Structural Mapping of Protein Complexes by Hydrogen/Deuterium Exchange

描述(申请人提供)：确定蛋白质复合体中的接触位置的最佳技术是X射线衍射和核磁共振。然而，由于样本量有限、聚集、不溶性、翻译后修饰的异质性、缺乏合适的晶体等原因，这些技术可能不适用于大的络合物。大多数替代方法基于将络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能力、交联剂等)。然后，接触表面可以被定位为在形成蛋白质复合体时连接在一起或变得不受化学反应影响的那些位置。最普遍适用的溶剂暴露措施是将主链酰胺氢交换为氢，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：在分离用于分析的蛋白水解物片段时，H向D的反向交换；质量分辨率太低，不能用质谱仪分离和鉴定部分氚化肽；胃蛋白酶裂解的不完全序列覆盖(胃蛋白酶通常在H/D交换被淬灭后在低pH下使用)；以及获取和解释数据的困难和持续时间。在这个项目中，我们将结合几项改进：消除反向交换的超临界流体层析；简化多肽鉴定的同位素耗竭和超高质量分辨率；一套不同蛋白分解特性的酶以更好地跨越序列；以及数据收集和数据分析的自动化。关联性。越来越明显的是，蛋白质复合体和组合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”，以及将基本成分输送到细胞或病毒中的生物“马达”。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部位。这个项目提出了几种新的方法来进行这种“映射”，以及一些建议的初步应用，这些应用可能指向未来的药物应用目标。这个项目是多学科的，汇集了几位杰出的合作者，他们有专注的研究项目，这些项目将直接受益于高分辨率质量分析的H/D交换。

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.