Ratiocinative screen of eukaryotic integral membrane protein expression and solubilization for structure determination.
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.
Hays
Franklin A
FA
Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA 94158-2517, USA. haysf@msg.ucsf.edu
Roe-Zurz
Zygy
Z
Li
Min
M
Kelly
Libusha
L
Gruswitz
Franz
F
Sali
Andrej
A
Stroud
Robert M
RM
D000818:Animals; D018360:Crystallography, X-Ray; D030562:Databases, Protein; D023281:Genomics; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry*; D011487:Protein Conformation; D020543:Proteome / Q000032:analysis; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism
D008565:Membrane Proteins; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins