Membrane Protein Crystallization In Meso: Lipid Type-Tailoring of the Cubic Phase

Abstract

Hydrated monoolein forms the cubic-Pn3m mesophase that has been used for in meso crystallization of membrane proteins. The crystals have subsequently provided high-resolution structures by crystallographic means. It is possible that the hosting cubic phase created by monoolein alone, which itself is not a common membrane component, will limit the range of membrane proteins crystallizable by the in meso method. With a view to expanding the range of applicability of the method, we investigated by x-ray diffraction the degree to which the reference cubic-Pn3m phase formed by hydrated monoolein could be modified by other lipid types. These included phosphatidylcholine (PC), phosphatidylethanolamine, phosphatidylserine, cardiolipin, lyso-PC, a polyethylene glycol-lipid, 2-monoolein, oleamide, and cholesterol. The results show that all nine lipids were accommodated in the cubic phase to some extent without altering phase identity. The positional isomer, 2-monoolein, was tolerated to the highest level. The least well tolerated were the anionic lipids, followed by lyso-PC. The others were accommodated to the extent of 20-25 mol %. Beyond a certain concentration limit, the lipid additives either triggered one or a series of phase transitions or saturated the phase and separated out as crystals, as seen with oleamide and cholesterol. The series of phases observed and their order of appearance were consistent with expectations in terms of interfacial curvature changes. The changes in phase type and microstructure have been rationalized on the basis of lipid molecular shape, interfacial curvature, and chain packing energy. The data should prove useful in the rational design of cubic phase crystallization matrices with different lipid profiles that match the needs of a greater range of membrane proteins.

LCP-FRAP Assay for Pre-Screening Membrane Proteins for In Meso Crystallization

“…The effect of 10% cholesterol on the lattice parameter of the fully hydrated monoolein cubic Pn3m phase is negligible. 40 Nevertheless, cholesterol may modulate the response of the cubic phase lattice to addition of different ingredients from the crystallization cocktails. Diffusion of β 2 AR(E122W)-T4L in cholesterol-doped LCP at crystallization conditions was on average ~2 times slower in agreement with the rigidifying effect of cholesterol.…”

Crystallizing Membrane Proteins for Structure-Function Studies Using Lipidic Mesophases

“…Our findings indicate that the default cubic mesophase is remarkably resilient and retains its phase identity in the presence of a vast array of different additives. These include glycerolipids, cholesterol, free fatty acids, detergents, denaturants, glycerol and thiol-group-specific reagents [22][23][24][25][26][27][28][29][30]. Of course, for each there is a concentration beyond which the cubic phase is no longer stable nor, indeed, useful for crystallogenesis.…”

“…An alternative was to use monoolein as the hosting lipid and to augment it with typical membrane lipids, thereby creating a more native-like environment. Accordingly, the carrying capacity of the monoolein cubic phase for a number of different lipids was established using SAXS [23]. This amounted to approximately 20 mol% in the case of phosphatidylcholine, phosphadidylethanolamine and cholesterol, with lesser amounts of phosphatidylserine and cardiolipin being accommodated.…”

“…The sponge phase [24], with its open aqueous channels and flatter bilayer, should prove particularly useful in this regard. Using it in combination with novel hosting and additive lipid screens [23,42] will go a long way towards producing crystals and ultimately highresolution structures where interactions that are integral to human health are revealed. A cartoon representation of the various phase states is included in which coloured zones represent water.…”

Interactions of Cyclic AMP and Its Dibutyryl Analogue with a Lipid Layer in the Aqueous Mixtures of Monoolein Preparation and Dioleoyl Phosphatidylcholine as Probed by X-Ray Diffraction and Raman Spectroscopy

“…In more recent investigations of the aqueous 1-monooleoylglycerol/DOPC mixture [12,13] it has been demonstrated that the fully hydrated (≥ 60 wt.% of water) Pn3m phase of 1-monooleoylglycerol can accommodate up to 25 mol% of DOPC. However, the spectroscopic investigation of these MO/DOPC samples is complicated since two separate phases, aqueous and lipidic, coexist at the excess water condition.…”