Membrane Proteins. Crystallogenesis and X-ray Structure

描述(申请人提供)：解决结构以了解膜蛋白的功能仍然是所有生物学的重大挑战之一。鉴于三分之一的人类基因组编码的是膜蛋白，其中许多起着信号传递、结构和运输的作用，是致病和治疗药物的靶标，迎接挑战的健康后果是巨大的。将采取多方面的方法，通过生产用于大分子结晶学的结构级晶体来促进我们对膜蛋白功能的理解。重点放在在脂类中间相结晶被称为“在中间相”或立方相法。这被证明是膜蛋白结构测定的一种普遍有用的方法。用这种方法，结晶是在膜环境中进行的，而膜环境很可能有利于目标膜蛋白。我们已经建造了一个独特的、最先进的机器人，它以高通量的方式在介观结晶中执行，并且需要极少量的蛋白质、脂肪和沉淀剂。它将在目前的应用中用于生产3D晶体，用于对选定的一组膜蛋白质进行高分辨率结构测定。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-多肽复合体。一些目标蛋白质将在内部生产，一些将由个人合作者提供，而另一些将通过NIH结构蛋白质组学倡议提供。 根据美国国立卫生研究院结构生物学路线图，在拟议的结构研究的同时，将致力于改进晶体形成和扩大可用于结构确定的膜蛋白靶标的范围。这将通过实施一项合成计划来实现，根据该计划，可以生产出具有理想的物理化学特性的脂类，用于结晶试验。还将对晶体成核和生长的分子机制进行研究，以期更合理、更成功地进行晶体形成。成功获得晶体，并最终获得膜蛋白的原子结构，将增强我们对一些最基本的细胞功能过程的理解，这些过程是人类健康不可或缺的。

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.