Protein footprinting at cysteines: probing ATP-modulated contacts in cysteine-substitution mutants of yeast DNA topoisomerase II.
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.
Tu
B P
BP
Department of Molecular and Cellular Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA.
Wang
J C
JC
D019943:Amino Acid Substitution; D003545:Cysteine; D018983:DNA Footprinting; D004250:DNA Topoisomerases, Type II / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D005656:Fungal Proteins; D004250:DNA Topoisomerases, Type II; D003545:Cysteine