(5bv) 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).