Mechanisms of Membrane Protein Crystallization

In spite of the importance of membrane proteins (MPs) in many essential biochemical pathways, there are limited high-resolution structures of this class of proteins in the Protein Data Bank. This is mostly due to the difficulties in obtaining well-diffracting crystals, as crystallization phenomena are not well-understood and crystals are obtained largely by trial-and-error. Our approach for improving in surfo MP crystallization is focused on the critical micelle concentration (CMC) of surfactant monomers and their interaction with common precipitating agents, including poly(ethylene) glycol (PEG) and 2-methyl-2,4-pentanediol (MPD). The phenomena were investigated using two model MPs - reaction center from Rhodobacter sphaeroides and outer membrane protein X expressed in Escherichia coli. Protein-detergent complexes (PDCs) of these proteins were studied at molecular and nanoscopic length scales using multiple techniques, including NMR, isothermal titration calorimetry (ITC) and small-angle scattering (i.e., SANS and SAXS) under crystallization conditions. We found that surfactant microstructure changes significantly in the presence of precipitating agents, and the CMC of surfactant monomers increases due to the favorable interaction with precipitants and additives. ITC dilution measurements showed an increase in the CMC over a broad range of crystallization conditions reported in the literature, indicating that precipitants act as co-solvents of monomeric surfactants. Additionally, NMR and scattering results suggest the formation of a complex between precipitant and surfactant molecules and shrinkage in the detergent micelle dimensions, respectively. Our crystallization trials with model MPs indicate that optimal crystallization conditions were closely correlated to the detergent concentrations at or slightly below the measured detergent CMC in a protein-free environment; excess surfactant conditions (> CMC) tend to inhibit crystal formation, while lower surfactant conditions (< CMC) promote aggregation. We propose a rational design of crystallization trials can be made based on a priori knowledge of surfactant CMCs under crystallization conditions.