Fluorescence Recovery After Photobleaching in Lipidic Cubic Phase (LCP-FRAP): A Precrystallization Assay for Membrane Proteins.

Abstract

Crystallization of integral membrane proteins (MPs) is notoriously difficult, given their poor stability outside native membrane environment and due to the interference of detergent micelles with crystallization process. MP crystallization in a membrane mimetic matrix, known as lipidic cubic phase (LCP), has recently started to gain popularity, following successes in structure determination of G protein-coupled receptors (GPCRs), transporters, and enzymes. Unlike crystallization trials in aqueous solutions where protein molecules are free to move, diffusion of MPs in LCP is restricted, and, thus, a high level of protein mobility can serve as an early indication for subsequent crystallization success. Prompted by our initial observations that precipitant conditions can dramatically affect diffusion of GPCRs in LCP, we have developed a simple precrystallization assay, based on measuring protein diffusion at a number of different conditions by fluorescence recovery after photobleaching (LCP-FRAP). Over the last few years, the LCP-FRAP assay was incorporated in our GPCR structure determination pipeline and proved as a powerful technique allowing for a faster identification of crystallization conditions for many different receptors. The assay is used to screen for the best protein constructs, ligands, LCP host lipids, precipitants, and additives, thereby focusing subsequent crystallization trials on the most promising parts of the multidimensional crystallization phase diagram, substantially increasing the likelihood of finding the right crystallization condition. Here, we describe our LCP-FRAP protocols for guiding GPCR crystallization, which can be adapted to any other MP, and discuss some of the critical considerations related to application of this assay.