On the crystallization of membrane proteins in lipidic sponge and cubic phases

Abstract: Membrane proteins are involved in many important biological processes and in order to understand their mechanism, their three-dimensional structures need to be elucidated to high resolution by for example X-ray crystallography. However, there is only limited structural knowledge for membrane proteins which is partly explained by the difficulties in obtaining well-diffracting crystals. To facilitate production of such crystals, cubic phases of monoolein (MO) can be used, but the mechanism behind this process is still unclear and requires further exploration. In this thesis, an anionic lipid, distearoyl phosphatidyl glycerol (DSPG) was used to enlarge the water pores in the cubic phase since there were indications that the water pore diameter is crucial for inclusion and diffusion of membrane proteins. The phase behaviour was also investigated in the MO-DSPG-water system with small amounts of the membrane protein, bacteriorhodopsin (bR), and detergent, octyl glycoside (OG). It was shown that both bR and OG stabilized the lamellar phase in favour for the cubic phase. The phase behaviour was characterized by visual inspection and small-angle X-ray scattering (SAXS). It was also demonstrated that bR and another membrane protein, reaction centre from Rhodopseudomonas viridis (RCvir), could be crystallized from the swelled MO-DSPG cubic phase. A new crystallization method that makes use of a liquid analogue of the cubic phase, the sponge phase, is also presented in this thesis. The sponge phase facilitates a considerable increase in the allowed size of membrane proteins aqueous domains and is easily setup using a conventional vapour diffusion crystallization experiment. Crystals of the reaction centre from Rhodobacter sphaeroides (RCsph) and RCvir were obtained by this method and the appearance of the sponge phase was confirmed by visual inspection, SAXS and NMR spectroscopy. MO is susceptible to (ester) hydrolysis and the cubic phase of MO proved to be unstable at high pH. Therefore, the possibility to use ester-free lipids, phytantriol (PT) and selachyl alcohol was investigated. Detailed phase diagrams were made with solvents known to create sponge phases in the MO-water system and it was shown that PT and selachyl alcohol could be good alternatives for MO in applications were extreme pH is of importance.

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