A novel in meso crystallisation method has facilitated the structural determination of several biologically relevant integral membrane proteins (IMPs).[1] However, the method remains poorly understood as IMPs are difficult to express and handle. Analogous to solution based crystallisation, in meso crystallisation requires extensive screening of precipitant conditions.[2] Bicontinuous cubic mesophases are the most commonly used lipid phases for in meso crystallisation. The self-assembled lipid structures of the three different experimentally observed cubic phases are shown schematically in Figure 1. The effect of the IMPs on the cubic phase and the compatibility of the crystallisation screen used with the cubic phase are important; if the underlying 3-D cubic nanostructure is destroyed, the screen[3] or protein / lipid combination may not be suitable for in meso crystallisation experiments. The secondary structure of IMPs might also be altered upon incorporation into cubic phases with different bilayer thicknesses formed by different lipids. This effect needs to be investigated in order to confirm that the active form of the proteins is used for structure determination.

We looked at the impact of IMPs on the cubic mesophases formed by three different lipids: monoolein, monopalmitolein and phytantriol using high-throughput synchrotron Small-Angle X-ray Scattering (SAXS). We also studied the secondary structure of the IMPs in the cubic mesophases with different bilayer thicknesses using Circular Dichroism Spectroscopy (CD). An improved understanding of the effect of IMPs on the cubic mesophase and of the effect of lipid bilayer thickness on IMP secondary structure can lead to an improved understanding of in meso crystallisation success rates and facilitate the structural determination of more IMPs.

Figure 1. Schematic presentation of self-assembled lipid structures, (a) Primitive cubic phase, (b) Diamond cubic phase, (c) Gyroid cubic phase.[3]