274230 A Microfluidic Platform for In Meso Crystallization and In Situ Crystal X-Ray Diffraction of Membrane Proteins

Monday, October 29, 2012: 9:20 AM
Allegheny III (Westin )
Daria S. Khvostichenko, Jeremy M. Schieferstein, Ashtamurthy Pawate and Paul J.A. Kenis, Department of Chemical & Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL

Membrane proteins are key actors in a number of biological processes, which is highlighted by the fact that they account for ~60% of all available drug targets. To better understand functional mechanisms, high-resolution crystal structures of proteins are required. The success rate of membrane protein crystallization trials, however, is notoriously low, and growing diffraction-quality crystals of membrane proteins is a major bottleneck in structural biology.

In meso crystallization exploits properties of certain lipids that self-assemble into continuous bilayer systems, known as lipidic mesophases, when mixed with water or aqueous solutions. During in meso crystallization trials membrane protein molecules are incorporated into the bilayers of the mesophase and are thus maintained in a native-like environment. In spite of a number of recent impressive successes, the in meso method is not used widely in the structural biology community, presumably due to extra steps required in the crystallization protocol compared to traditional crystallization from solutions, and due to the difficulties of handling highly viscous lipidic mesophases. Similarly, harvesting fragile protein crystals from gel-like lipidic mesophases as necessary for crystal X-ray diffraction data collection is challenging and may compromise the quality of crystals.

We present a microfluidic platform for membrane protein crystallization in lipidic mesophases and in situ X-ray analysis. The multi-well platform requires 60 nL of the protein solution and 25 nL of lipid per well, with a potential for further reduction in the sample amount, and automates simultaneous formulation of multiple samples. The platform design is optimized for diffusional mixing of the protein solution with the lipid to formulate protein-loaded mesophases. For fluid metering and routing the platform relies on the multilayer polydimethylsiloxane (PDMS) architecture with monolithic pneumatic valves. For X-ray transparency thin layers of PDMS patterned with features for metering and routing are sandwiched between layers of cyclic olefin copolymer (COC), which provides rigidity and servers as an evaporation barrier. We validate our approach by crystallizing membrane protein photosynthetic reaction center (R. Sphaeroides) and collecting protein crystal X-ray diffraction data on-chip.


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