X-ray transparent microfluidic chips for high-throughput screening and optimization of in meso membrane protein crystallization
Abstract
Elucidating and clarifying the function of membrane proteins ultimately requires atomic resolution structures as determined most commonly by X-ray crystallography. Many high impact membrane protein structures have resulted from advanced techniques such as in meso crystallization that present technical difficulties for the set-up and scale-out of high-throughput crystallization experiments. In prior work, we designed a novel, low-throughput X-ray transparent microfluidic device that automated the mixing of protein and lipid by diffusion for in meso crystallization trials. Here, in this work, we report X-ray transparent microfluidic devices for high-throughput crystallization screening and optimization that overcome the limitations of scale and demonstrate their application to the crystallization of several membrane proteins. Two complementary chips are presented: (1) a high-throughput screening chip to test 192 crystallization conditions in parallel using as little as 8 nl of membrane protein per well and (2) a crystallization optimization chip to rapidly optimize preliminary crystallization hits through fine-gradient re-screening. We screened three membrane proteins for new in meso crystallization conditions, identifying several preliminary hits that we tested for X-ray diffraction quality. Further, we identified and optimized the crystallization condition for a photosynthetic reaction center mutant and solved its structure to a resolution of 3.5 Å.
- Authors:
-
[1];
[1]
- Univ. of Illinois at Urbana-Champaign, IL (United States)
- Univ. of Toronto, ON (Canada)
- Publication Date:
- Research Org.:
- Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
- Sponsoring Org.:
- USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities Division; National Institutes of Health (NIH); German Research Foundation (DFG); Canada Excellence Research Chairs (CERC)
- OSTI Identifier:
- 1355039
- Alternate Identifier(s):
- OSTI ID: 1361839
- Grant/Contract Number:
- AC02-06CH11357; R01 GM086727; ACB-12002; AGM-12006; 1S10OD012289-01A1; BR 5124/1-1
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Biomicrofluidics
- Additional Journal Information:
- Journal Volume: 11; Journal Issue: 2; Journal ID: ISSN 1932-1058
- Publisher:
- American Institute of Physics (AIP)
- Country of Publication:
- United States
- Language:
- ENGLISH
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
Citation Formats
Schieferstein, Jeremy M., Pawate, Ashtamurthy S., Sun, Chang, Wan, Frank, Sheraden, Paige N., Broecker, Jana, Ernst, Oliver P., Gennis, Robert B., and Kenis, Paul J. A. X-ray transparent microfluidic chips for high-throughput screening and optimization of in meso membrane protein crystallization. United States: N. p., 2017.
Web. doi:10.1063/1.4981818.
Schieferstein, Jeremy M., Pawate, Ashtamurthy S., Sun, Chang, Wan, Frank, Sheraden, Paige N., Broecker, Jana, Ernst, Oliver P., Gennis, Robert B., & Kenis, Paul J. A. X-ray transparent microfluidic chips for high-throughput screening and optimization of in meso membrane protein crystallization. United States. https://doi.org/10.1063/1.4981818
Schieferstein, Jeremy M., Pawate, Ashtamurthy S., Sun, Chang, Wan, Frank, Sheraden, Paige N., Broecker, Jana, Ernst, Oliver P., Gennis, Robert B., and Kenis, Paul J. A. Mon .
"X-ray transparent microfluidic chips for high-throughput screening and optimization of in meso membrane protein crystallization". United States. https://doi.org/10.1063/1.4981818. https://www.osti.gov/servlets/purl/1355039.
@article{osti_1355039,
title = {X-ray transparent microfluidic chips for high-throughput screening and optimization of in meso membrane protein crystallization},
author = {Schieferstein, Jeremy M. and Pawate, Ashtamurthy S. and Sun, Chang and Wan, Frank and Sheraden, Paige N. and Broecker, Jana and Ernst, Oliver P. and Gennis, Robert B. and Kenis, Paul J. A.},
abstractNote = {Elucidating and clarifying the function of membrane proteins ultimately requires atomic resolution structures as determined most commonly by X-ray crystallography. Many high impact membrane protein structures have resulted from advanced techniques such as in meso crystallization that present technical difficulties for the set-up and scale-out of high-throughput crystallization experiments. In prior work, we designed a novel, low-throughput X-ray transparent microfluidic device that automated the mixing of protein and lipid by diffusion for in meso crystallization trials. Here, in this work, we report X-ray transparent microfluidic devices for high-throughput crystallization screening and optimization that overcome the limitations of scale and demonstrate their application to the crystallization of several membrane proteins. Two complementary chips are presented: (1) a high-throughput screening chip to test 192 crystallization conditions in parallel using as little as 8 nl of membrane protein per well and (2) a crystallization optimization chip to rapidly optimize preliminary crystallization hits through fine-gradient re-screening. We screened three membrane proteins for new in meso crystallization conditions, identifying several preliminary hits that we tested for X-ray diffraction quality. Further, we identified and optimized the crystallization condition for a photosynthetic reaction center mutant and solved its structure to a resolution of 3.5 Å.},
doi = {10.1063/1.4981818},
journal = {Biomicrofluidics},
number = 2,
volume = 11,
place = {United States},
year = {Mon Apr 24 00:00:00 EDT 2017},
month = {Mon Apr 24 00:00:00 EDT 2017}
}
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