METHODOLOGY DEVELOPMENT FOR NMR STUDIES OF BIOMOLECULES

该项目的目标是开发和评估用于研究生物分子结构和动力学的新的溶液状态核磁共振方法。核磁共振光谱学是研究溶液中生物分子的一种非常强大的工具，因为它能够在原子水平上提供详细的信息。核磁共振波谱在多大程度上可用于确定生物分子的结构，研究药物/受体、酶/底物和抗体/抗原复合体等系统中的分子间相互作用，以及生物分子动力学的表征，取决于操纵相关核磁共振活性核以产生所需信息的能力。核磁共振光谱学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，这在很大程度上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。该提案针对溶液状态核磁共振光谱学的严格一般领域，其中将追求一些具体目标：1)探索利用偶极耦合的相干转移实验的发展。随着最近在分子翻滚中实现轻微各向异性的方法的引入，从这些部分排列的样品中产生的残余耦合的开发中潜在地有了丰富的新的结构信息。2)将研究通过开发优化的绝热混合序列来改进TOCSY实验。这项工作在超高场核磁共振应用中具有重要意义，因为它有可能改善相干传输带宽，同时最大限度地减少射频样品加热。3)将在侧重于在分子或络合物的构象柔性区域获得有价值的结构信息的应用中审查低温探针技术的使用，在这些应用中，化学交换拓宽通常会阻碍常规测量。4)一种研究化学和构象交换效应的新技术将应用于研究RNA发夹中的动力学过程。要了解RNA分子的功能，需要对其内部动力学有详细的了解。将探索进一步发展这项技术的可能性，以便将其应用于完全标记13C的分子。5)我们将改进现有的模拟软件，以确定在射频辐照、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这项提议中描述的实验技术和正确解释将要记录的大部分数据是必不可少的。对核磁共振数据的正确解释关键取决于对实验潜在的自旋物理有一个详细的了解。

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.