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Enzymatic and mass spectral analysis of mutant and wild‐type selenomethionyl‐dihydrofolate reductase
Author(s) -
Broderick Kathleen Marie,
Boles Jeffrey O
Publication year - 2011
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.25.1_supplement.lb64
Subject(s) - dihydrofolate reductase , cyanogen bromide , chemistry , biochemistry , amino acid , methionine , enzyme , trypsin , mutant , wild type , leucine , peptide sequence , gene
The biosynthetic incorporation of unnatural amino acids, primarily selenomethionine, has been used for over a decade to facilitate structural determination of proteins. 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 (Se‐Met DHFR) and wild type DHFR have been analyzed for catalytic activity and stability. The sensitivity to cyanogen bromide and trypsin for mass spectral analysis will be presented. A methionine to leucine mutation at position 6, also subjected to these analyses for both the wild‐type and selenoprotein, will be presented.