A novel approach to the site-selective dual labelling of a protein via chemoselective cysteine modification

Local protein microenvironment is used to control the outcome of reaction between cysteine residues and 2,5-dibromohexanediamide. The differential reactivity is exploited to introduce two orthogonal reactive handles onto the surface of a double cysteine mutant of superfolder green fluorescent protein in a regioselective manner. Subsequent elaboration with commonly used thiol and alkyne containing reagents affects site-selective protein dual labelling.