Structural Mapping of Protein Complexes by Hydrogen/Deuterium Exchange

通过氢/氘交换绘制蛋白质复合物的结构图

• 批准号: 7135456
• 负责人: ALAN G MARSHALL
• 金额: $ 31.48万
• 依托单位: FLORIDA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7135456
• 关键词: active sites; amides; automated data processing; binding sites; conformation; deuterium; electrospray ionization mass spectrometry; enzyme activity; epitope mapping; gene expression; high performance liquid chromatography; hydrogen transport; ligands; nuclear magnetic resonance spectroscopy; peptide structure; posttranslational modifications; protein localization; protein protein interaction; protein purification; protein structure; protein structure function; proteolysis; robotics

DESCRIPTION (provided by applicant): The best techniques for identifying sites of contact in protein complexes are x-ray diffraction and nuclear magnetic resonance. However, those techniques may not be available for large complexes, due to limited sample amount, aggregation, insolubility, posttranslational modification heterogeneity, lack of suitable crystals, etc. Most alternative methods are based on exposing the complex to some sort of chemical perturbation (e.g., chemical reactivity toward general or specific agents, cross-linking, and the like). The contact surfaces may then be located as those sites that are linked or become protected against chemical reactivity on formation of the protein complex. The most generally applicable measure of solvent exposure is exchange of backbone amide hydrogens for deuteriums, because it is least dependent on the amino acid sequence. Major current difficulties for the H/D exchange method include: back-exchange of H for D during separation of proteolytic fragments for analysis; mass resolving power too low to separate and identify partially deuterated peptides by mass spectrometry; incomplete sequence coverage by pepsin cleavage (pepsin is usually used due to its activity at low pH after H/D exchange has been quenched); and the difficulty and duration of acquiring and interpreting the data. In this project, we shall combine several improvements: supercritical fluid chromatography to eliminate back-exchange; isotopic depletion and ultrahigh mass resolving power to simplify identification of peptides, a suite of enzymes of different proteolytic specificity to better span the sequence; and automation of data collection and data analysis. Relevance. It is becoming increasingly evident that protein complexes and assemblies play a key role in many human diseases: e.g., the protein "capsid" that protects RNA in the AIDS virus and in the biological "motors" that transport essential components into a cell or virus. A first step in understanding (and eventually controlling) those functions is to identify the sites of contact that hold proteins together. This project presents several new approaches for such "mapping", along with some suggested initial applications that could point to future targets with pharmaceutical applications. This project is multidisiplinary and pulls together several distinguished collaborators with focused research projects that will benefit directly from H/D exchange with high-resolution mass analysis.

描述（由申请人提供）：识别蛋白质复合物中接触位点的最佳技术是 X 射线衍射和核磁共振。然而，由于样品量有限、聚集、不溶性、翻译后修饰异质性、缺乏合适的晶体等，这些技术可能不适用于大型复合物。大多数替代方法基于将复合物暴露于某种化学扰动（例如，对一般或特定试剂的化学反应性、交联等）。然后可以将接触表面定位为在蛋白质复合物形成时连接或变得免受化学反应的那些位点。最普遍适用的溶剂暴露测量是将主链酰胺氢交换为氘，因为它对氨基酸序列的依赖性最小。目前H/D交换方法的主要困难包括：在分离蛋白水解片段进行分析的过程中将H反向交换为D；质量分辨率太低，无法通过质谱法分离和识别部分氘化的肽；胃蛋白酶切割导致的序列覆盖不完整（通常使用胃蛋白酶，因为 H/D 交换淬灭后其在低 pH 下具有活性）；以及获取和解释数据的难度和持续时间。在这个项目中，我们将结合多项改进：超临界流体色谱消除反交换；同位素消耗和超高质量分辨能力可简化肽的鉴定，一套具有不同蛋白水解特异性的酶可更好地跨越序列；以及数据收集和数据分析的自动化。关联。越来越明显的是，蛋白质复合物和组装体在许多人类疾病中发挥着关键作用：例如，保护艾滋病病毒中 RNA 的蛋白质“衣壳”，以及将重要成分输送到细胞或病毒的生物“发动机”中的蛋白质“衣壳”。理解（并最终控制）这些功能的第一步是确定将蛋白质结合在一起的接触位点。该项目提出了这种“映射”的几种新方法，以及一些建议的初步应用，这些应用可能会指出未来的制药应用目标。该项目是多学科的，汇集了几位杰出的合作者和重点研究项目，这些项目将直接受益于高分辨率质量分析的 H/D 交换。