Structure and Folding of Integral Membrane Proteins

描述（由申请人提供）：膜蛋白，其中许多是重要的药物靶点，约占人类蛋白质组的30%。虽然结构基因组学计划正在顺利进行，以解决大量可溶性蛋白的高分辨率结构，但技术还没有进步到足以采取类似的大规模并行方法来解决大量完整膜蛋白的结构。一个瓶颈涉及到膜蛋白成功再折叠的困难。我们最近在这一领域取得了实质性进展，实现了可逆的重折叠条件，并阐明了存在于革兰氏阴性菌、线粒体和叶绿体外膜的-桶膜蛋白的折叠机制。我们最近也通过溶液核磁共振解出了OmpA的结构。这是第一个用这种方法解决的更大的整体膜蛋白结构。在此基础上，我们建议以OmpA为模型进一步阐明膜蛋白折叠机制和膜蛋白稳定性的能量学，并将我们的经验扩展到其他完整的膜蛋白，即孔蛋白OmpG和异戊酰半胱氨酸羧甲基转移酶（ICMT） Ste14p，后者与翻译后修饰Ras的人类ICMT具有高度的序列同源性。我们还建议进一步完善溶液核磁共振方法来求解结构和阐明洗涤剂/脂质胶束中膜蛋白的动力学，并将这些方法应用于结构和动力学性质尚不清楚的OmpG和Ste14p。

DESCRIPTION (provided by applicant): Membrane proteins, many of which are important drug targets, constitute about 30 % of the human proteome. While structural genomics initiatives are well underway to solve the high-resolution structures of scores of soluble proteins, technologies have not advanced far enough to undertake a similar massive parallel approach to solve the structures of a very large number of integral membrane proteins. One bottleneck concerns difficulties with the successful refolding of membrane proteins. We have recently made substantial progress in this area by achieving reversible refolding conditions and elucidating folding mechanisms of beta-barrel membrane proteins, which are present in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. We have also recently solved the structure of OmpA by solution NMR. This was the first structure of a larger integral membrane protein that has been solved by this method. Building on this success, we propose to further elucidate mechanisms of membrane protein folding and the energetics of membrane protein stability using OmpA as a model and to extend our experience to other integral membrane proteins, namely the porin OmpG, and the isoprenylcysteine carboxyl methyltransferase (ICMT) Ste14p, which shares high sequence homology with the human ICMT that post-translationally modifies Ras. We also propose to further refine solution NMR methods for solving structures and elucidating the dynamics of membrane proteins in detergent/lipid micelles and to apply these methods to OmpG and Ste14p, whose structures and dynamical properties are not yet known.