Dynamics of membrane proteins unraveled by time-resolved serial crystallography

摘要 膜蛋白对人类健康极其重要，人类蛋白质组的30%由 膜蛋白，在重要的细胞过程中起关键作用，控制和调节 细胞之间的相互作用，调节细胞内和细胞外的运输，也是生物能源的主要参与者 包括呼吸作用在内的转化；目前所有药物中有40%针对膜蛋白。然而， 关于它们在原子水平上的结构和功能的知识是稀疏的，因为少于800个的结构是独一无二的 到目前为止，已经确定了膜蛋白。此外，这些结构中的大多数只显示静态的 膜蛋白的图片，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膜蛋白的“分子电影”，特别是 细胞色素氧化酶催化循环动力学的测定及构象研究 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 以时间分辨飞秒(Fs)X射线结晶学和XFELs为重点的方法的组合。财政司司长 由我们的团队在之前的资助周期中开创的结晶学已经使X射线发生了革命性的变化 结晶学。它克服了辐射损伤，使生物大分子的结构分析能够在 基于新的结构确定系列方法的生理条件下的室温 在那里，从完全水合的流中收集了数万个X射线衍射快照 蛋白质的纳米/微晶，与自由电子激光的飞秒X射线脉冲相互作用。新动向 在纳米晶体生长和表征以及新注射器技术和数据的开发方面 评估方法基于这一结果以非常快的速度推进了SFX的新方法 该项目已经发表了70篇论文，其中29篇发表在影响较大的期刊上，并获得了专利(已接受和 提交的临时申请)。此R01续订是基于前一工作的成功，但探索了 通过将主要重点从概念验证转移到改进和应用来实现新的领域 两种重要的膜蛋白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。 氧化酶是一种大的多蛋白膜蛋白复合体，也是呼吸作用中的关键酶，它催化4 电子-4-质子将氧还原为2个水分子，其详细机理仍是研究的热点。 辩论。目的2主要研究β-肾上腺素能受体及其与arrestin和arrestin的复合体的动力学。 G蛋白。在这个目标中，我们将结合新的SFX方法开发(包括触发GPCR 通过光，以允许非常快速的动力学被检测)与时间分辨的低温电子显微镜研究的GPCR 使我们能够在几秒的时间尺度上解开运动的复杂结构。

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.