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: 8945800
• 负责人: Lin X Chen
• 金额: $ 31.63万
• 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/8945800
• 关键词: Active Sites; Address; Binding Proteins; Biochemical Reaction; Biological; Biological Process; Catalysis; Cells; Data Collection; Dependence; Detection; Electron Transport; Electronics; Enzymes; Event; Geometry; Kinetics; Lasers; Lead; 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; Solutions; Source; Spectrum Analysis; Staging; Structural Protein; Structure; Synchrotrons; System; Time; Transport Process; Work; absorption; catalase; cytochrome c; data acquisition; density; detector; electronic structure; free-electron laser; hybrid hemoglobins; improved; inhibitor/antagonist; innovation; insight; instrumentation; metalloenzyme; oxidation; programs; protein folding; protein function; protein structure; public health relevance; research study

 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，酶和信号转移蛋白的储存和运输。在这些金属蛋白中，金属氧化态/配位几何形状与整体蛋白质构象之间的相互作用在其功能中起着重要作用。拟议研究的长期目标是通过高分辨率简单的结构“快照”在不同的生物学相关过程中，例如电子转移，配体结合和蛋白质折叠，从而通过高分辨率简单的结构“快照”获得了金属活性位点结构及其功能之间的相关性的新见解。拟议的研究旨在构建仪器，以在多个空间和临时尺度上绘制反应轨迹。金属蛋白的瞬时电子和核结构将使用X射线瞬态绝对（XTA或瞬态X射线抽象光谱）光谱捕获，而时间分辨率从10-13秒（100 fs）到更长的时间分辨率，而蛋白质构型则沿反应坐标可以通过X射线变化捕获蛋白质结构变化。该方法使用激光脉冲泵触发生物学反应，该反应可以直接在金属活性位点进行配体/抑制剂或光诱导的氧化还原反应，该反应随后触发蛋白质构象的变化，以及X射线脉冲以探测蛋白质的活性位点结构和蛋白质的延迟时间与lase pulser and lase and x-ray and x-ray and rase and rase and rase and rase and rase and rase and rase。拟议的研究重点是构建一种便携式和多功能样品室/检测设置，以使酶促反应期间对金属蛋白的局部/远程结构进行简单检测。拟议的设置将建立在现有的X射线设施上，该设施具有高级探测器和激光系统，以满足蛋白质样品的特定需求。一旦构建了仪器，它将成为用于在其他光源中进行实验的Portabl仪器的一部分，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。将使用通过拟议的工作构建的仪器研究三种金属蛋白系统：1）在折叠/重折叠过程中，金属结合位点结构和细胞色素C的蛋白质构象； 2）杂交血红蛋白电子传递的会议门控； 3）过氧化氢酶中的瞬态活性位点结构。这些结构性结果与其他瞬时光谱测量的反应动力学结合在一起，将在酶促反应过程中对蛋白质内部能量转移以及通过活性位点调节蛋白质功能的指导提供更深入的了解，这将导致综合功能增强，催化计算，以及该蛋白质的进步。