Blocking S-adenosylmethionine synthesis in yeast allows selenomethionine incorporation and multiwavelength anomalous dispersion phasing.
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.
Malkowski
Michael G
MG
Center for Pediatric Biomedical Research and Departments of Environmental Medicine and Biochemistry and Biophysics, University of Rochester Medical School, Rochester, NY 14642, USA.
Quartley
Erin
E
Friedman
Alan E
AE
Babulski
Julie
J
Kon
Yoshiko
Y
Wolfley
Jennifer
J
Said
Meriem
M
Luft
Joseph R
JR
Phizicky
Eric M
EM
DeTitta
George T
GT
Grayhack
Elizabeth J
EJ
D018360:Crystallography, X-Ray; D012436:S-Adenosylmethionine / Q000037:antagonists & inhibitors* / Q000096:biosynthesis* / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000378:metabolism*; D012645:Selenomethionine / Q000737:chemistry / Q000493:pharmacokinetics*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D012436:S-Adenosylmethionine; D012645:Selenomethionine