Membrane Protein Co- Crystallization with Highly Crystalline and Soluble Proteins

描述（由申请人提供）：膜蛋白质混淆了旨在解决其结构的异常英勇的尝试。膜蛋白过表达、纯化和结晶的常规方法通常由于不溶性和折叠的问题而失败。该项目利用可溶性和高度结晶蛋白质的大型文库来鉴定膜蛋白的结合伴侣。来自这些文库的选择剂将提供用于膜蛋白共表达、亲和纯化和共结晶的亲和试剂。与结合伴侣的共表达可以帮助避免膜蛋白聚集，并允许蛋白折叠发生。结合伴侣的亲和层析旨在辅助膜蛋白纯化，共结晶旨在减缓晶体形成期间的蛋白聚集和沉淀。 第一个具体目标 专注于设计和构建噬菌体展示的蛋白质文库，用于高亲和力结合膜蛋白。策略性地选择用于文库形成的蛋白质，如高度可结晶的蛋白质溶菌酶和异常可溶的蛋白质S-晶状体蛋白，用于噬菌体展示将有助于确保该项目的成功;专门为G-蛋白偶联受体（GPCR）定制的另外的文库包括G-蛋白和GPCR配体的变体。为了获得高亲和力结合、热稳定性、溶解性和其他性质，第二个具体目标是 具有选择和屏幕的流程。在第三个具体目标中，将来自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-功能研究提供强有力的工具。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来定义蛋白质工程和分子识别的新方法。 公共卫生相关性：膜蛋白代表了与许多疾病相关的非常重要的一类蛋白质，并且被大约40%的治疗药物靶向。然而，尽管它们的生物医学重要性，膜蛋白的结构是相对罕见的，由于其不溶性和结晶性的问题。该建议利用已知表现出很大溶解性和结晶性的蛋白质类作为设计用于结合、稳定和加速膜蛋白结构测定的试剂。

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. PUBLIC HEALTH RELEVANCE: Membrane proteins represent an extraordinarily important class of proteins associated with many diseases and targeted by roughly 40% of therapeutics. Yet, despite their biomedical importance, structures of membrane proteins are relatively rare due to problems with their insolubility and crystallizability. This proposal harnesses classes of proteins known to exhibit great solubility and crystallizability as reagents designed to bind, stabilize, and expedite determination of membrane protein structures.