Microfluidic Platforms for the Study of in Meso Membrane Protein Crystallization

Microfluidic technology originally gained prominence because of the ability to work at very small volumes with precise control.  Research in this field has advanced to the point where an idea of a complete “lab on a chip” is a realistic goal, even to the extent of performing clinical and diagnostic tests.  Microfluidic approaches also enable the efficient handling of a large number of individual trials simultaneously.  In the field of crystallization and structural biology this kind of high throughput screening capability is critical to identify conditions at which biological macromolecules will crystallize so their structure can be determined. 

Membrane proteins play a critical role in a variety of biological processes including signaling and material or energy transduction.  As such they are prime targets for pharmacological treatments. Knowledge of their structure and function would be of tremendous fundamental and medical benefit.  Despite their importance, the precise structure of a disproportionate number of membrane proteins is still unknown.  One of the key bottlenecks in this process is creating and maintaining membrane protein functionality, not only during protein expression and purification, but also during crystallization studies for structural characterization.  To counter this difficulty the in meso (lipidic cubic phase, LCP) method was developed, allowing membrane proteins to remain in a membranous environment during crystallization [1].  This method has had marked successes with membrane proteins such as bacteriorhodopsin that were not successfully crystallized by traditional methods. More recently, the crystallization and structure determination of two human G protein-coupled receptors has been reported [2,3].   Despite its successes, the biggest challenge of this method is the preparation of the highly viscous lipid mesophase used as the crystallization medium, at sub-microliter volumes to enable screening of many potential crystallization conditions. 

In my graduate research I have developed a method for preparing in meso crystallization trials at the 20 nL level using multilayer microfluidic technology in polydimethylsiloxane (PDMS).  Within these chips, complex patterns of fluid flow for mixing can be achieved by pneumatic actuation of integrated valves and pumps.  A novel multi-chamber design enables the mixing of highly viscous lipids with an inviscid aqueous protein solution to prepare the lipid mesophase needed for in meso crystallization trials.  Arrays of these mixing and crystallization elements enable the screening of a wide range of crystallization conditions.  We demonstrated feasibility with the successful on-chip crystallization of the previously characterized membrane proteins bacteriorhodopsin [4] and photosynthetic reaction center.  Presently we are extending application of these microfluidic in meso crystallization chips to novel proteins for which no structure is known.

A secondary challenge in the structure determination of proteins is the harvesting and mounting of fragile and potentially tiny crystals, a task that is exacerbated when trying to harvest a crystal from a tiny microfluidic compartment.  The ability to perform in situ X-ray analysis of crystals grown on-chip would circumvent these issues.  To accomplish this, we created an X-ray transparent, PDMS / polyimide hybrid device architecture that retains the ability to perform complex fluid handling while allowing for on-chip X-ray analysis.   Testing of these all-integrated on-chip crystallization and in situ X-ray analysis platforms is in progress.

With these microfluidic platforms now in hand, I am also pursuing studies of the phase behavior of novel lipid/water systems for use with in meso crystallization trials as well as mechanistic studies of how in meso crystallization occurs.  All of this work together has the potential to develop the scientific understanding of in meso protein crystallization to the point where some level of intelligent design could be incorporated into future crystallization trials, leading to improved rates of success.

References:

[1]  Landau, E. M.; Rosenbusch, J. P., P Natl Acad Sci USA 1996, 93, 14532-14535.

[2]  Cherezov, V.; Rosenbaum, D. M.; Hanson, M. A.; Rasmussen, S. G. F.; Thian, F. S.; Kobilka, T. S.; Choi, H. J.; Kuhn, P.; Weis, W. I.; Kobilka, B. K.; Stevens, R. C., Science 2007, 318, 1258-1265.

[3]  Jaakola, V. P.; Griffith, M. T.; Hanson, M. A.; Cherezov, V.; Chien, E. Y. T.; Lane, J. R.; IJzerman, A. P.; Stevens, R. C., Science 2008, 322, 1211-1217.

[4]  Perry, S. L.; Roberts, G. W.; Tice, J. D.; Gennis, R. B.; Kenis, P. J. A., Cryst Growth Des 2009 (in press, DOI: 10.1021/cg900289d).