NMR Investigation of Protein Hydration and Dynamics under Nanoconfinement

描述（由申请人提供）：溶剂驱动力，如疏水堆积，是决定蛋白质结构的主要推动力，蛋白质结构决定了蛋白质的大部分功能。尽管蛋白质-溶剂相互作用的重要性是已知的，但尚未进行溶剂动力学的全局位点分辨测量。建议的工作的第一个目的是确定在蛋白质内部的结构沃茨和蛋白质表面上的水合沃茨的性质。高分辨率NMR将用于测量蛋白质-水偶极-偶极相互作用，如核Overhauser效应（NOE）所示。在本体水溶液中，蛋白质表面附近的溶剂动力学太快，无法测量蛋白质表面上大量的蛋白质-溶剂相互作用。我们将使用反胶束来封装这些实验的蛋白质。反胶束限制减缓溶剂动力学高达两个数量级，允许测量蛋白质表面上的蛋白质-水相互作用的数十，同时保持封装蛋白质的结构保真度。通过测量蛋白质-水NOE的泛素，细胞色素c，和黄素氧还蛋白在反胶束中，我们将能够检查蛋白质的表面特征，氧化态，和配体结合的位置和时间尺度上的蛋白质和它们的溶剂化环境之间的特定相互作用的影响。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影响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。人们广泛认识到，电池的边界呈现出动态变化的并且比本体水溶液的溶剂化环境复杂得多的溶剂化环境。因此，改变溶剂化动力学的影响下，纳米限制蛋白质动力学的根本利益。拟议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。使用反胶束作为限制介质，我们将使用高分辨率NMR测量的骨干和甲基松弛，以评估蛋白质动力学的差异，作为纳米限制的结果。泛素，细胞色素c，和flavodoxin将分别进行检查，允许比较表面静电特性，氧化态和配体结合溶剂动力学和蛋白质动力学之间的相互作用的影响。解释蛋白质和它们的溶剂化环境之间的基本关系对于药物治疗学的发展的改进是至关重要的。

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.