(303d) Microfluidic Platforms for in Meso Membrane Protein Crystallization

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 therapies and
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 method was highlighted with the
crystallization and structure determination of two human G protein-coupled
receptors [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. 

We 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.  The ability to efficiently set up and analyze a
large number of crystallization trials while minimizing external influences,
such as those from sample manipulation and crystal harvesting has the potential
to clarify the science of macromolecular crystallization such that 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).