(335h) Microfluidic Platforms for Screening Membrane Protein Crystallization Conditions
Membrane proteins play a crucial role in many pivotal biological processes such as energy and material transduction across cellular membranes, molecular recognition, immune response and interaction between intra and extra cellular environment. Their malfunction has been linked to various diseases and therefore they are important pharmacological targets. Membrane proteins have proven to be extremely difficult to crystallize due to their amphiphilicity and tendency to denature and unfold when moved out of their native environment.
The in meso method for crystallization uses lipids to maintain the membrane proteins in a more native environment. The membrane proteins are reconstituted into a cubic mesophase of bicontinuous water channels and highly curved lipid bilayers. Crystallogenesis can occur upon addition of a precipitant solution, which is proposed to induce a phase change that drives crystal growth. This method has proved successful in growing crystals of membrane proteins such as bacteriorhodopsin where traditional methods have failed. The recent success in obtaining high quality crystals of two G Protein Coupled Receptors has highlighted the potential of this method [1, 2]. The in meso method involves mixing of lipid which is thirty times more viscous than water with an inviscid protein solution to form a mixture that is nearly five orders of magnitude more viscous than water. The microfluidic platforms allows variation of screening conditions over a much wider range as compared to traditional syringe based methods which allow only a single concentration of the protein solution to be mixed
Prediction of crystallization conditions a priori is currently not possible. Hence, intensive screening of various salts and precipitants is necessary to determine successful crystallization conditions. We report here the design, fabrication and testing of a microfluidic chip which enables screening for crystallization conditions of membrane proteins . We screen for crystallization conditions of two model systems, bacteriorhodopsin and photosynthetic reaction center, using our microfluidic chips and compare the outcomes with those obtained with traditional crystallization plates. The crystallization of proteins of unknown structure, specifically members of the heme copper oxidase family, is presently ongoing. Our focus on this superfamily stems from the fact that specific members of a subfamily of these proteins are present in many pathogenic bacteria, such as Helicobacter pylori, the organism responsible for ulcers, and hence are potential drug targets.
1. Cherezov V., Rosenbaum D.M., Hanson M.A., Rasmussen S.G., Thian F.S., Kobilka T.S., Choi H.J., Kuhn P., Weis W.I., Kobilka B.K. and Stevens R.C., 2007, Science, 318, 1258?1265
2. Jaakola V.P., Griffith M.T., Hanson M.A., Cherezov V., Chien E.Y.T., Lane J.R., Ijzerman A.P. and Stevens R.C., 2008, Science, 322, 1211?1217
3. Perry S.L., Roberts G.W., Tice J.D., Gennis R.B. and Kenis P.J.A., 2009, Crystal Growth and Design (in press, DOI: 10.1021/cg900289d)