Abstract
Cysteine-substitution mutants of yeast DNA topoisomerase II were used to test footprinting of the enzyme by 2-nitro-5-thiocyanobenzoate, which cyanylates exposed cysteines in a native protein for peptide cleavage at the cyanylated sites upon unfolding and incubating the protein at pH 9. For a mutant enzyme containing a single cysteine, the extent of peptide cleavage was found to reflect the accessibility of the residue in the native protein. For proteins with multiple cysteines, however, such a correlation was obscured by the transfer of cyano groups from modified to unmodified cysteines during incubation of the unfolded protein at pH 9; accessibilities of the cysteinyl residues in a native protein could be assessed only if cyano shuffling was prevented by blocking uncyanylated sulfhydryls with a second thiol reagent. The successive use of two reagents in cysteine footprinting was applied in probing the ATP-modulated formation of contacts in yeast DNA topoisomerase II.
Original language | English (US) |
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Pages (from-to) | 4862-4867 |
Number of pages | 6 |
Journal | Proceedings of the National Academy of Sciences of the United States of America |
Volume | 96 |
Issue number | 9 |
DOIs | |
State | Published - Apr 27 1999 |
Keywords
- Ligand-induced protein contacts
- Mutagenesis
- Thiol cyanylation
ASJC Scopus subject areas
- General