Protein Hydrogen Exchange: Mechanism and Interpretation

尽管氢交换(HX)已经成为分子生物物理研究中最强大、发展最快的方法，但遗憾的是，整个HX企业所依据的基础知识仍然不安全，这严重阻碍了对HX结果的解释。为了确保这些基础，该项目将进行广泛的努力，结合实验和理论分析，研究替代的HX机制和决定HX行为的蛋白质因素的作用。潜在的HX机制包括无阻碍的表面HX、溶剂渗透、局部波动、协作性次全局和全局展开。潜在的蛋白质因素包括氢键、空间位阻、埋藏深度、二级结构类型、静电场、相互作用密度。该项目的目标是通过最适合预期的HX时间尺度(快速HX的Cleanex-PM，较慢HX的标准HSQC)和蛋白质大小(新开发的用于较大蛋白质的快速3D核磁共振)的核磁共振技术，在各种测试条件下获得广泛的现场分辨HX测量。同时，理论分析将用于研究表面空间、动力学和局部波动过程(MD和能量精化)、展开反应(Corex)和静电效应(Delphi)。基于四种蛋白质的特殊优势(大小、稳定性、高分辨结构、电荷密度、深埋、已知的核磁共振时间尺度动力学、预先存在的HX信息)，本研究将使用四种蛋白质。此外，已发表的大量关于其他蛋白质的HX数据提供了一个有价值的参考数据库。对许多单个氢的重要机制和确定蛋白质因子的检查将巩固HX知识和理解，并将建立预测能力。广泛的影响HX机制的阐述将为正确解释许多正在进行的研究提供坚实的基础，这些研究现在将HX方法作为主要工具。所取得的进展将通过研究文献中的出版物和在科学会议上的陈述加以通报。详细的数据和结果将在实验室网站上公布，供蛋白质社区进一步分析。该项目规划的科学工作将继续为青年科学家提供良好的培训经验。在过去的几年里，这个实验室培养了一大批科学家，他们现在在学术、政府和工业中心任职。今年，来自宾夕法尼亚大学校区的四名研究生在这个实验室接受培训，准备进入研究职业生涯，这个实验室是其他一些共享实验室设施和智力互动的人的中心。在过去的几年里，这个实验室的三名研究生被宾夕法尼亚大学评为年度最佳博士论文。此外，这个实验室一直支持一批高中生和本科生，并让他们参与一线研究。在过去的一年里，五名高中生参加了实验室研究项目。更广泛地说，这个实验室的当前研究为研究生水平的讲座/讨论课程提供了信息，在过去一年中，该课程在宾夕法尼亚大学医学院生物医学研究生研究计划的32门课程中被学生评价为最高的课程。该协会是生物化学与分子生物物理学研究生项目招生委员会的主席，并监督了越来越多未被充分代表的少数族裔学生的入学申请，包括过去两年中的9名URM申请者。该项目由分子和细胞生物科学部的分子生物物理学和化学部的生命过程化学计划共同资助。

Although hydrogen exchange (HX) has become the most powerful and fastest growing method for the study of molecular biophysics, the foundational knowledge on which the entire HX enterprise is based is still unfortunately insecure, which seriously hinders the interpretation of HX results. To secure those foundations, this project will pursue a wide ranging effort, integrating experimental and theoretical analysis, to examine the role of alternative HX mechanisms and the protein factors that determine HX behavior. Potential HX mechanisms include unhindered surface HX, solvent penetration, local fluctuations, cooperative subglobal and global unfolding. Potential protein factors include H-bonding, steric blocking, depth of burial, secondary structural type, electrostatic field, interaction density. The objective of this project is to obtain extensive site-resolved HX measurements under various test conditions by NMR techniques best suited for the intended HX time scale (Cleanex-PM for fast HX, standard HSQC for slower HX) and protein size (newly developed fast 3D NMR for larger proteins). In parallel, theoretical analyses will be used to study surface sterics, dynamics, and local fluctuational processes (MD and energy refinement), unfolding reactions (COREX), and electrostatic effects (DelPhi). Four proteins will be used for this study based on their special advantages (size, stability, high resolution structure, charge density, deep burial, known NMR time scale dynamics, preexisting HX information). In addition the large body of published HX data on other proteins provides a valuable reference data base. Examination of the important mechanisms and determining protein factors for many individual hydrogens will solidify HX knowledge and understanding, and will build toward a predictive capability.Broader ImpactsThe elaboration of HX mechanisms will provide a firm foundation for correctly interpreting the very many ongoing research studies that now use HX approaches as a major tool. Progress made will be communicated through publications in the research literature and in presentations at scientific meetings. Detailed data and results will be published on the laboratory web site for further analysis by the protein community. The scientific work planned in this project will continue to provide an excellent training experience for young scientists. In past years this laboratory has trained a large number of scientists who now hold positions at academic, governmental, and industrial centers. In this year four graduate students from across the University of Pennsylvania campus are training in this lab to enter research careers and this lab serves as a center for a number of others who share lab facilities and intellectual interactions. In past years three graduate students from this lab have been recognized for the Best PhD Dissertation of the Year at the University of Pennsylvania. In addition this lab has always supported a complement of high school and undergraduate college students and involved them in front line research. During the past year five high school students have participated in laboratory research projects. More broadly the current research of this laboratory informs the graduate level lecture/discussion course of the PI which this past year was ranked highest by student evaluation among 32 courses in the Biomedical Graduate Studies program at the U Penn School of Medicine. The PI chairs the Admissions Committee of the Biochemistry & Molecular Biophysics graduate program and has overseen an increasing entry of under represented minority students, including 9 URM applicants in the past two years. This project is jointly funded by Molecular Biophysics in the Division of Molecular and Cellular Bioscience and the Chemistry of Life Processes program in the Chemistry division.