NMR Determination of Biomolecular Structural Dynamics Using Residual Dipolar Couplings Measured in Multiple Alignment Media

本项目的目标是开发基于残余偶极耦合（RDC）的核磁共振方法，用于测定生物分子结构和动力学。该方法将基于在不同各向异性溶剂环境下对多组rdc的测量。为了提高数据采集的效率，将开发新的方法来调节各向异性溶剂环境的性质。特别是，这些研究将集中在利用复合材料的对齐力之间观察到的干扰，机械有序的介质，如噬菌体Pf1颗粒嵌入在应变聚丙烯酰胺凝胶基质中。由主酰胺N-H键的PI证明的解释rdc的DIDC方法将扩展到侧链构象和动力学的确定。此外，这些基于多序列rdc的方法将适用于蛋白质脑膜炎奈瑟菌血红素加氧酶，其动力学已被认为是其功能所必需的。这些研究将作为使用基于rdc的方法详细表征中等大小蛋白质（24 kDa）结构动力学的能力的测试案例。预计这些研究将为未来的核磁共振研究奠定基础，并为蛋白质结构动力学与功能之间的关系提供新的见解。这项研究将为表征生物分子系统的迁移性和结构提供有力的工具，从而将对在原子分辨率上表征生物分子功能的力学方面的能力产生深远的影响。该项目还将广泛影响生物分子核磁共振波谱学的本科教学和研究，由于高场核磁共振仪器的费用和地理集中，本科生往往无法获得这项技术。为了增加本科生在生物分子核磁共振方面的研究机会，已经设立了一个本科生暑期研究职位，主要面向约翰霍普金斯大学社区以外的学生。正在作出持续努力，以促进合作和促进当地生物物理和生物化学研究人员的培训，使他们能够利用最先进的生物分子核磁共振光谱技术

The goal of this project is the development of residual dipolar coupling (RDC)-based NMR methods for the determination of biomolecular structure and dynamics. This approach will be based on the measurement of multiple sets of RDCs under conditions of different anisotropic solvent environments. Novel methods for modulating the nature of the anisotropic solvent environment will be developed in order to improve the efficiency of data acquisition. In particular, these studies will focus on the exploitation of the interference observed between aligning forces for composite, mechanically ordered media such as bacteriophage Pf1 particles embedded within a strained polyacrylamide gel matrix. The DIDC approach to the interpretation of RDCs, demonstrated by the PI for backbone amide N-H bonds, will be extended to the determination of side-chain conformation and dynamics. Furthermore, these multi-alignment RDC-based approaches will be adapted for application to the protein Neisseria meningitidis heme oxygenase, for which dynamics have been implicated as necessary for its function. These studies will serve as a test case for the ability to characterize in detail the structural dynamics of a moderately sized protein (24 kDa) using RDC-based methods. It is anticipated that these studies will lay a foundation for future NMR investigations and provide novel insights into the relationship between protein structural dynamics and function. This research will provide powerful tools for the characterization of mobility and structure of biomolecular systems and thus will have a far reaching impact on the ability to characterize the mechanical aspects of biomolecular function at atomic resolution. This project will also broadly impact undergraduate teaching and research in biomolecular NMR spectroscopy, a technique often inaccessible to undergraduates due to the expense and geographical concentration of high field NMR instrumentation. In order to increase undergraduate research opportunities in biomolecular NMR, one undergraduate summer research position has been established, primarily intended for students outside of the Johns Hopkins community. Sustained efforts are being made to stimulate collaboration and facilitate training of local biophysical and biochemical researchers such that they can employ state of the art biomolecular NMR spectroscopic techniques to advantage