Capturing Transient Protein Structures on Multiple Spatial and Temporal Scales

在多个空间和时间尺度上捕获瞬态蛋白质结构

• 批准号: 9297321
• 负责人: Lin X Chen
• 金额: $ 33.31万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9297321
• 关键词: Active Sites; Address; Binding Proteins; Biochemical Reaction; Biological; Biological Process; Catalysis; Cells; Data Collection; Dependence; Detection; Electron Transport; Enzymes; Event; Geometry; Kinetics; Lasers; Ligands; Ligation; Light; Maps; Measurement; Metabolism; Metal Binding Site; Metalloproteins; Metals; Methods; Modification; Molecular; Molecular Conformation; Nuclear; Nuclear Structure; Optics; Oxidation-Reduction; Pathway interactions; Photons; Physiologic pulse; Physiological Processes; Play; Process; Prosthesis; Protein Conformation; Protein Dynamics; Proteins; Pump; Reaction; Research; Research Personnel; Resolution; Respiration; Roentgen Rays; Role; Sampling; Series; Signal Transduction; Signaling Protein; Site; Source; Spectrum Analysis; Structural Protein; Structure; Synchrotrons; System; Time; Transact; Transport Process; Work; absorption; catalase; cytochrome c; data acquisition; density; detector; electronic structure; experimental study; hybrid hemoglobins; improved; inhibitor/antagonist; innovation; insight; instrumentation; metalloenzyme; oxidation; portability; programs; protein folding; protein function; protein structure; public health relevance; x-ray free-electron laser

 DESCRIPTION: Metalloproteins have many different functions in cells, such as storage and transport of small molecular substrates, proteins, enzymes and signal transduction proteins. In these metalloproteins, the interplay between the metal oxidation state/coordination geometry and the overall protein conformation plays important roles in their functions. The long term objective of the proposed research is to gain new insight into correlations between metal active site structures of a metalloenzyme and their functions through high resolution simultaneous structural "snapshots" during different biological relevant processes, such as electron transfer, ligand binding and protein folding. The proposed research aims at building instrumentation for mapping reaction trajectories on multiple spatial and temporal scales. Transient electronic and nuclear structures of metal centered active sites in a series of metalloproteins will be simultaneously captured using X-ray transient absorption (XTA, or transient X-ray absorption spectroscopy) spectroscopy with time resolutions from 10-13 second (100 fs) to longer, while the protein conformation change along the reaction coordinates can be captured by X-ray transient scattering (XTS). The method uses a laser pulse pump to trigger a biological reaction, which can be direct photodissociation of ligands/inhibitors or photoinduced redox reaction at metal active sites that subsequently triggers protein conformation changes, and an X-ray pulse to probe the active site structures and the conformations of proteins as a function of the delay time between the laser and X-ray pulses. The proposed research focuses on building a portable and multifunctional sample chamber/detection setup to enable simultaneous detection of the local/long range structures of metalloproteins during enzymatic reactions. The proposed setup will be built upon an existing X-ray facility with advanced detectors and laser systems to fulfill specific needs for protein samples. Once the instrumentation is built, it will be a part of portabl instrumentation for conducting experiments in other light sources, such as the linear coherence light source (LCLS) with femtosecond pulsed X-rays, and a part of shared instrumentation with other users. Three metalloprotein systems will be investigated using the instrumentation built through the proposed work: 1) metal binding site structures and protein conformations of cytochrome c during folding/refolding; 2) conformation gating in electron transfer of hybrid hemoglobins; and 3) transient active site structures in catalase. These structural results combined with those of reaction kinetics from other transient spectroscopic measurements will provide much deeper understanding of energy transduction inside the proteins during enzymatic reactions and guidance for modulating protein functions via structural modifications around the active sites, which will lead to advances in enzymatic function enhancement, catalysis, as well as theoretical calculations.

 描述：金属蛋白在细胞中具有许多不同的功能，例如小分子底物、蛋白质、酶和信号转导蛋白的储存和运输，在这些金属蛋白中，金属氧化态/配位几何结构与整体蛋白质构象之间的相互作用起着重要作用。拟议研究的长期目标是通过高分辨率同步结构获得对金属酶的金属活性位点结构与其功能之间的相关性的新见解。不同生物相关过程（例如电子转移、配体结合和蛋白质折叠）的“快照”旨在构建在多个空间和时间尺度上绘制反应轨迹的仪器。将使用 X 射线瞬态吸收（XTA 或瞬态 X 射线吸收光谱）光谱同时捕获一系列金属蛋白，时间分辨率从 10-13 秒 (100 fs) 到更长，而通过X射线瞬态散射（XTS）可以捕获沿反应坐标的蛋白质构象变化，该方法使用激光脉冲泵触发生物反应，该反应可以是配体/抑制剂的直接光解离或金属活性位点的光诱导氧化还原反应。随后触发蛋白质构象变化，并用 X 射线脉冲探测活性位点结构和蛋白质构象，作为激光和 X 射线脉冲之间延迟时间的函数。拟议的研究重点是构建便携式多功能装置。样本腔室/检测装置能够在酶反应过程中同时检测金属蛋白的局部/长距离结构。拟议的装置将建立在现有的 X 射线设施的基础上，配备先进的探测器和激光系统，以满足蛋白质样品的特定需求。仪器建成后，它将成为便携式仪器的一部分，用于在其他光源下进行实验，例如带有飞秒脉冲X射线的线性相干光源（LCLS），以及与其他用户共享仪器的一部分。将使用通过拟议工作建立的仪器来研究金属蛋白系统：1）折叠/重折叠过程中细胞色素c的金属结合位点结构和蛋白质构象；2）混合血红蛋白电子转移中的构象门控；3）这些结构结果与其他瞬态光谱测量的反应动力学结果相结合，将提供对酶反应过程中蛋白质内部能量转导的更深入的了解，并指导通过围绕过氧化氢酶的结构修饰来调节蛋白质功能。活性位点，这将导致酶功能增强、催化以及理论计算方面的进步。