Site-resolved hydration dynamics of PDZ domains

PDZ 域的位点解析水合动力学

• 批准号: 9189626
• 负责人: Christine Jorge
• 金额: $ 3.03万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9189626
• 关键词: Allosteric Regulation; Amino Acids; Binding; Binding Proteins; Biochemical; Buffers; C-terminal; Charge; Chemistry; Computer Simulation; Coupling; Encapsulated; Entropy; Event; Family; Fright; Genomics; Global Change; Goals; Homologous Protein; Human; Hydration status; Hydrogen; Left; Ligand Binding; Ligands; Measures; Membrane Proteins; Methodology; Methods; Micelles; Molecular; Morphologic artifacts; Motion; NMR Spectroscopy; NOESY; Nature; Nuclear; Nuclear Magnetic Resonance; PDZ protein; Peptides; Plague; Positioning Attribute; Protein Dynamics; Protein Family; Proteins; ROESY; Reporting; Resolution; Role; Sequence Homology; Signal Pathway; Signal Transduction; Site; Sodium; Solvents; Source; Structure; Surface; Surveys; System; Tail; Temperature; Tertiary Protein Structure; Testing; Thermodynamics; Tight Junctions; Ubiquitin; Water; Work; chemical property; member; molecular recognition; nanoscale; novel; protein function; protein protein interaction; protein structure; public health relevance; residence; surfactant; time use

 DESCRIPTION (provided by applicant): Water contributes to many aspects of protein function; however, difficulties associated with experimentally measuring protein-water interactions with site-resolution has left our understanding of the nature of protein hydration rather unclear. Though solution nuclear magnetic resonance (NMR) spectroscopy has been proposed as a means to characterize these interactions, several features of the interaction of water with the protein render the approach insensitive and susceptible to artifacts. We have developed reverse micelle encapsulation as a means to overcome these limitations, thereby permitting the unambiguous differentiation of ratios of the nuclear Overhauser effect (NOE) and rotating frame Overhauser effect (ROE) to characterize hydration water dynamics. Recently, we have shown that this approach can be used to survey the surface hydration dynamics of proteins using ubiquitin encapsulated in sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse micelles. We find that surface hydration dynamics are heterogeneous and clustered suggesting that local factors contribute to differential hydration dynamics on the protein surface. Furthermore, there is a correlation between surfaces having slow hydration water and whether that surface participates in protein- protein interactions involving a dry interface suggesting tha the hydration shell may contribute to molecular recognition events. In this proposal we extend our analysis to measure the hydration dynamics of representative members of the PSD-95/disc-large/zonula occludens-1 (PDZ) domain family. PDZ domains are recognition modules in protein signaling pathways. These small, structurally homologous proteins have been extensively characterized biochemically and biophysically. These factors make PDZ domains an ideal system for measuring hydration dynamics using high-resolution NMR spectroscopy. The goals of this proposal are two-fold: 1) To understand what aspects of the protein surface correspond to differential hydration dynamics and 2) to investigate the role of hydration dynamics on ligand binding. We will use high resolution NOESY- HSQC and ROESY-HSQCs to extract site specific NOE/ROE ratios that report on surface hydration. We will measure surface hydration dynamics of several unliganded PDZ domains to understand how local amino acid chemistry may contribute to strength of water-protein interactions. Additionally, we will analyze the surface hydration dynamics of unliganded and liganded states to determine whether hydration dynamics are conserved between structurally related proteins. Together we seek to enhance our understanding of the molecular underpinnings of the protein hydration shell and how it may contribute to molecular recognition.

 描述（由申请人提供）：水有助于蛋白质功能的许多方面;然而，与实验测量蛋白质-水相互作用和位点解析相关的困难使我们对蛋白质水合作用的性质的理解相当不清楚。虽然溶液核磁共振（NMR）光谱已被提出作为一种手段来表征这些相互作用，水与蛋白质的相互作用的几个功能，使该方法不敏感，容易受到文物。我们已经开发了反胶束封装作为一种手段，以克服这些限制，从而允许明确区分的比率的核奥弗豪泽效应（NOE）和旋转框架奥弗豪泽效应（ROE），以表征水合水动力学。最近，我们已经表明，这种方法可以用来调查的表面水化动力学的蛋白质使用泛素封装在钠双（2-乙基己基）磺基琥珀酸酯（AOT）反胶束。我们发现，表面水化动力学是异质性和集群表明，当地的因素有助于差异水化动力学的蛋白质表面。此外，具有缓慢水合水的表面与该表面是否参与涉及干燥界面的蛋白质-蛋白质相互作用之间存在相关性，这表明水合壳可能有助于分子识别事件。在这个建议中，我们扩展我们的分析，以测量的PSD-95/光盘大/小带occludens-1（PDZ）域家族的代表性成员的水合动力学。PDZ结构域是蛋白质信号传导途径中的识别模块。这些小的，结构同源的蛋白质已被广泛的生化和生物药理学特征。这些因素使得PDZ域成为使用高分辨率NMR光谱测量水化动力学的理想系统。该提案的目标是双重的：1）了解蛋白质表面的哪些方面对应于差分水合动力学和2）研究水合动力学对配体结合的作用。我们将使用高分辨率NOESY-HSQC和ROESY-HSQC来提取现场特定的NOE/ROE比率，以报告表面水合作用。我们将测量几个unliganded PDZ结构域的表面水化动力学，以了解当地的氨基酸化学可能有助于水-蛋白质相互作用的强度。此外，我们将分析表面水化动力学的unliganded和liganded状态，以确定是否水化动力学结构相关的蛋白质之间的保守。我们一起寻求提高我们的蛋白质水化壳的分子基础的理解，以及它如何可能有助于分子识别。