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Mass Spectral Analysis of Mutant and Wild‐Type Selenomethionyl‐Dihydrofolate Reductase
Author(s) -
Boles Jeffrey Oakley,
Broderick Kathleen M,
Broderick Molly
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.519.2
Subject(s) - dihydrofolate reductase , cyanogen bromide , chemistry , methionine , amino acid , wild type , mutant , biochemistry , trypsin , enzyme , peptide sequence , gene
The biosynthetic incorporation of unnatural amino acids, primarily selenomethionine, has been increasingly used for over a decade to facilitate structural determination of proteins through the utilization of multi‐wavelength anomalous diffraction (MAD). This substitution has been shown to lead to instability and incomplete incorporation in some target proteins. In this study, the stability, level of incorporation and catalytic activity of selenomethionyl dihydrofolate reductase (SeMet‐DHFR) and wild type DHFR have been analyzed for catalytic activity and stability. The sensitivity to cyanogen bromide and trypsin for mass spectral analysis has been examined. A methionine to leucine mutation at position 6, also subjected to these analyses for both the wild‐type and selenoprotein, suggests subtle but potentially significant perturbations which likely go undetected if the wild type structure is not also solved.