Solubilization, purification, and crystallization of memebrane-associated protein

膜相关蛋白的溶解、纯化和结晶

• 批准号: 7147853
• 负责人: DAVID G. MYSZKA
• 金额: $ 27.66万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7147853
• 关键词: affinity chromatography; biosensor device; biotinylation; cell surface receptors; conformation; crystallization; developmental neurobiology; diabetes mellitus; human immunodeficiency virus; immune response; intermolecular interaction; ligands; method development; monoclonal antibody; protein purification; protein structure function; receptor binding; solubility; virus protein

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.

描述（由申请人提供）：许多膜蛋白，例如G蛋白偶联受体（GPCR），在介导众多生理过程中发挥着关键作用，这使得它们成为药物干预的重要靶点。虽然人类基因组序列迄今为止已揭示了超过 800 个假定的 GPCR 的存在，但只有其中一种受体（视紫红质）的高分辨率结构已得到解决。无法获得更多这些蛋白质的结构信息，部分原因在于它们的丰度较低，以及一旦它们从膜环境中分离出来，就难以维持其活性。拟议研究的目标是改进受体溶解和纯化方案，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。为了成功进行 GPCR 的结构分析，这项工作将由生物化学家、生物物理学家和晶体学家合作进行，并将重点关注与 HIV 生物学、免疫反应、神经发育和糖尿病有关的受体。具体目标集中在 1) 优化活性受体的分离，2) 识别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，以及 5) 解决动力学问题和配体/受体复合物的构象状态。总的来说，这些目标将导致分离出易于进行结构分析的高质量受体，并将促进更系统的结晶方法。简化晶体学工作，同时发现受体功能的细节，将成为开发阻止、控制或治愈 GPCR 相关疾病和病症的药物的基础。