Tu B P, Wang J C
Department of Molecular and Cellular Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA.
Proc Natl Acad Sci U S A. 1999 Apr 27;96(9):4862-7. doi: 10.1073/pnas.96.9.4862.
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.
酵母DNA拓扑异构酶II的半胱氨酸替代突变体被用于测试2-硝基-5-硫氰基苯甲酸对该酶的足迹分析，在pH 9条件下展开并孵育蛋白质时，该试剂会使天然蛋白质中暴露的半胱氨酸氰化，以便在氰化位点进行肽段切割。对于含有单个半胱氨酸的突变酶，发现肽段切割的程度反映了天然蛋白质中该残基的可及性。然而，对于含有多个半胱氨酸的蛋白质，在pH 9条件下孵育未折叠蛋白质时，氰基会从修饰的半胱氨酸转移到未修饰的半胱氨酸，从而掩盖了这种相关性；只有通过用第二种硫醇试剂封闭未氰化的巯基来防止氰基转移，才能评估天然蛋白质中半胱氨酰残基的可及性。两种试剂在半胱氨酸足迹分析中的连续使用被应用于探究酵母DNA拓扑异构酶II中ATP调节的接触形成。
