Development of an Automated High Throughput LCP-FRAP Assay to Guide Membrane Protein Crystallization in Lipid Mesophases.

Crystallization in lipidic mesophases (in meso) has been successfully used to obtain a number of high-resolution membrane protein structures including challenging members of the human G protein-coupled receptor (GPCR) family. Crystallogenesis in arguably the most successful mesophase, lipidic cubic phase (LCP), critically depends on the ability of protein to diffuse in the LCP matrix and to form specific protein-protein contacts to support crystal nucleation and growth. The ability of an integral membrane protein to diffuse in LCP is strongly affected by the protein aggregation state, the structural parameters of LCP, and the chemical environment. In order to satisfy both requirements of diffusion and specific interactions, one must balance multiple parameters, such as identity of LCP host lipid, composition of precipitant solution, identity of ligand, and protein modifications. Screening within such multi-dimensional crystallization space presents a significant bottleneck in obtaining initial crystal leads. To reduce this combinatorial challenge, we developed a pre-crystallization screening assay to measure the diffusion characteristics of a protein target in LCP. Utilizing the Fluorescence Recovery After Photobleaching (FRAP) technique in an automated and high throughput manner, we were able to map conditions that support adequate diffusion in LCP using a minimal amount of protein. Data collection and processing protocols were validated using two model GPCR targets: the β(2)-adrenergic receptor and the A(2A) adenosine receptor.