Synthesis of 3,3′-bis(sulfonato)-4,4′-bis(chloroacetamido) azobenzene and cysteine cross-linking for photo-control of protein conformation and activity

This protocol describes a procedure for the synthesis of 3,3′-bis(sulfonato)-4,4′-bis(chloroacetamido)azobenzene (BSBCA), a water-soluble, thiol-reactive, photo-switchable cross-linker. In addition, a protocol is outlined for installing the cross-linker in an intramolecular fashion onto proteins bearing two surface-exposed Cys residues. BSBCA is designed to be used as an in vitro activity switch that operates by exerting temporal and reversible photo-control over α-helix content within synthetic peptides and recombinant proteins. Synthesis of the cross-linker requires approximately 4.5 d, and cross-linking can be performed in 10-12 h.