Stabilization of native structure by the closed disulfide loop of staphylococcal enterotoxin B

Abstract The extent and rate of reduction of aqueous staphylococcal enterotoxin B by dithiothreitol in the absence or presence of the denaturant guanidine hydrochloride were directly proportional to the extent and rate of enterotoxin B unfolding. Thus, the single disulfide bond of native enterotoxin B must be positioned in a region of tertiary structure. Reduction followed by alkylation with iodoacetamide or iodoacetic acid of enterotoxin B unfolded in 8 M urea resulted, after refolding in dilute alkaline buffer, in aggregation of the alkylated monomer with a significant increase in viscosity. Also, the stability of alkylated enterotoxin B toward denaturation by guanidine hydrochloride was greatly diminished as compared to unmodified enterotoxin B. However, the refolded, alkylated enterotoxin derivatives retained the ability to completely precipitate rabbit antibody specific for native toxin. Tryptic hydrolysis of a single peptide bond in the 92–112 disulfide loop also significantly lowered the stability of the protein to guanidine denaturation. It is proposed, therefore, that the closed disulfide loop of enterotoxin B stabilizes a minor domain of structure whose disruption increases the conformational flexibility of the toxin molecule without extensive denaturation and loss of biologic specificity.