Protein Hydrogen Exchange: Mechanism and Interpretation

蛋白质氢交换：机制和解释

• 批准号: 1020649
• 负责人: S. Englander
• 金额: $ 82.99万
• 依托单位: University of Pennsylvania
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1020649&HistoricalAwards=false
• 关键词: Protein; Hydrogen; Exchange; Mechanism; Interpretation

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.

尽管氢交换（HX）已经成为分子生物物理学研究中最强大、发展最快的方法，但遗憾的是，整个HX事业所基于的基础知识仍然是不安全的，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，整合实验和理论分析，以研究替代HX机制的作用和决定HX行为的蛋白质因素。潜在的HX机制包括无阻碍的表面HX，溶剂渗透，局部波动，合作亚全球和全球展开。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HX时间尺度（Cleanex-PM用于快速HX，标准HSQC用于较慢HX）和蛋白质大小（新开发的快速3D NMR用于较大蛋白质）的NMR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和能量细化），展开反应（COREX）和静电效应（德尔菲）。四种蛋白质将根据其特殊优势（大小、稳定性、高分辨率结构、电荷密度、深埋、已知的NMR时间尺度动力学、预先存在的HX信息）用于本研究。此外，大量已发表的其他蛋白质的HX数据提供了有价值的参考数据库。许多个别氢的重要机制和决定蛋白质因素的检查将巩固HX知识和理解，并将建立一个预测能力。更广泛的影响HX机制的阐述将提供一个坚实的基础，正确解释非常多的正在进行的研究，现在使用HX方法作为一个主要工具。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发言来传播。详细的数据和结果将在实验室网站上公布，供蛋白质界进一步分析。该项目计划开展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，这个实验室培养了大量的科学家，他们现在在学术，政府和工业中心担任职务。今年，来自宾夕法尼亚大学校园的四名研究生在这个实验室接受培训，以进入研究职业，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。在过去的几年中，该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，该实验室一直支持高中和本科生的补充，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，该实验室目前的研究为PI的研究生水平讲座/讨论课程提供了信息，在过去的一年里，PI在宾夕法尼亚大学医学院生物医学研究生课程的32门课程中被学生评价最高。PI主持了生物化学分子生物物理学研究生课程的招生委员会，并监督了越来越多的少数民族学生的入学，包括过去两年中的9名URM申请人。 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项目共同资助。