Dynamics of membrane proteins unraveled by time-resolved serial crystallography

时间分辨系列晶体学揭示膜蛋白的动力学

基本信息

批准号：
9887557
负责人：
PETRA FROMME
金额：
$ 39.59万
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 2024-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9887557
关键词：
Adrenergic Agents Agonist Area Arrestins Binding Biological Biology Cell physiology Cells Cellular Metabolic Process Code Complex Computer Simulation Cryoelectron Microscopy Crystallization Crystallography Data Development Devices Disease Drug Targeting Electrons Energy Supply Enzymes Evaluation Funding G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors GTP-Binding Protein alpha Subunits, Gs GTP-Binding Proteins Genes Grant Growth Health Human Hydration status Image Journals Knowledge Lasers Lead Legal patent Ligand Binding Ligands Light Lipids Mediating Medicine Membrane Membrane Proteins Methods Microfluidics Mitochondria Modeling Molecular Molecular Conformation Monitor Motion Oxidases Oxidation-Reduction Oxygen Paste substance Pharmaceutical Preparations Phase Physiologic pulse Physiological Play Porifera Process Protein Dynamics Proteins Proteome Proton Pump Protons Publications Radiation induced damage Reaction Regulation Respiration Rhodopsin Roentgen Rays Role Sampling Side Signal Transduction Spectrum Analysis Stream Structural Biologist Structure Techniques Technology Temperature Time Tooth structure Water Work X ray diffraction analysis X-Ray Crystallography base beta-adrenergic receptor cytochrome c oxidase design free-electron laser insight macromolecule method development molecular dynamics movie nano nanocrystal novel protein complex success tool

项目摘要

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.

抽象的膜蛋白对人类健康极其重要，人类蛋白质组的 30% 由膜蛋白，在控制和介导细胞的重要细胞过程中发挥着关键作用细胞之间的相互作用，调节细胞进出细胞的运输，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% 是针对膜蛋白的。对它们在原子水平上的结构和功能的了解很少，因为只有不到 800 个独特的结构此外，迄今为止，大多数膜蛋白仅表现出静态。膜蛋白的图片，而它们在细胞中的功能是高度动态的。开发新方法来确定膜蛋白“工作”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构象该提案旨在开发和使用 β-肾上腺素能 G 蛋白偶联受体的动力学。专注于时间分辨飞秒 (fs) X 射线晶体学与 XFEL 的方法组合。我们的团队在之前的融资周期中首创的晶体学彻底改变了 X 射线它克服了辐射损伤，使得生物大分子的结构分析成为可能。基于新的串行结构测定方法的生理条件下的室温，从完全水合的水流中收集了数以万计的 X 射线衍射快照蛋白质的纳米/微晶体，与自由电子激光器的飞秒 X 射线脉冲相互作用。纳米晶体生长和表征，以及新注射器技术和数据的开发评估方法，基于此结果以非常快的速度开发了SFX的新方法该项目已发表 70 篇出版物，其中 29 篇发表于高影响力期刊以及专利（已接受和该 R01 更新基于先前工作的成功，但进行了探索。通过将主要重点从概念验证转移到改进和应用来开拓新领域—— 两种重要膜蛋白功能动力学的分子电影：细胞色素c氧化酶和在目标 1 中，我们将研究细胞色素 c 催化循环的动力学。氧化酶是一种大型多蛋白膜蛋白复合物，也是呼吸作用的关键酶，催化 4 电子4质子将氧还原成2个水分子，其详细机制仍然是研究的热点辩论的目标 2 集中于 β-肾上腺素受体及其与抑制蛋白和复合物的动力学。在这个目标中，我们将结合新的 SFX 方法开发（包括触发 GPCR）。通过光来检测非常快的动态）以及 GPCR 的时间分辨冷冻电镜研究复合体将使我们能够在秒的时间尺度上解开运动。