Membrane Protein Co- Crystallization with Highly Crystalline and Soluble Proteins

描述(由申请人提供)：膜蛋白使任何旨在解决其结构的异常英勇的尝试都感到困惑。传统的膜蛋白过度表达、纯化和结晶的方法通常会因为不溶性和折叠的问题而失败。该项目利用大量的可溶性和高结晶性蛋白质文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的共表达、亲和纯化和共结晶提供亲和剂。与结合伙伴共表达有助于避免膜蛋白聚集，并允许蛋白质折叠发生。亲和层析的目的是辅助膜蛋白的纯化，而共结晶的目的是减缓蛋白质在晶体形成过程中的聚集和沉淀。第一个具体目标 重点是设计和构建噬菌体展示蛋白文库，用于与膜蛋白的高亲和力结合。战略性地选择蛋白质用于文库形成，例如高度结晶的蛋白质溶菌酶和特殊可溶性的S晶体蛋白，用于噬菌体展示将有助于确保项目的成功；其他专门为Forg-蛋白质偶联受体(GPCR)量身定做的文库包括G-蛋白质和GPCR配体的变体。为了获得高亲和力结合、热稳定性、溶解性等性质，第二个具体目标 以选择和屏幕的流动路径为特色。第三个具体目的是将噬菌体展示的亲和试剂应用于膜蛋白的生产和结晶。通过结合并冻结膜蛋白的特定构象，亲和试剂可以为膜蛋白的结构生物学和其他结构功能研究提供强大的工具。总之，这项提议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。

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.