Core facility infrastructure for optimizing conditions for lipidic cubic phase-based membrane protein crystallization

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Membrane proteins perform a variety of biological functions that are critical to the survival of bacteria, plants, and eukaryotes. Crystal structures of these membrane proteins have shed enormous insights into the functions of transporters, signal transduction receptors, and enzymes. However, crystallization of membrane proteins remains a major bottleneck for structure determination. The use of lipid mesophases, such as lipid cubic phase (LCP), has been highly successful in obtaining high-resolution crystal structures. One of the major factors for successful membrane protein crystallization in LCP is the ability of the protein to diffuse within the lipid bilayer. The diffusion rate of the protein is influenced by protein aggregation, LCP composition, and environment, and can be measured by fluorescence recovery after photobleaching (FRAP). FRAP is able to measure the amount of time required for the fluorescence intensity of a tagged protein to recover within the LCP drop after photobleaching. FRAP allows immediate screening of crystallization conditions, without having to wait weeks to determine whether the crystallization conditions are amenable for crystal growth. The Formulatrix FRAP is a standalone imager capable of high-throughput FRAP to bleach and image a 96-well plate in under 50 minutes. This system will allow rapid screening of LCP lipid components, precipitant composition, ligand and protein modifications. The Formulatrix FRAP system is not available anywhere in Canada and will become an important resource for the entire structural biology community in Toronto. Moreover, the Formulatrix FRAP will expand the current capabilities of an integrated, multi-department UToronto Membrane Protein Crystallization Facility directed by Profs. Jeffrey Lee and Trevor Moraes.