NMR Investigation of Protein Hydration and Dynamics under Nanoconfinement

纳米限制下蛋白质水合和动力学的 NMR 研究

• 批准号: 8259313
• 负责人: NATHANIEL V NUCCI
• 金额: $ 3.65万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8259313
• 关键词: Active Sites; Affect; Behavior; Binding; Cells; Characteristics; Complement; Complex; Crystallography; Data; Detection; Development; Electrostatics; Encapsulated; Environment; Equilibrium; Flavodoxin; Goals; Hydration status; Hydrogen Bonding; Investigation; Ligand Binding; Location; Measurable; Measurement; Measures; Mediating; Membrane; Membrane Proteins; Methods; Micelles; Modeling; Motion; Nature; Nuclear; Pharmacologic Substance; Process; Property; Protein Dynamics; Proteins; Relaxation; Research; Resolution; Set protein; Side; Site; Solutions; Solvents; Structure; Surface; System; Temperature; Therapeutic; Time; Ubiquitin; Vertebral column; Water; Work; aqueous; cofactor; cytochrome c; driving force; in vivo; infancy; interest; meetings; nanoscale; oxidation; protein function; protein structure; protein structure function; research study; residence; success; surfactant

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.

描述（申请人提供）：溶剂驱动力，例如疏水性堆积，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。尽管蛋白质-溶剂相互作用的重要性众所周知，但尚未进行溶剂动力学的全局位点解析测量。拟议工作的首要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-水偶极子-偶极子相互作用，如核奥弗豪瑟效应 (NOE) 所示。在大量水溶液中，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。反向胶束限制可将溶剂动力学减慢多达两个数量级，从而允许测量蛋白质表面上数十种蛋白质-水相互作用，同时保持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 和黄素氧还蛋白的蛋白质-水 NOE，我们将能够检查蛋白质表面特征、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。人们普遍认为，细胞的范围呈现出动态变化且比本体水溶液复杂得多的溶剂化环境。因此，纳米级限制下溶剂化动力学改变对蛋白质动力学的影响具有根本意义。该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，我们将使用主链和甲基松弛的高分辨率核磁共振测量来评估纳米限制导致的蛋白质动力学差异。将分别检查泛素、细胞色素 c 和黄素氧还蛋白，从而比较表面静电特征、氧化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。解释蛋白质及其溶剂化环境之间的基本关系对于改进药物治疗的开发至关重要。

项目成果

Reply to Kitahara and Mulder: An ensemble view of protein stability best explains pressure effects in a T4 lysozyme cavity mutant.

回复 Kitahara 和 Mulder：蛋白质稳定性的整体观点最好地解释了 T4 溶菌酶空腔突变体中的压力效应。

• DOI: 10.1073/pnas.1424002112
• 发表时间: 2015
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Wand,AJoshua;Nucci,NathanielV
• 通讯作者: Nucci,NathanielV

Site-resolved measurement of water-protein interactions by solution NMR.

• DOI: 10.1038/nsmb.1955
• 发表时间: 2011-02
• 期刊: Nature structural & molecular biology
• 影响因子: 16.8

Mapping the hydration dynamics of ubiquitin.

• DOI: 10.1021/ja202033k
• 发表时间: 2011-08-17
• 期刊: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
• 影响因子: 15
• 作者: Nucci, Nathaniel V.;Pometun, Maxim S.;Wand, A. Joshua
• 通讯作者: Wand, A. Joshua