膜蛋白的功能动力学的分子电影：细胞色素c氧化酶和 β-肾上腺素能受体在目标1中，我们将研究细胞色素c催化循环的动力学 氧化酶是一种多蛋白质的膜蛋白复合体，是呼吸作用的关键酶，它催化4 电子4质子还原氧2水分子，其详细机制仍然是一个热门话题， 辩论目的2是研究β-肾上腺素能受体及其与抑制蛋白复合物的动力学， G蛋白在这个目标中，我们将结合联合收割机新的SFX方法的发展（包括使GPCR触发 利用GPCR的时间分辨冷冻-EM研究 这些复合体将使我们能够在秒的时间尺度上解开运动。

ABSTRACT Membrane proteins are of extreme importance for human health and 30% of the human proteome consist of membrane proteins, which are key players in important cellular processes controlling and mediating the interaction between cells, regulate transport in and out of the cells and are also the major players in bioenergy conversion including respiration; 40% of all current drugs being targeted to membrane proteins. However knowledge of their structure and function at the atomic level is sparse, as the structures for less 800 unique membrane proteins has been determined to date. Furthermore, most of these structures show only a static picture of the membrane protein, while their function in the cell is highly dynamic. This proposal aims to develop novel methods to determine “molecular movies” of membrane proteins “at work,” and specifically, to determine the dynamics of the catalytic cycle of the cytochrome oxidase and to study the conformational dynamics of the beta-adrenergic G-protein coupled receptor. The proposal aims to develop and use a combination of methods that focus on time-resolved femtosecond (fs) X-ray crystallography with XFELs. Fs crystallography, pioneered by our team in the previous funding cycles, has revolutionized X-ray crystallography. It overcomes radiation damage, enabling structure analysis of biological macromolecules at room temperature under physiological conditions based on the new serial approach for structure determination, where tens of thousands of X-ray diffraction snapshots are collected from a stream of fully hydrated nano/microcrystals of proteins, interacting with fs X-ray pulses from a Free Electron Laser. New developments in nanocrystal growth and characterization, together with development of new injector technology and data evaluation methods, have progressed the new method of SFX at a very fast pace based on results from this project, which has led to 70 publications, 29 of them in high impact journals as well as patents (accepted and provisional applications filed). This R01 Renewal is based on the success of the previous work but explores new areas by shifting the major focus from the proof-of-concept to their improvement and applicationtowards molecular movies of the functional dynamics of two important membrane proteins: cytochrome c oxidase and the beta-adrenergic receptor. In Aim 1 we will study the dynamics of the catalytic cycle of cytochrome c oxidase, a large-multi-protein membrane protein complex and the key enzyme in respiration, catalyzing the 4 electron 4 proton reduction of oxygen to 2 water molecules, whose detailed mechanisms is still a hot topic of debate. Aim 2 is focused on the dynamics of the beta-adrenergic receptor and its complexes with arrestin and G-protein. In this Aim, we will combine new SFX method developments (including making the GPCR triggered by light to allow for very fast dynamics to be detected) with the time resolved cryo-EM studies of the GPCR complexes that will allow us to unravel motions on a time scale of seconds.