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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。项目V: Robinson（将于2006年6月终止）整体膜蛋白在细胞功能中起着关键作用，是市场上和开发中的大多数药物的靶标。然而，对它们的结构和功能的理解以及我们生产和研究它们的能力远远落后于可溶性蛋白质。对于控制整体膜蛋白的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，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区域的环境。在平行研究中，我们描述了螺旋倾向、内在稳定性和七个螺旋的关联，以开发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理解，并为这些有价值的蛋白质的结构研究、生化分析和药物发现应用提供更有效的方法。在鉴定A2a受体折叠途径的中间体方面已经取得了实质性进展，并且表征为非通路失活状态。我们正在启动诱变研究，以阐明每种状态下存在的结构接触。我们已经证明A2a受体的7个TM螺旋对应的肽是螺旋状的，并且正在研究它们之间的相互作用，使我们能够开发折叠途径的模型。

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.