STRUCTURAL BASIS OF BACTERIORHODOPSIN BIOGENESIS

描述（由申请人提供）：拟议的长期目标 研究是了解膜蛋白生物合成的结构基础。的 焦点是多位（多跨）膜蛋白，细菌视紫红质， 一种由古菌盐生盐杆菌产生的完整的膜蛋白。 这种蛋白质折叠形成一束七个跨膜α-螺旋， 视网膜和组装在一个二维晶格称为 紫膜细菌视紫红质是理解膜的模型 蛋白质的插入、折叠和组装，因为它的三维结构 已知在天然膜环境中具有高分辨率。客观 在这个建议中，是在体内测试细菌视紫红质是否适合两阶段 跨膜片段之间的相互作用是主要的模型 折叠和组装的决定因素。 具体的目的是（1）通过饱和度来在整个BR中引入突变， 在初步研究中实施的诱变方法;（II）鉴定那些 在这些残基处可以容忍取代而不影响 从BO和视黄醛形成BR;和（III）确定 取代可以被容忍而不影响BR的组装 晶格分析结果将与 BR的三维结构，以测试是否预测的 两阶段模型得到证实。这些研究预计将产生重要的 关于膜蛋白质折叠的结构基础的新信息 在细胞中组装。

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is understand the structural basis of membrane protein biogenesis. The focus is on the polytopic (multispanning) membrane protein, bacteriorhodopsin, an integral membrane protein produced by the archaeon Halobacteriurn salinarum. This protein folds to form a bundle of seven transmembrane alpha-helices, binds retinal and assembles in a two-dimensional crystalline lattice known as the purple membrane. Bacteriorhodopsin is a model for understanding membrane protein insertion, folding and assembly because its three-dimensional structure is known at high resolution in the native membrane environment. The objective in this proposal is to test in vivo whether bacteriorhodopsin fits a two-stage model in which interactions between transmembrane segments are the primary determinant of folding and assembly. The specific aims are (1) to introduce mutations throughout BR by a saturation mutagenesis approach implemented in preliminary studies; (II) to identify those residues at which substitutions can be tolerated without affecting the formation of BR from BO and retinal; and (III) to determine residues at which substitutions can be tolerated without affecting the assembly of the BR lattice. The results from this analysis will be compared with the three-dimensional structure of BR to test whether the predictions of the two-stage model are confirmed. These studies are expected to yield important new information on the structural basis of integral membrane protein folding and assembly in the cell.