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 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.
Burns
Darcy C
DC
Worsfold Water Quality Centre, Trent University, 1600 West Bank Drive, Peterborough, ON, Canada K9J 7B8. darcyburns@trentu.ca
Zhang
Fuzhong
F
Woolley
G Andrew
GA
D001391:Azo Compounds / Q000138:chemical synthesis*; D003432:Cross-Linking Reagents / Q000138:chemical synthesis*; D003545:Cysteine / Q000737:chemistry; D015394:Molecular Structure; D010777:Photochemistry / Q000379:methods*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet
D001391:Azo Compounds; D003432:Cross-Linking Reagents; D011506:Proteins; C009850:azobenzene; D003545:Cysteine