291394 Protein Structure Determination Using X-RAY Compatible Microfluidic Platforms

Tuesday, October 30, 2012
Hall B (Convention Center )
Sudipto Guha, Ashtamurthy Pawate, Sarah L. Perry and Paul J.A. Kenis, Department of Chemical & Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL

Microfluidics has found extensive use in fields as diverse as energy conversion, medical and clinical diagnostics, point-of-care medical devices, proteomics and protein crystallization. Microfluidics allows for ease of sample preparation, small sample sizes, ease of scale-up, as well as integration of various sample analysis techniques on a single platform1,2.

Here, we focus on the application of microfluidic platforms for membrane protein crystallization. Membrane proteins (MPs) play a crucial role in many important biological processes including energy and material transduction across cellular membranes, molecular recognition and immune response. They are amphiphilic, structurally diverse, and extremely responsive to the surrounding environment. Efforts to understand the structure-function aspects of these proteins through X-ray crystallography have been hampered by difficulty in obtaining high quality crystals3. Identifying the optimal crystallization condition(s) requires systematic screening of a huge chemical space (salts, buffers and precipitants), which requires a lot of protein sample that is generally not available. Additionally, current methods of structure determination are not optimized for handling small and fragile crystals that MPs tend to form.

We present an array-based, X-ray compatible microfluidic platform comprised of cyclic olefin copolymer (COC) and a thin polydimethylsiloxane (PDMS) membrane needed for valve actuation. The array-based design allows for screening for upto 96 conditions using < 6μL of protein solution. After screening, a similar chip is used for structure determination of the protein by merging data from multiple crystals grown on-chip. The use of X-ray compatible materials allows on-chip analysis, thus preventing damage to fragile protein crystals that might occur during manual harvesting and mounting of crystals. As proof of concept, we have screened crystallization conditions for a soluble protein, phosphonoacetate hydrolase(PhnA). After obtaining the optimal condition and using anomalous data collected solely on-chip, wedetermined its structure to a resolution of 1.99 Å.

We are now in the process of validating our platform by screening and solving the structure of two MPs - photosynthetic reaction center (RC) and cytochrome ba3-type oxidase. We have collected data on RC to 3.0 Å and are currently optimizing the crystallization conditions and chip design to get higher quality data.

(1) Hansen, C.; Quake, S. R. Current Opinion in Structural Biology 2003, 13, 538-544.

(2) Li, L.; Ismagilov, R. F. Annual Review of Biophysics 2010, 39, 139-158.

(3) Stevens, R. C. Current Opinion in Structural Biology 2000, 10, 558-563.

Extended Abstract: File Not Uploaded