Membrane Proteins. Crystallogenesis and X-ray Structure

描述（由申请人提供）：从理解膜蛋白功能的角度来解决膜蛋白的结构仍然是所有生物学中的重大挑战之一。鉴于三分之一的人类基因组编码膜蛋白，其中许多起信号转导、结构和运输作用，并且是疾病病原体和治疗剂的目标，因此迎接挑战的健康后果是巨大的。通过生产用于大分子晶体学的结构级晶体，将采取多方面的方法来提高我们对膜蛋白功能的理解。通过所谓的“中介相”或立方相方法，重点放在脂质中间相的结晶上。这被证明是一种普遍有用的膜蛋白结构测定方法。用这种方法，结晶发生在靶膜蛋白可能喜欢的膜环境中。我们已经建立了一个独特的，最先进的机器人，以高通量的方式进行中介晶化，只需要极少量的蛋白质，脂质和沉淀剂。它将在目前的应用中用于生产3d晶体，用于确定一组膜蛋白的高分辨率结构。目标群体涵盖了广泛的膜蛋白类型，包括真核生物和原核生物，整体和外周，单体和多聚体，以及蛋白质-蛋白质和蛋白质-肽复合物。一些目标蛋白质将由内部生产，一些将由个人合作者提供，而其他将通过NIH结构蛋白质组学计划提供。

DESCRIPTION (provided by applicant): Solving the structure with a view to understanding the function of membrane proteins remains one of the grand challenges in all of Biology. Given that a third of the human genome codes for membrane proteins, many of which serve signal transducing, structural and transport roles and are targets of disease causative and treatment agents, the health consequences of meeting the challenge are great. A multifaceted approach will be taken to advance our understanding of membrane protein function by producing structure grade crystals for use in macromolecular crystallography. Emphasis is placed on crystallization in lipidic mesophases by what is referred to as the 'in meso' or cubic phase method. This is proving to be a generally useful approach for membrane protein structure determination. With this method crystallization takes place in a membrane environment that is likely to be favored by the target membrane protein. We have built a unique, state-of-the-art robot that performs in meso crystallization in high-throughput fashion and that requires miniscule amounts of protein, lipid and precipitant. It will be used in the current application to produce 3-D crystals for the high-resolution structure determination of a select group of membrane proteins. The target group covers a broad range of membrane protein types including eukaryote and prokaryote, integral and peripheral, monomeric and multimeric, as well as protein-protein and protein-peptide complexes. Some of the target proteins will be produced in-house, some will be provided by individual collaborators, while others will be supplied through the NIH Structural Proteomics Initiative. In line with the NIH Structural Biology Road Map, and in parallel with the proposed structure study, effort will be devoted to improving crystallogenesis and to broadening the range of membrane protein targets that yield to structure determination. This will be achieved by implementing a synthesis program whereby lipids with desirable physico-chemical characteristics are produced for use in crystallization trials. The molecular mechanism of crystal nucleation and growth will be studied too with a view to more rational and successful crystallogenesis. Success in obtaining crystals, and ultimately the atomic structure of membrane proteins, will enhance our understanding of some of the most fundamental processes underlying cellular function that are integral to human health.