Protein footprinting at cysteines: Probing ATP-modulated contacts in cysteine-substitution mutants of yeast DNA topoisomerase II

Benjamin P. Tu, James C. Wang

Research output: Contribution to journalArticle

11 Scopus citations

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 languageEnglish (US)
Pages (from-to)4862-4867
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume96
Issue number9
DOIs
StatePublished - Apr 27 1999

Keywords

  • Ligand-induced protein contacts
  • Mutagenesis
  • Thiol cyanylation

ASJC Scopus subject areas

  • General

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