Circular dichroism of diphtheria toxin, Pseudomonas aeruginosa exotoxin A, and various derivatives

Abstract

We have recorded the near- and far-ultraviolet circular dichroism spectra of diphtheria toxin, Pseudomonas aeruginosa exotoxin A, and derivatives of these toxins. The far-ultraviolet spectra of various forms of diphtheria toxin were virtually identical, implying that no major changes in secondary structure accompany proteolytic nicking or dimerization of toxin, or binding of the endogenous dinucleotide, adenylyl-(3′,5′)-uridine 3′-monophosphate Ado PUrd P). Alpha-helix content was estimated to be 29%, as compared with 8% for fragment A. Near-ultraviolet spectra were identical between nicked and intact diphtheria toxin. A broad negative transition with a minimum at 304 nm was assigned to the intrachain disulfide bridge within the B moiety. Dimeric diphtheria toxin showed perturbations of aromatic residues. Binding of Ado PUrd P to monomeric diphtheria toxin or of adenylyl-(3′,5′)-uridine (Ado PUrd) to fragment A perturbed one or more tryptophans. The latter results correlate with evidence for involvement of a trytophan in NAD binding. Native exotoxin A was estimated to have 16% α-helix, and the activated form of exotoxin A, 11%. An enzymatically active, 31 kDa proteolytic fragment of exotoxin A showed similar α-helix content (7%) to that of diphtheria toxin fragment A.