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Ratiocinative screen of eukaryotic integral membrane protein expression and solubilization for structure determination |
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| Authors: | Franklin A Hays Zygy Roe-Zurz Min Li Libusha Kelly Franz Gruswitz Andrej Sali Robert M Stroud |
| Institution: | (1) Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA 94158-2517, USA;(2) Membrane Protein Expression Center, University of California at San Francisco, San Francisco, CA 94158-2517, USA;(3) Graduate Group in Bioinformatics, University of California at San Francisco, San Francisco, CA 94158-2517, USA;(4) Center for the Structure of Membrane Proteins, University of California at San Francisco, San Francisco, CA 94158-2517, USA;(5) Department of Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, CA 94158-2517, USA;(6) California Institute for Quantitative Biosciences, University of California at San Francisco, San Francisco, CA 94158-2517, USA;(7) Department of Bioengineering and Therapeutic Sciences, University of California at San Francisco, San Francisco, CA 94158-2517, USA |
| Abstract: | Persistent hurdles impede the successful determination of high-resolution crystal structures of eukaryotic integral membrane proteins (IMP). We designed a high-throughput structural genomics oriented pipeline that seeks to minimize effort in uncovering high-quality, responsive non-redundant targets for crystallization. This “discovery-oriented” pipeline sidesteps two significant bottlenecks in the IMP structure determination pipeline: expression and membrane extraction with detergent. In addition, proteins that enter the pipeline are then rapidly vetted by their presence in the included volume on a size-exclusion column—a hallmark of well-behaved IMP targets. A screen of 384 rationally selected eukaryotic IMPs in baker’s yeast Saccharomyces cerevisiae is outlined to demonstrate the results expected when applying this discovery-oriented pipeline to whole-organism membrane proteomes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Franklin A. Hays and Zygy Roe-Zurz have contributed equally to this work. |
| Keywords: | Discovery-oriented screen Membrane protein structure Structural genomics Eukaryotic integral membrane protein Saccharomyces cerevisiae |
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