Protein structure and function by hydrogen exchange mass spectrometry analysis

描述(申请人提供)：蛋白质化学家的主要挑战是解释蛋白质如何实现其有限的结构和生物物理性质，以 产生了它们无数的功能。与任何其他实验室方法不同，氢交换(HX)行为以氨基酸分辨率编码关于产生蛋白质功能的生物物理因素--结构、结构变化、相互作用、动力学和能量学--的详细定量信息。以前版本的NIH赠款通过使用各种核磁共振方法在HX领域的开发中发挥了重要作用，但常规的核磁共振分析仅限于大量和高浓度的小而高溶解的蛋白质。一项正在开发的氢交换-质谱学技术(HX-MS)有望将这一经过验证的能力扩展到更大、更复杂的蛋白质系统，这些系统使生物工作，同时只需要亚微摩尔浓度的微摩尔蛋白质。本申请建议完善HX-MS技术，并将其应用于重要蛋白质系统的研究。它们是：1)载脂蛋白E3和E4，当无脂和脂结合时，对心血管疾病和阿尔茨海默病很重要；2)获得性TTP疾病中自身免疫抗体表位的定义，与抗体治疗和更广泛的蛋白质相互作用相关；3)HSP104蛋白解聚酶，与蛋白质聚集和淀粉样蛋白加工控制有关。HX MS的全面发展及其与困难但核心重要的蛋白质系统的示范将提供一种独特而强大的方法学，广泛适用于蛋白质结构-功能问题的研究。

DESCRIPTION (provided by applicant): The major challenge for the protein chemist is to explain how proteins implement their limited repertoire of structural and biophysical properties to produce their myriad functions. Unlike any other laboratory method, hydrogen exchange (HX) behavior encodes detailed quantitative information, at amino acid resolution, on the biophysical factors that produce protein function -- structure, structure change, interactions, dynamics, and energetics. Previous versions of this NIH grant have been instrumental in developing the HX field by using a variety of NMR methods, but routine NMR analysis is limited to small highly soluble proteins in substantial quantity and high concentration. A developing hydrogen exchange - mass spectrometry technology (HX MS) promises to extend this proven capability to the larger and more complex protein systems that make biology work while requiring only picomoles of protein at sub-micromolar concentrations. This application proposes to perfect the HX MS technology and apply it in studies of important protein systems. They are: 1) apolipoproteins E3 and E4, when lipid- free and lipid-bound, important for cardiovascular and Alzheimer's diseases; 2) the definition of autoimmune antibody epitopes in acquired TTP disease, relevant to antibody therapeutics and to protein interactions more generally; 3) the Hsp104 protein disaggregase, relevant to the control of protein aggregation and amyloid processing. The full development of HX MS and its demonstration with difficult but centrally important protein systems will provide a uniquely powerful methodology that is widely applicable to the study of protein structure - function problems.