NMR Investigation of Protein Hydration and Dynamics under Nanoconfinement

描述(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，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，但还没有进行全局位置分辨的溶剂动力学测量。这项拟议工作的第一个目标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-偶极相互作用，如核Overhauser效应(NOE)所示。在大块水溶液中，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的大量蛋白质-溶剂相互作用。我们将使用反胶束来包裹这些实验中的蛋白质。反向胶束限制使溶剂动力学减慢达两个数量级，允许测量蛋白质表面的数十个蛋白质-水相互作用，同时保持被包裹蛋白质的结构保真度。通过测量反胶束中泛素、细胞色素c和黄还蛋白的蛋白质-水NOES，我们将能够检测蛋白质表面性质、氧化状态和配体结合对蛋白质与其溶解环境之间特定相互作用的位置和时间尺度的影响。近年来，蛋白质的动态运动是其功能的一个重要方面，这一点已经变得很清楚。我们对蛋白质动态运动及其含义的理解还处于初级阶段，还需要进一步阐明这种动态过程的基本方面。人们普遍认为，电池的边界呈现出一个动态变化的溶剂化环境，而且比大块水溶液的溶剂化环境复杂得多。因此，在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非常重要的。这项拟议研究的第二个目标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，我们将使用骨架和甲基松弛的高分辨率核磁共振测量来评估由于纳米限制而导致的蛋白质动力学的差异。将分别检测泛素、细胞色素c和黄毒素，以便比较表面静电特性、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于改进药物疗法的发展至关重要。

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.