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Proc. Natl. Acad. Sci. U.S.A. Apr (2007); 104(16):6678-83
Blocking S-adenosylmethionine synthesis in yeast allows selenomethionine incorporation and multiwavelength anomalous dispersion phasing-
Malkowski MG, Quartley E, Friedman AE, Babulski J, Kon Y, Wolfley J, Said M, Luft JR, Phizicky EM, DeTitta GT, Grayhack EJ
Center for Pediatric Biomedical Research and Departments of Environmental Medicine and Biochemistry and Biophysics, University of Rochester Medical School, Rochester, NY 14642, USA-
Abstract: Saccharomyces cerevisiae is an ideal host from which to obtain high levels of posttranslationally modified eukaryotic proteins for x-ray crystallography- However, extensive replacement of methionine by selenomethionine for anomalous dispersion phasing has proven intractable in yeast- We report a general method to incorporate selenomethionine into proteins expressed in yeast based on manipulation of the appropriate metabolic pathways- sam1--- sam2--- mutants, in which the conversion of methionine to S-adenosylmethionine is blocked, exhibit reduced selenomethionine toxicity compared with wild-type yeast, increased production of protein during growth in selenomethionine, and efficient replacement of methionine by selenomethionine, based on quantitative mass spectrometry and x-ray crystallography- The structure of yeast tryptophanyl-tRNA synthetase was solved to 1-8 A by using multiwavelength anomalous dispersion phasing with protein that was expressed and purified from the sam1--- sam2--- strain grown in selenomethionine- Six of eight selenium residues were located in the structure-
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Chris Stark, Bobby-Joe Breitkreutz, Teresa Reguly, Lorrie Boucher, Ashton Breitkreutz, Mike Tyers.
Nucleic Acids Res. Jan 1;34:D535-9.