For the optimization of membrane protein crystal growth, it can make a huge difference if the 'proper' lipids are present in the crystallization experiment.
How do you add lipids to a crystallization trial? Lipids don't readily dissolve in water. There are several ways to get these amphipathic molecules to participate in the crystal formation process. When dealing with a standard vapor diffusion crystallization optimization, where the membrane protein detergent complex is combined with a precipitant solution, the lipid can be added in detergent micellar form. Better yet use less detergent and prepare a lipid film inside a glass container that is then solubilized by the detergent that is present in the solublized membrane protein sample.
This makes it very simple to change the lipid composition in every single experiment. A systematic approach to study the effect of lipids at comparably high concentrations has recently been described as HiLiDe:
Gourdon, P., Andersen, J., Hein, K., Bublitz, M., Pedersen, B., Liu, X., Yatime, L., Nyblom, M., Nielsen, T., Olesen, C., Møller, J., Nissen, P., & Morth, J. (2011). HiLiDe—Systematic Approach to Membrane Protein Crystallization in Lipid and Detergent Crystal Growth & Design, 11 (6), 2098-2106 DOI: 10.1021/cg101360d
The described re-lipidation is reminiscent of reconstitution of membrane proteins into bilayer membranes, while avoiding the second detergent removal step. I talked to the lead author Pontus earlier this year at the Keystone Conference and he explained to me that he thinks the lipid/detergent mixtures at high concentrations may form a generic crystallization environment for membrane proteins, somewhat reminiscent of lipidic cubic phases or sponge phases. The results (crystallization of rKv1.2-beta2, E.coli Complex I and T.thermophilus Complex I and similar, previously reported crystallizations with high lipid composition such as pea LHC-II, bovine rhodopsin, bovine Cyt bc1, SERCA1a, pig Na/K-ATPase, Na/K-ATPase) speak for themselves.
On the other hand, if the crystallization is carried out with the use of lipids that can spontaneously form a range of lipidic materials such as sponge or lipidic cubic phases, additional lipids can be added to the matrix lipid for crystal growth optimization. Let's say for instance, the matrix lipid is monoolein (good choice, by the way). This lipid has a melting temperature of 37C and in its liquid form can dissolve other membrane components, such as Cholesterol. How can this be done practically? To test three different Cholesterol concentrations, one can prepare a 20% Cholesterol in Monoolein mix by melting (i.e. 80 mg) Monoolein to 40C and dissolving the dry Cholesterol (20 mg) in it, obtaining a 20% (w/w) mixture. Combining this mixture with neat liquid Monoolein at a 50/50 ratio, one would obtain a 10% Cholesterol content (etc.). Fortunately, Monoolein and lipid mixtures with Monoolein can be supercooled (i.e. remain liquid at room temperature for many minutes), allowing simple manipulation with pipettors or syringes prior to mixing with the membrane protein to form a lipidic cubic phase (or other lipidic materials).
In case you're still not convinced about the utility of amphiphilic compounds in membrane protein crystallization, a good case is made here:
NOLLERT, P. (2005). Membrane protein crystallization in amphiphile phases: practical and theoretical considerations Progress in Biophysics and Molecular Biology, 88 (3), 339-357 DOI: 10.1016/j.pbiomolbio.2004.07.006
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Regards,
Peter