Solubilization, purification, and crystallization of memebrane-associated protein

描述(申请人提供)：许多膜蛋白，如G蛋白偶联受体(GPCRs)，在调节许多生理过程中发挥关键作用，这使它们成为药物干预的重要靶点。虽然到目前为止，人类基因组序列已经揭示了800多个假定的GPCR的存在，但只有一个受体(视紫红质)的高分辨率结构已经被解决。无法获得更多这些蛋白质的结构信息的部分原因是它们的丰度较低，以及一旦它们从膜环境中分离出来，就很难保持它们的活性。拟议研究的目标是改进受体的增溶和纯化方案，并确定不会对受体活性的各个方面产生不利影响的结晶条件。为了成功地进行GPCRs的结构分析，这项工作将作为生物化学家、生物物理学家和结晶学家的合作进行，并将重点放在参与艾滋病毒生物学、免疫反应、神经发育和糖尿病的受体上。具体目标集中在1)优化活性受体的分离，2)鉴定用于共结晶的构象敏感抗体，3)开发亲和层析方案，4)评估结晶条件对受体活性的影响，5)解决配体/受体复合体的动力学和构象状态。总而言之，这些目标将导致分离出易于结构分析的更高质量的受体，并将促进更系统的方法来实现它们的结晶。简化结晶学的工作，同时发现受体功能的细节，将成为开发阻碍、控制或治愈GPCR相关疾病和疾病的药物的基础。

DESCRIPTION (provided by applicant): Many membrane proteins, such as G-protein-coupled receptors (GPCRs), play key roles in mediating numerous physiological processes, which makes them important targets for pharmaceutical intervention. While the human genome sequence has revealed the existence of more than eight hundred putative GPCRs to date, the high-resolution structure of only one of these receptors (rhodopsin) has been solved. The inability to derive structural information for more of these proteins stems in part from their low abundance, as well as from difficulties associated with maintaining their activity once they are isolated from their membrane environments. The goal of the proposed research is to improve receptor solubilization and purification protocols and to identify crystallization conditions that do not adversely affect various aspects of receptor activity. To succeed in the the structural analysis of GPCRs, the work will be performed as a collaboration between biochemists, biophysicists, and crystallographers and will focus on receptors involved in HIV biology, the immune response, neural development, and diabetes. The specific aims center on 1) optimizing the isolation of active receptors, 2) identifying conformationally sensitive antibodies for co- crystalization, 3) developing affinity chromatography protocols, 4) evaluating the effects of crystallization conditions on receptor activity, and 5) resolving the kinetics and conformational states of ligand/receptor complexes. Collectively, these aims will lead to the isolation of higher-quality receptors readily amenable to structural analysis and will foster a more systematic approach toward their crystallization. Streamlining crystallography efforts while simultaneously discovering details about receptor function will serve as the basis for developing agents that thwart, control, or cure GPCR-related diseases and conditions.