DEVELOPMENT OF MEMBRANE GLYCOPROTEIN STRUCTURE

本研究的长期目标是了解折叠信息在膜蛋白一级序列中编码的方式，以及细胞在信息传递过程失败时识别的方法。由该基金资助的囊性纤维化跨膜传导调节因子（CFTR）的研究进入了第四个年头，为了解导致大多数囊性纤维化病例的AF508折叠突变的性质以及与CFTR短暂相互作用以协助有效折叠的蛋白质提供了相关的基本信息。未来的研究将集中于阐明关键折叠中间体的构象，识别这些中间体的机制，以及折叠的后期步骤，即CFTR与其他蛋白质结合在膜上形成大分子复合物。为此目的，五个具体目标是：1。表征AF508突变改变的CFTR foldina中间体的结构。已建立的生物物理方法将用于表征关键折叠中间体的结构特征。2. 确定识别折叠中间体所需的蛋白质机制，并描述其作用机制。识别体内关键折叠中间体所需的蛋白质将使用我们最近开发的体外蛋白水解试验进行分离，该试验可在折叠过程中区分突变型和野生型蛋白质。3. 确定CFTR第一跨膜结构域的折叠途径及致病突变的影响。CFTR跨膜的多肽模型和体外翻译系统将用于研究野生型和突变型跨膜结构域的膜整合和螺旋结合步骤。4. 鉴定识别错误折叠跨膜结构域的蛋白质并确定其作用机制。位点特异性交联和高强度免疫沉淀将用于鉴定与CFTR错误折叠突变体跨膜结构域相互作用的蛋白质。5. 表征CFTR与其他蛋白质在成熟过程中形成超分子复合物的相互作用。基于我们最近的发现，CFTR激活了表达CFTR的细胞和组织中的阴离子交换器，我们将测试这些蛋白质在折叠的最后步骤中结合形成一个大的四分之一结构的假设。为了实现这些目标，将采用生化，生物物理，免疫化学，分子和细胞生物学方法的组合，所有这些方法都是在这个实验室建立的。这些研究对于详细了解膜蛋白折叠的机制是必要的和基础的。

The long-range objectives of this study are to understand the manner in which folding information is encoded in the primary sequence of membrane proteins and the means by which the cell recognizes when this process of information transfer has failed. The studies of the cystic fibrosis transmembrane conductance regulator (CFTR) supported by this grant entering its fourth year, have provided fundamental information relevant to understanding the nature of the AF508 folding mutant responsible for most cases of cystic fibrosis and the proteins which transiently interact with CFTR to assist efficient folding. Future studies will focus on elucidation of the conformation of critical folding intermediates, the machinery that recognizes these intermediates, and the later steps in folding, namely the association of CFTR with other proteins to form macromolecular complexes at the membrane. To this end the five specific aims are to: 1. Characterize the structure of the CFTR foldina intermediate altered bv the AF508 mutation. Established biophysical methods will be employed to characterize the structural features of the critical folding intermediate. 2. Identify the protein machinery required for recognition of the folding intermediate and characterize their mechanism of action. The proteins required for recognition of the critical folding intermediate in vivo will be isolated using our recently developed in vitro proteolysis assay which distinguishes mutant from wild type protein during folding. 3. Determine the folding pathway of the first transmembrane domain of CFTR and the effect of disease-causing mutations. Peptide models of the transmembrane spans of CFTR and an in vitro translation system will be utilized to study the membrane-integration and helical-association steps of the wild type and mutant transmembrane domains. 4. Identity the proteins that recognize misfolded transmembrane domains and determine their mechanism of action. Site specific-crosslinking and high stringency immunoprecipitations will be used to identify proteins that interact with misfolded mutant transmembrane domains of CFTR. 5. Characterize the interaction of CFTR with other proteins during maturation to form a supramolecular complex. Based on our recent finding that CFTR activates the anion exhanger in CFTR expressing cells and tissues, we will test the hypothesis that these proteins associate to form a large quartemary structure during the last steps of folding. To accomplish these goals, a combination of biochemical, biophysical, immunochemical, molecular and cell biological approaches, all established in this laboratory, will be employed. These studies are necessary for and fundamental to a detailed understanding of the mechanisms by which membrane proteins fold.