Membrane Protein Co- Crystallization with Highly Crystalline and Soluble Proteins

描述（由申请人提供）：膜蛋白会混淆任何旨在解决其结构的异常英勇的尝试。传统的膜蛋白过表达、纯化和结晶方法通常由于不溶性和折叠问题而失败。该项目利用大量的可溶性和高结晶性蛋白库来确定膜蛋白的结合伙伴。这些文库中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。与结合伙伴的共表达可以帮助避免膜蛋白聚集，并允许蛋白质折叠发生。结合伙伴亲和层析的目的是协助膜蛋白纯化，共结晶的目的是减缓晶体形成过程中蛋白质的聚集和沉淀。第一个具体目标

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.