Femtosecond nano-crystallography of membrane proteins

描述（由申请人提供）：本提案的目的是开发用于膜蛋白结构测定的飞秒（fs）晶体学方法，其中X射线结构分析基于来自膜蛋白的完全水合纳米/微晶流的数十万个X射线衍射图案，这些图案是在斯坦福大学的LCLS使用新的高能fs X射线激光收集的。LCLS于2009年秋季开始运行，提供的飞秒脉冲强度超过第三代同步辐射源12个数量级。膜蛋白在所有活细胞中都非常重要，因为它们催化呼吸，光合作用，运输和细胞通讯等重要功能。所有人类蛋白质的30%是膜蛋白，并且超过60%的药物靶向于膜蛋白。尽管它们极其重要，但由于缺乏结构信息，对其分子功能的理解受到阻碍;虽然X射线晶体学和NMR已经解决了60，000多个可溶性蛋白质的结构，但迄今为止确定的不同膜蛋白质结构不到250个。迄今为止解决的膜蛋白结构的测定通常涉及耗时的过程，其中需要数年（或有时甚至数十年）来生长适合于X射线结构测定的大的、良好有序的晶体。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，特别是当它们含有金属和/或氧化还原活性辅因子时。X射线引起的辐射损伤对微晶的X射线衍射施加了限制，即使在低温条件下。该建议是基于2009年12月在LCLS使用fs X射线脉冲收集的膜蛋白光系统I的纳米/微晶上的300万个衍射图案的fs-纳米晶体照相术的第一个原理证明。作为模型系统的光系统I具有1，056，000道尔顿的分子量，并且由非共价结合的36种蛋白质和381种辅因子组成，使得光系统I成为迄今为止已结晶的最复杂的膜蛋白质之一。这些实验已经证明，2006年首次展示的蚀刻到硝酸硅薄膜中的图像的“破坏前衍射原理”（Chapman 2006，Nature Physics）可以直接扩展到迄今为止存在的最脆弱的蛋白质晶体之一，其含有78%的溶剂和仅4个涉及晶体接触的盐桥。该提案旨在为膜蛋白晶体学开辟一条令人兴奋的新途径，在室温下，使用母液中的完全水合纳米/微晶，可以在几分钟的时间内收集数十万个衍射图案，X射线激光脉冲非常短，X射线诱导的辐射损伤仅在数据收集后开始。这种新的方法也有可能获得激发态的分子结构相结合的飞秒X射线数据收集在未来的光学激光激发。由于该提案开辟了新的未开发领域，它涉及方法开发，从筛选最佳微晶和微晶的定义生长到高通量数据筛选，数据评估和相确定的新方法开发。 公共卫生相关性：该提案的目的是开发一种用于膜蛋白结构测定的新方法。这使用超短飞秒X射线脉冲，由第一个硬X射线激光器（斯坦福大学的“LCLS”）提供，从连续的完全水合的膜蛋白纳米晶体流中收集X射线衍射数据。脉冲是如此短暂，以至于它们在辐射损伤过程开始之前就终止了。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: The aim of this proposal is to develop a new method for the structure determination of membrane proteins. This uses ultra-short femtosecond X-ray pulses, provided by the first hard-X-ray laser (the "LCLS" at Stanford) to collect X-ray diffraction data from a continuous stream of fully-hydrated membrane protein nanocrystals. The pulses are so brief that they terminate before radiation damage processes can begin.