UDE COBRE: DETERMINANTS OF STABILITY AND ASSEMBLY OF INTEGRAL MEMBRANE PROTEINS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。项目V：Robinson(将于2006年6月停产)完整的膜蛋白在细胞功能中发挥关键作用，是市场上和正在开发的大多数药物的靶标。然而，对它们的结构和功能以及我们生产和研究它们的能力的了解远远落后于可溶性蛋白质。人们对控制完整膜蛋白稳定性和组装的力和原理知之甚少。我们的目标是确定蛋白质稳定性的关键决定因素，即G蛋白偶联受体超家族成员的折叠和组装途径。我们打算测量受体在天然、中间和非活性状态下的突变效应和侧链相互作用，以确定直接折叠和错误折叠的接触。我们将使用本征荧光来监测折叠过程中特定结构域的环境。在平行研究中，我们正在表征七个螺旋的螺旋倾向、内在稳定性和关联性，以开发一个折叠过程中螺旋形成和关联的模型。这些研究将增加对膜蛋白折叠的了解，并导致更有效的方法来生产这些有价值的蛋白质，用于结构研究、生化分析和药物开发应用。在鉴定A2a受体折叠途径中的中间体方面取得了实质性进展，并表征了一种非途径非活性状态。我们正在启动突变研究，以阐明每个状态中存在的结构联系。我们已经证明了与A2a受体的7个TM螺旋相对应的多肽是螺旋的，并正在研究它们的相互作用，以使我们能够开发一个折叠途径的模型。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Project V: Robinson (To be discontinued for June 2006) Integral membrane proteins play critical roles in cell function, and are targets for a majority of drugs on the market and in development. However, understanding of their structures and functions and our ability to produce and study them lag far behind soluble proteins. Little is known about the forces and principles governing stability and assembly for integral membrane proteins. Our goal is to identify key determinants of protein stability, pathways for the folding and assembly of a member of the G-protein-coupled receptor superfamily. We intend to measure mutational effects and side-chain interactions in native, intermediate, and inactive states of the receptor, to identify contacts that direct folding and misfolding. We will use intrinsic fluorescence to monitor environments of specific domains during folding. In parallel studies we are characterizing the helical propensity, intrinsic stabilities, and association of the seven helices to develop a model for helix formation and association during folding. These studies will increase understanding of membrane protein folding, and lead to more effective methods for production of these valuable proteins for structural studies, biochemical analysis, and drug discovery application. Substantial progress has been made in identifying an intermediate in the folding pathway of the A2a receptor, and characterized an off-pathway inactive state. We are initiating mutagenesis studies to elucidate the structural contacts present in each state. We have shown that peptides corresponding the 7 TM helices of A2a receptor are helical, and are studying their interactions, to enable us to develop a model for the folding pathway.