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 watersoluble, 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 alpha-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.