Femtosecond nano-crystallography of membrane proteins

摘要 这项建议的目的是发展飞秒(飞秒)结晶学的方法 膜蛋白的结构测定，X射线结构分析是其基础 关于完全水合的蒸汽产生的数十万个X射线衍射图 用新的高能飞秒X射线收集膜蛋白的纳米/微晶 雷瑟在斯坦福的LCLS工作。联营公司于2009年秋季开始运作，并提供 强度超过第三代同步加速器源12个数量级的FS脉冲 震级。 膜蛋白在所有活细胞中都是极其重要的，因为它们催化 呼吸、光合作用、运输和细胞通讯等功能。30%的人 人类蛋白质是膜蛋白质，超过60%的药物针对的是 膜蛋白。尽管它们极其重要，但对它们的理解 分子功能因缺乏结构信息而受阻；而超过 用X射线结晶学和X射线衍射法研究了6万种可溶性蛋白质的结构。 到目前为止，还不到250种不同的膜蛋白结构被确定。 到目前为止，膜蛋白结构的测定通常需要一段时间- 耗费数年(有时甚至数十年)才能壮大的过程 适用于X射线结构测定的有序晶体。此外，X射线诱导的 辐射损伤是许多膜蛋白晶体的主要问题，尤其是 当它们含有金属和/或氧化还原活性辅因时。X射线辐射 即使在低温条件下，损伤也会对微晶体的X射线衍射造成限制 条件。这项提议是基于对FS的第一个原则性证明 通过收集300万个纳米级衍射图进行纳米晶学研究 2009年12月LCLS上膜蛋白光系统I的微晶体，使用 FS X射线脉冲。作为模型系统的光系统I具有一个分子 重量为1,056,000道尔顿，由36种蛋白质和381种非 共价结合，使光系统I成为最复杂的膜蛋白之一 到目前为止，这一点已经具体化。这些实验已经证明， 《先绕射后破坏原理》于2006年首次上映，用于蚀刻成 硝酸硅薄膜，(Chapman 2006，自然物理)，可以直接扩展到 到目前为止存在的最脆弱的蛋白质晶体，它含有78%的溶剂，只有4% 与水晶接触有关的盐桥。这项提议旨在开启一个令人振奋的新 膜蛋白结晶学的大道，那里有数十万 使用完全水合的Nano/可以在几分钟的时间内收集衍射图 微晶在它们的母液中，在室温下，用X射线激光脉冲 如此之短，以至于X射线辐射损伤只有在数据收集之后才会开始。新的 该方法还有可能通过以下方式获得分子的激发态结构 将光学激光激发与飞秒X射线数据采集相结合是未来的发展方向。作为 提案进入了新的未知领域，它涉及到一系列方法开发 从最佳微晶体的筛选和微晶体的定义生长到 用于高通量数据筛选、数据评估和阶段的新方法开发 决心。

Summary The aim of this proposal is to develop the method of femtosecond (fs) crystallography for the structure determination of membrane proteins, where X-ray structure analysis is based on hundreds of thousands of X-ray diffraction patterns from a steam of fully hydrated nano/ microcrystals of membrane proteins, collected using the new high energy fs X-ray laser at LCLS in Stanford. The LCLS started its operation in the fall of 2009 and provides fs-pulses of an intensity that exceeds third-generation synchrotron sources by 12 orders of magnitude. Membrane proteins are of extreme importance in all living cells as they catalyze vital functions like respiration, photosynthesis, transport, and cell communication. 30% of all human proteins are membrane proteins and more than 60% of all drugs are targeted to membrane proteins. Despite their extreme importance, the understanding of their molecular function is hampered by the lack of structure information; while more than 60,000 structures of soluble proteins have been solved by X-ray crystallography and NMR, less than 250 different membrane protein structures have so far been determined. The determination of membrane protein structures solved to date often involved a time- consuming process where it took years (or sometimes even decades) to grow large, well- ordered crystals suitable for X-ray structure determination. Furthermore, X-ray-induced radiation damage is a major problem for many membrane protein crystals, especially when they contain metals and/or redox active cofactors. The X-ray-induced radiation damage imposes a limitation for X-ray diffraction on microcrystals, even under cryogenic conditions. This proposal is based on the first proof of principle for fs- nanocrystallography by the collection of 3 million diffraction patterns on nano/ microcrystals of the membrane protein Photosystem I in December 2009 at LCLS, using fs X-ray pulses. Photosystem I, which served as the model system, has a molecular weight of 1,056,000 Daltons and consists of 36 proteins and 381 cofactors that are non- covalently bound, making Photosystem I one of the most complex membrane proteins that has been crystallized to date. These experiments have already proven that the "diffraction before destroy principle," first shown in 2006 for an image etched into a silicon-nitrate film, (Chapman 2006, Nature Physics), can be directly extended to one of the most fragile protein crystals that exists to date, which contain 78% solvent and only 4 salt bridges involved in crystal contact. This proposal aims to open an exciting new avenue for membrane protein crystallography, where hundreds of thousands of diffraction patterns can be collected in a time frame of minutes using fully hydrated nano/ microcrystals in their mother liquor, at room temperature, with X-ray laser pulses that are so short that X-ray-induced radiation damage only starts after data collection. The new method has also the potential to obtain structures of excited states of the molecules by combining optical laser excitation with fs X-ray data collection in the future. As the proposal breaks into new unexplored grounds, it involves method developments ranging from the screening for the best microcrystals and the defined growth of microcrystals to new method developments for high throughput data screening, data evaluation and phase determination.