X-ray transparent microfluidic chips for high-throughput screening and optimization of in meso membrane protein crystallization

Jeremy M. Schieferstein, Ashtamurthy S. Pawate, Chang Sun, Frank Wan, Paige N. Sheraden, Jana Broecker, Oliver P. Ernst, Robert B. Gennis, Paul J.A. Kenis

Research output: Contribution to journalArticle

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, 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 3.5Å.

Original languageEnglish (US)
Article number024118
JournalBiomicrofluidics
Volume11
Issue number2
DOIs
StatePublished - Mar 1 2017

ASJC Scopus subject areas

  • Biomedical Engineering
  • Materials Science(all)
  • Condensed Matter Physics
  • Fluid Flow and Transfer Processes
  • Colloid and Surface Chemistry

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