A lipidic-sponge phase screen for membrane protein crystallization.

A major current deficit in structural biology is the lack of high-resolution structures of eukaryotic membrane proteins, many of which are key drug targets for the treatment of disease. Numerous eukaryotic membrane proteins require specific lipids for their stability and activity, and efforts to crystallize and solve the structures of membrane proteins that do not address the issue of lipids frequently end in failure rather than success. To help address this problem, we have developed a sparse matrix crystallization screen consisting of 48 lipidic-sponge phase conditions. Sponge phases form liquid lipid bilayer environments which are suitable for conventional hanging- and sitting-drop crystallization experiments. Using the sponge phase screen, we obtained crystals of several different membrane proteins from bacterial and eukaryotic sources. We also demonstrate how the screen may be manipulated by incorporating specific lipids such as cholesterol; this modification led to crystals being recovered from a bacterial photosynthetic core complex.

Aqueous self-assembly of phytantriol in ternary systems: effect of monoolein, distearoylphosphatidylglycerol and three water-miscible solvents.

The aqueous phase behavior of phytantriol (PT) in mixtures of monoolein (MO), distearoylphosphatidylglycerol (DSPG), propylene glycol (PG), polyethylene glycol 400 (PEG 400) and 2-methyl-2,4-pentanediol (MPD) was investigated by visual inspection, polarized light microscopy and small angle X-ray diffraction at room temperature. The phase diagrams of PT and MO in water are qualitatively very similar and PT/MO mixtures in excess water form one cubic phase of space group Pn3m irrespective of mixing ratio. The addition of the charged membrane lipid DSPG to the PT system gives rise to a considerable water swelling of the cubic phases as well as the occurrence of a cubic phase of space group Im3m. Whereas all three solvents studied give rise to a sponge (L3) phase in the MO-water system, this phase was only found when MPD was added to the PT-water system. The results are discussed with respect to the chemical differences between PT and MO.

Cubic, sponge, and lamellar phases in the glyceryl monooleyl ether-propylene glycol-water system.

The phase behavior of 1-glyceryl monooleyl ether (GME) in mixtures of propylene glycol (PG) and water was investigated by visual inspection, polarization microscopy, small-angle X-ray diffraction, and conductance measurements. A phase diagram, based on over 200 samples of the ternary system GME-PG-water, was constructed at 20 degrees C. Without PG, GME forms a reverse micellar phase with up to 10 wt % water and a reverse hexagonal liquid-crystalline phase between 10 and 25 wt % water, a phase that can coexist with excess water. If PG is added in amounts exceeding about 10 wt %, then cubic and lamellar liquid-crystalline phases start to form. A cubic phase, belonging to space group Pn3m, can coexist with excess PG-water mixtures. If even more PG is added, then the cubic phase is transformed into a sponge phase. A lamellar phase forms at water contents between 10 and 15 wt % and with widely differing PG/GME weight ratios. We postulate that the phase behavior is caused by the fact that PG makes the interfacial region between self-assembled GME and PG-water less negatively curved, which in turn allows for the formation of the new phases. The phase behavior obtained for the GME system shows a striking similarity with the phase behavior of the corresponding system in which the GME has been replaced by the ester, 1-glycerol monooleate (GMO), differing only in one extra carbonyl oxygen. The major difference is the lower amount of water present in the GME phases, an effect that is mainly due to the more hydrophobic character of GME compared to that of GMO.