Detergents Destabilize the Cubic Phase of Monoolein: Implications for Membrane Protein Crystallization

Abstract

The in meso method for membrane protein crystallization uses a lipidic cubic phase as the hosting medium. The cubic phase provides a lipid bilayer into which the protein presumably reconstitutes and from which protein crystals nucleate and grow. The solutions used to spontaneously form the protein-enriched cubic phase often contain significant amounts of detergents that were employed initially to purify and to solubilize the membrane protein. By virtue of their surface activity, detergents have the potential to impact on the phase properties of the in meso system and, by extension, the outcome of the crystallization process. The purpose of this study was to quantify the effects that a popular series of nonionic detergents, the n-alkyl-beta-D-glucopyranosides, have on the phase behavior of hydrated monoolein, the lipid upon which the in meso method is based. Phase identity and phase microstructure were characterized by small-angle x-ray diffraction on samples prepared to mimic in meso crystallization conditions. Measurements were made in the 0-40 degrees C range. Samples prepared in the cooling direction allow for the expression of metastability, a feature of liquid crystalline phases that might be exploited in low-temperature crystallization. The results show that the cubic phase is relatively insensitive to small amounts of alkyl glucosides. However, at higher levels the detergents trigger a transition to the lamellar phase in a temperature- and salt concentration-dependent manner. These effects have important implications for in meso crystallization. A diffraction-based method for assaying detergents is presented.

Lipid cubic phase as a membrane mimetic for integral membrane protein enzymes

“…It may also have to do with the changing characteristics of the mesophase that come about as the concentration of decyl maltoside (DM) detergent, brought along by the protein, rises. This will tend toward a lamellar and away from the cubic mesophase at higher surfactant levels (33,34). As noted, the bulk lamellar phase is not compatible with the diffusion of water-soluble substrate and product of the type needed to support activity that can be monitored by coupled enzyme assay.…”

“…As noted, the bulk lamellar phase is not compatible with the diffusion of water-soluble substrate and product of the type needed to support activity that can be monitored by coupled enzyme assay. For reference, a loading of 10 mg DgkA/mL corresponds to a DM concentration of approximately 0.1 M in meso, which is still well below the level needed to trigger a transition in meso (33,34). Parenthetically, 10 mg∕mL is within the concentration range used for in meso crystallization trials.…”

Membrane protein crystallization in lipidic mesophases. A mechanism study using X-ray microdiffraction

“…Co-crystallization of the protein and its native lipid is shown in this illustration. As much as possible, the dimensions of the lipid (light brown oval with tail), detergent (pink oval with tail), native membrane lipid (purple oval with tails), protein (blue; outer membrane Vitamin B 12 transporter, BtuB; PDB code 1NQE), bilayer and aqueous channels (purple) have been drawn to scale. The lipid bilayer is approximately 40 Å thick.…”

Ice breaking in GPCR structural biology

“…Several groups have tried to build a computational model, but they had little success [78,79] . The reason for failure might be that there are several components in the crystallization trials, quite a few of which, such as salt, PEGs and trace amounts of detergent, could alter the dimensions or even the phase behav-www.chinaphar.com Zhao Q et al Acta Pharmacologica Sinica npg ior of the whole system [80,81] (Figure 4D).…”