NMR Investigation of Protein Hydration and Dynamics under Nanoconfinement

描述（由申请人提供）：溶剂驱动的力，如疏水填料，是决定蛋白质结构的主要动力，而蛋白质结构决定了蛋白质的大部分功能。尽管已知蛋白质-溶剂相互作用的重要性，但尚未进行溶剂动力学的全局位点分辨测量。提出的工作的第一个目的是确定蛋白质内部结构水和蛋白质表面水合水的性质。高分辨率核磁共振将用于测量蛋白质-水偶极子-偶极子相互作用，如核Overhauser效应（NOE）所示。在散装水溶液中，蛋白质表面附近的溶剂动力学速度太快，无法测量蛋白质表面上大量的蛋白质-溶剂相互作用。在这些实验中，我们将使用反胶束来封装蛋白质。反胶束约束使溶剂动力学减慢了两个数量级，允许在蛋白质表面测量数十种蛋白质-水相互作用，同时保持被封装蛋白质的结构保真度。通过测量反胶束中泛素、细胞色素c和黄氧还毒素的蛋白质-水NOEs，我们将能够检查蛋白质表面特性、氧化态和配体结合对蛋白质及其溶剂化环境之间特定相互作用的位置和时间尺度的影响。近年来，人们已经清楚地认识到蛋白质的动态运动是其功能的一个重要方面。我们对蛋白质动态运动及其含义的理解还处于起步阶段，需要进一步阐明这种动态过程的基本方面。人们普遍认识到，细胞的边界呈现出一个动态变化的、比体积水溶液复杂得多的溶剂化环境。因此，纳米尺度限制下改变的溶剂化动力学对蛋白质动力学的影响是一个重要的研究方向。本研究的第二个目的是确定纳米限制对蛋白质动力学的影响。使用反胶束作为约束介质，我们将使用高分辨率核磁共振测量骨架和甲基松弛来评估纳米约束导致的蛋白质动力学差异。泛素、细胞色素c和黄氧还素将分别被检查，允许比较表面静电特性、氧化状态和配体结合对溶剂动力学和蛋白质动力学之间相互作用的影响。解释蛋白质与其溶剂化环境之间的基本关系对改善药物治疗学的发展至关重要。

DESCRIPTION (provided by applicant): Solvent-driven forces, such as hydrophobic packing, are the primary impetuses which determines protein structure, and protein structure determines much of protein function. Despite the known importance of protein-solvent interactions, global site-resolved measurement of solvent dynamics has not been performed. The first aim of the proposed work is to determine the nature of structural waters in the protein interior and of hydration waters on the protein surface. High-resolution NMR will be used to measure protein-water dipole-dipole interactions, as manifested in the nuclear Overhauser effect (NOE). In bulk aqueous solution, solvent dynamics near the protein surface are too fast to measure large numbers of protein-solvent interactions on the protein surface. We will use reverse micelles to encapsulate the proteins for these experiments. Reverse micellar confinement slows solvent dynamics by up to two orders of magnitude, permitting measurement of tens of protein-water interactions on the protein surface, while maintaining the structural fidelity of the encapsulated protein. By measuring the protein-water NOEs for ubiquitin, cytochrome c, and flavodoxin in reverse micelles, we will be able to examine the effect of protein surface character, oxidation state, and ligand binding on the location and timescale of specific interactions between proteins and their solvating environment. In recent years, it has become clear that the dynamic motions of proteins are a vital aspect of their function. Our understanding of protein dynamic motions and their implications is in its infancy, and further elucidation of the fundamental aspects of such dynamic processes is needed. It is widely recognized that the confines of the cell present a dynamically altered and vastly more complex solvation environment than that of the bulk aqueous solutions. The effects of the altered solvation dynamics under nanoscale confinement on protein dynamics is thus of fundamental interest. The second aim of the proposed research is to determine the impact of nanoconfinement on protein dynamics. Using reverse micelles as the confining medium, we will use high-resolution NMR measurements of backbone and methyl relaxation to evaluate the differences in protein dynamics as a result of nanoconfinement. Ubiquitin, cytochrome c, and flavodoxin will each be examined, allowing comparison of the effects of surface electrostatic character, oxidation state, and ligand binding on the interplay between solvent dynamics and protein dynamics. Explanation of the fundamental relationship between proteins and their solvating environment is crucial to improvements in the development of pharmaceutical therapeutics.