Femtosecond nano-crystallography of membrane proteins

膜蛋白的飞秒纳米晶体学

基本信息

批准号：
8027697
负责人：
PETRA FROMME
金额：
$ 30万
依托单位：
ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-30 至 2014-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8027697
关键词：
Binding Biological Models Cell Communication Cells Collection Complex Crystallography Data Data Collection Data Set Development Drug Delivery Systems Evaluation Film Future Generations Growth Human Image Individual Lasers Life Membrane Proteins Metals Methods Molecular Molecular Weight Mothers Nature Nitrates Optics Oxidation-Reduction Pattern Phase Photosynthesis Photosystem I Physics Physiologic pulse Powder Diffraction Process Proteins Radiation Radiation induced damage Research Activity Respiration Roentgen Rays Screening procedure Silicon Sodium Chloride Solvents Source Steam Stream Structure Synchrotrons Techniques Temperature Time Work X ray diffraction analysis X-Ray Crystallography X-Ray Diffraction base cofactor cryogenics dalton distilled alcoholic beverage falls indexing method development nano nanocrystal novel operation protein structure research study

项目摘要

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.

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完全水合的膜蛋白纳米/微晶体蒸汽，是使用斯坦福大学 LCLS 的新型高能飞秒 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加速器源 12 个数量级。膜蛋白在所有活细胞中都极其重要，因为它们催化呼吸、光合作用、运输和细胞通讯等重要功能。 30% 的人类蛋白质是膜蛋白，超过 60% 的药物都是针对膜蛋白的。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。尽管 X 射线晶体学和 NMR 已经解析了 60,000 多种可溶性蛋白质的结构，但迄今为止确定的不同膜蛋白结构还不到 250 种。迄今为止，膜蛋白结构的测定通常涉及一个耗时的过程，需要数年（有时甚至数十年）才能生长出适合 X 射线结构测定的大型且有序的晶体。此外，X射线引起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，即使在低温条件下也是如此。该提案基于 2009 年 12 月在 LCLS 上使用飞秒 X 射线脉冲收集了膜蛋白光系统 I 的纳米/微晶体上的 300 万个衍射图案，从而对飞秒纳米晶体学原理进行了首次验证。作为模型系统的光系统 I 的分子量为 1,056,000 道尔顿，由 36 个蛋白质和 381 个非共价结合的辅因子组成，使光系统 I 成为迄今为止结晶的最复杂的膜蛋白之一。这些实验已经证明，“破坏前衍射原理”首次在 2006 年针对蚀刻到硝酸硅薄膜的图像中展示（Chapman 2006，《自然物理学》），可以直接扩展到最脆弱的蛋白质晶体之一，迄今为止，它含有 78% 的溶剂，并且只有 4 个参与晶体接触的盐桥。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 X 射线激光，使用母液中完全水合的纳米/微晶体，可以在几分钟的时间内收集数十万个衍射图案脉冲非常短，以至于 X 射线引起的辐射损伤仅在数据收集后才开始。该新方法未来还有可能通过将光学激光激发与飞秒X射线数据收集相结合来获得分子激发态的结构。由于该提案突破了新的未探索领域，它涉及方法开发，从最佳微晶的筛选和微晶的确定生长到高通量数据筛选、数据评估和物相测定的新方法开发。公共健康相关性：该提案的目的是开发一种确定膜蛋白结构的新方法。该技术使用第一台硬 X 射线激光器（斯坦福大学的“LCLS”）提供的超短飞秒 X 射线脉冲，从连续的完全水合膜蛋白纳米晶体流中收集 X 射线衍射数据。这些脉冲非常短暂，以至于在辐射损伤过程开始之前就终止了。