METHODOLOGY DEVELOPMENT FOR NMR STUDIES OF BIOMOLECULES

该项目的目标是开发和评估新的溶液态核磁共振方法，用于研究生物分子的结构和动力学。核磁共振波谱是一种非常强大的工具，用于研究溶液中的生物分子，因为它能够提供原子水平上的详细信息。核磁共振光谱学在生物分子结构测定、药物/受体、酶/底物和抗体/抗原复合物等系统中分子间相互作用的研究以及生物分子动力学表征方面的应用程度，取决于操纵相关核磁共振活性核以产生所需信息的能力。核磁共振波谱在过去十年中对生物分子和生物物理过程的研究产生了巨大的影响，这主要是由于从感兴趣的系统中提取有用数据的新技术或改进技术的不断发展。该提案针对溶液态核磁共振波谱学的严峻一般领域，其中将追求一些具体目标：1)将探索利用偶极耦合的相干转移实验的发展。随着最近引入的影响分子翻滚中轻微各向异性的方法，从这些部分排列样品中产生的残余耦合的开发中可以获得大量新的结构信息。2)通过开发优化的绝热混合序列，将研究TOCSY实验的改进。这项工作在超高场核磁共振应用中具有重要意义，因为它有可能提高相干传输带宽，同时最大限度地减少射频样品加热。3)低温探针技术的应用将被研究，重点是在分子或复合物的构象柔性区域获得有价值的结构信息，在这些区域，化学交换展宽通常阻碍了传统的测量。4)一种研究化学和构象交换效应的新技术将应用于RNA发夹动力学过程的研究。了解RNA分子的功能需要对其内部动力学有详细的了解。这项技术进一步发展的可能性，例如它可以应用于完全13c标记的分子，将被探索。5)我们现有的模拟软件将得到改进，以确定在射频辐射、自旋弛豫和交换过程影响下的核自旋动力学。这些理论工具对于发展本提案中描述的实验技术和正确解释将被记录的许多数据是必不可少的。对核磁共振数据的正确解释关键取决于对实验中潜在的自旋物理的详细理解。

The goal of this project is the development and evaluation of new, solution-state NMR methodologies for use in studies of structure and dynamics of biomolecules. NMR spectroscopy is an extremely powerful tool for the study of biomolecules in solution, due to its ability to provide detailed information at an atomic level. The extent to which NMR spectroscopy can be exploited for the structure determination of biomolecules, the investigation of intermolecular interactions in systems such as drug/receptor, enzyme/substrate and antibody/antigen complexes, and the characterization of biomolecular dynamics, depends on the ability to manipulate the relevant NMR-active nuclei to yield the desired information. The enormous impact that NMR spectroscopy has made during the last decade in the investigation of biomolecules and biophysical processes is due largely to the continuing development of new or improved techniques for extracting useful data from the systems of interest. The proposal targets severe general areas of solution-state NMR spectroscopy in which a number of specific goals will be pursued: 1) Development of coherence transfer experiments that utilize dipolar couplings will be explored. With the recent introduction of methods for effecting slight anisotropies in molecular tumbling, there is a wealth of new structural information potentially available from the exploitation of the residual couplings that are generated in these partially aligned samples. 2) Improvements in TOCSY experiments via the development of optimized, adiabatic mixing sequences will be investigated. This work is of significant interest in ultra-high field NMR applications due to the possibility of improving coherence transfer -bandwidths while minimizing RF sample-heating. 3) The use of cryogenic probe technology will be examined in applications that focus on obtaining valuable structural information in conformationally flexible regions of a molecule or complex, where chemical exchange broadening normally impedes conventional measurements. 4) A new technique for investigating chemical and conformational exchange effects will be applied to the study of dynamical processes in an RNA hairpin. Understanding how RNA molecules function will require detailed knowledge of their internal dynamics. The possibility for further developments of this technology, such that it could be applied to fully 13C-labeled molecules, will be explored. 5) Our existing simulation software will be improved to allow a determination,of nuclear spin dynamics under the influence of RF irradiation, spin relaxation and exchange processes. Such theoretical tools are essential for developing the experimental techniques described in this proposa1 and for correctly interpreting much of the data that will be recorded. The correct interpretation of NMR data depends critically on having a detailed understanding of the underlying spin physics of the experiments.