Structure and Folding of Integral Membrane Proteins

描述(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，以解决数十种可溶性蛋白质的高分辨率结构，但技术还没有进步到采取类似的大规模并行方法来解决非常大量的完整膜蛋白的结构。一个瓶颈是膜蛋白成功重折叠的困难。最近，我们在这一领域取得了实质性进展，实现了可逆复性条件，并阐明了存在于革兰氏阴性细菌、线粒体和叶绿体外膜中的β-桶膜蛋白的折叠机制。我们最近还用溶液核磁共振技术解决了OmpA的结构问题。这是第一个用这种方法解决的更大的完整膜蛋白的结构。在此基础上，我们建议以OmpA为模型进一步阐明膜蛋白折叠的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Porin OmpG和异丙基半胱氨酸羧甲基转移酶(ICMT)Ste14p，它与翻译后修饰RAS的人ICMT有很高的序列同源性。我们还建议进一步改进溶液核磁共振方法，以解决洗涤剂/脂胶束中膜蛋白的结构和动力学问题，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.