Membrane Protein Co- Crystallization with Highly Crystalline and Soluble Proteins

膜蛋白与高度结晶和可溶性蛋白质共结晶

• 批准号: 8536875
• 负责人: Gregory A. Weiss
• 金额: $ 26.23万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8536875
• 关键词: Affinity; Affinity Chromatography; Antibodies; Area; Binding; Binding Proteins; Binding Sites; Biological Assay; Caveolins; Cells; Chimeric Proteins; Collaborations; Crystal Formation; Crystallization; Detergents; Development; Disease; Exhibits; Foundations; Freezing; G Protein-Coupled Receptor Genes; GTP-Binding Proteins; Generations; Goals; Laboratories; Letters; Libraries; Ligands; Membrane Proteins; Methods; Molecular Conformation; Muramidase; Nature; Phage Display; Precipitation; Production; Property; Protein Engineering; Protein Overexpression; Protein S; Proteins; Publishing; Reagent; S-crystallin; Solubility; Specificity; Structural Biologist; Structure; System; Techniques; Therapeutic; Thermodynamics; Variant; Work; base; biological systems; design; design and construction; innovation; molecular recognition; novel strategies; overexpression; protein aggregation; protein folding; protein function; protein purification; protein structure; receptor coupling; research study; structural biology; success; tool

DESCRIPTION (provided by applicant): Membrane proteins confound anything less than exceptionally heroic attempts aimed at solving their structures. The conventional approaches to membrane protein overexpression, purification, and crystallization typically fail due to problems with insolubility and folding. This project leverages large libraries of soluble and highly crystallizable proteins to identify binding partners for membrane proteins. Selectants from these libraries will provide affinity reagents for membrane protein co-expression, affinity purification and co-crystallization. Co-expression with the binding partner could help avoid membrane protein aggregation, and allow protein folding to take place. Affinity chromatography with the binding partner is aimed at assisting membrane protein purification, and co-crystallization aims to slow protein aggregation and precipitation during formation of crystals. The first specific aim focuses on design and construction of phage-displayed protein libraries for high affinity binding to membrane proteins. Strategic choice of proteins for library formation, such as the highly crystallizable protein lysozyme and the exceptionally soluble protein S-crystallin, for phage display will help insure the success of the project; additional libraries specifically tailored forG-protein coupled receptors (GPCRs) include variants of G- proteins and GPCR ligands. To obtain high affinity binding, thermal stability, solubility, and other properties, the second specific aim features a flow path of selections and screens. In the third specific aim, the affinity reagents from phage display are applied to the production of membrane proteins and their crystallization. By binding to and essentially freezing specific conformations of the membrane protein, the affinity reagents could offer powerful tools both for structural biology, but also other structure-function studies of membrane proteins. In summary, this proposal will define new approaches to protein engineering and molecular recognition, through development of new fusion proteins and their use in the recognition of membrane proteins.

描述（由申请人提供）：膜蛋白混淆了任何旨在解决其结构的异常英勇的尝试。膜蛋白过表达、纯化和结晶的传统方法通常会因不溶性和折叠问题而失败。该项目利用大型可溶性和高度结晶蛋白库来识别膜蛋白的结合伴侣。来自这些文库的选择物将为膜蛋白共表达、亲和纯化和共结晶提供亲和试剂。与结合伴侣共表达可以帮助避免膜蛋白聚集，并允许蛋白质折叠发生。结合配偶体的亲和层析旨在协助膜蛋白纯化，共结晶旨在减缓晶体形成过程中的蛋白质聚集和沉淀。 第一个具体目标 专注于设计和构建噬菌体展示蛋白库，以与膜蛋白高亲和力结合。用于文库形成的蛋白质的战略选择，例如用于噬菌体展示的高度结晶的蛋白质溶菌酶和极易溶解的蛋白质S-晶状体蛋白，将有助于确保该项目的成功；其他专为 G 蛋白偶联受体 (GPCR) 定制的文库包括 G 蛋白和 GPCR 配体的变体。为了获得高亲和力结合、热稳定性、溶解度等性能，第二个具体目标 具有选择和屏幕的流程路径。第三个具体目标是将噬菌体展示的亲和试剂应用于膜蛋白的生产及其结晶。通过结合并基本上冻结膜蛋白的特定构象，亲和试剂可以为结构生物学以及膜蛋白的其他结构功能研究提供强大的工具。总之，该提案将通过开发新的融合蛋白及其在膜蛋白识别中的应用，定义蛋白质工程和分子识别的新方法。