Determinants of Membrane Protein Topology

描述(由申请人提供)： 大肠杆菌乳糖渗透酶是一种由十二个跨膜结构域组成的蛋白质，它的拓扑结构随膜脂组成的不同而变化。拓扑是动态的，随着脂质环境的变化，翻译后和插入后都会发生变化。跨膜螺旋第七号似乎是这些转变的一个铰链点，可能是因为它相对亲水的性质，以及它之前的非正则膜外环。在这项提案中，我们概述了将识别乳糖渗透酶中有助于拓扑灵活性的特定残基的工作。导致脂质非依赖性拓扑或新的脂质依赖性拓扑图的氨基酸变化将通过定点突变和遗传筛选来鉴定。脂类与这些新发现的蛋白质突变体的相互作用将被评估。通过这些研究，我们希望找出在决定初始拓扑和拓扑灵活性方面重要的脂质相关因素。控制乳糖渗透酶折叠的一般原理将适用于大肠杆菌和真核生物中的其他膜蛋白，从而有助于寻找有效的治疗阿尔茨海默病和囊性纤维化等膜蛋白疾病的方法。

DESCRIPTION (provided by applicant): E. coli lactose permease is a twelve-transmembrane domain protein that displays variable topology dependent on the lipid composition of the membrane. The topology is dynamic, changing posttranslationally and post-insertionally with changes in the lipid environment. Transmembrane helix number VII seems to be a hinge point for these transitions, likely because of its relatively hydrophilic nature, and the noncanonical extramembrane loops that precede it. In this proposal, we outline work that will identify specific residues in lactose permease that contribute to topological flexibility. Amino acid changes that lead to either lipid-independent topologies or new lipid-dependent topology profiles will be identified by both site directed mutagenesis and genetic screens. Lipid interactions with these newly identified protein mutants will be assessed. From these studies, we hope to identify lipid-related factors important in determining initial topology and topological flexibility. The general principles governing the folding of lactose permease will apply to other membrane proteins in both E. coli and eukaryotes, and thereby aid in the search for effective therapies of such membrane protein disorders as Alzheimer's Disease and cystic fibrosis.