Site-resolved hydration dynamics of PDZ domains

 描述(申请人提供)：水有助于蛋白质功能的许多方面；然而，与用现场分辨率实验测量蛋白质-水相互作用相关的困难使我们对蛋白质水化的本质的理解相当不清楚。尽管溶液核磁共振波谱已被提出作为表征这些相互作用的一种手段，但水与蛋白质相互作用的几个特征使得该方法不敏感且容易受到人工制品的影响。我们开发了反胶束包裹作为克服这些限制的一种手段，从而允许明确区分核Overhauser效应(NOE)和旋转框架Overhauser效应(ROE)的比率来表征水化水动力学。最近，我们已经证明，这种方法可以用来研究包裹在双(2-乙基己基)琥珀酸磺酸钠(AOT)反胶束中的泛素的蛋白质表面水化动力学。我们发现表面水化动力学是异质性和聚集性的，这表明局部因素对蛋白质表面不同的水化动力学有贡献。此外，具有缓慢水化水的表面与该表面是否参与了涉及干燥界面的蛋白质-蛋白质相互作用之间存在关联，这表明水化壳层可能有助于分子识别事件。在这个提案中，我们将我们的分析扩展到测量PSD-95/Disk-Large/Zonula occludens-1(PDZ)结构域家族的代表性成员的水化动力学。PDZ结构域是蛋白质信号通路中的识别模块。这些小的、结构上同源的蛋白质已经被广泛地表征为生化和生物物理。这些因素使PDZ结构域成为使用高分辨率核磁共振光谱测量水化动力学的理想系统。这个建议的目标有两个：1)了解蛋白质表面的哪些方面对应于不同的水化动力学；2)研究水化动力学在配体结合中的作用。我们将使用高分辨率NOESY-HSQC和ROESY-HSQCS来提取报告表面水化的站点特定NOE/ROE比率。我们将测量几个未连接的PDZ结构域的表面水化动力学，以了解局部氨基酸化学如何有助于水-蛋白质相互作用的强度。此外，我们将分析未连接和连接状态的表面水化动力学，以确定结构相关蛋白质之间的水化动力学是否保守。我们一起寻求加强我们对蛋白质水合壳的分子基础以及它如何有助于分子识别的理解。

 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.