Membrane Protein Co- Crystallization with Highly Crystalline and Soluble Proteins

University Of California-Irvine, Irvine CA

Investigators

Linked publications & trials

Abstract

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.