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，例如在这些金属蛋白中，小分子基条信号转导蛋白，金属氧化状态/坐标几何形状和整体蛋白质概要在提议的长期目标中起重要作用研究将在不同的生物相关性的电子传递，配体结合和蛋白质折叠的目标中，通过高分辨率同时“快照”与AF的金属活性结构与函数之间的相关性。 X射线瞬态吸收（XTA）或X射线吸收光谱均可以通过X射线瞬态散射（XTS）捕获X射线瞬时吸收（XTA）。反应可以是在金属ctive位点的配体/抑制剂或光诱导的反应的反应，随后触发蛋白质构型会随着激光和X射线脉冲之间的ASA时间而改变蛋白质。同时检测酶促反应期间金属蛋白的局部/远距离结构。端口的线性相干光源（LCLS），具有X射线X射线，以及与其他仪器的一部分。 c在/重新折叠期间； 2）杂交血红蛋白的电子转移； 3）catala se。通过结构修饰调节功能h活动位点H将导致酶促功能增强，催化和理论化的进步。