Host Lipid and Temperature as Important Screening Variables for Crystallizing Integral Membrane Proteins in Lipidic Mesophases. Trials with Diacylglycerol Kinase

Abstract

A systematic study of the crystallization of an α-helical, integral membrane enzyme, diacylglycerol kinase, DgkA, using the lipidic cubic mesophase or in meso method is described. These trials have resulted in the production of blocky, rhombohedron-shaped crystals of diffraction quality currently in use for structure determination. Dramatic improvements in crystal quality were obtained when the identity of the lipid used to form the mesophase bilayer into which the protein was reconstituted as a prelude to crystallogenesis was varied. These monoacylglycerol lipids incorporated fatty acyl chains ranging from 14 to 18 carbon atoms long with cis olefinic bonds located toward the middle of the chain. Best crystals were obtained with a lipid that had an acyl chain 15 carbon atoms long with the double bond between carbons 7 and 8. It is speculated that the effectiveness of this lipid derives from hydrophobic mismatch between the target integral membrane protein and the bilayer of the host mesophase. Low temperature (4 °C) worked in concert with the short chain lipid to provide high quality crystals. Recommended screening strategies for crystallizing membrane proteins that include host lipid type and low temperature are made on the basis of this and related in meso crystallization trials.