Linked thermal and solute perturbation analysis of cooperative domain interactions in proteins. Structural stability of diphtheria toxin

Abstract

The temperature and guanidine hydrochloride (GuHCl) dependence of the structural stability of diphtheria toxin has been investigated by high-sensitivity differential scanning calorimetry. In 50 mM phosphate buffer at pH 8.0 and in the absence of GuHCl, the thermal unfolding of diphtheria toxin is characterized by a transition temperature (Tm) of 54.9 degrees C, a calorimetric enthalpy change (delta H) of 295 kcal/mol, and a van't Hoff to calorimetric enthalpy ratio of 0.57. Increasing the GuHCl concentration lowers the transition temperature and the calorimetric enthalpy change. At the same time, the van't Hoff to calorimetric enthalpy ratio increases until it reaches a value of 1 at 0.3 M GuHCl and remains constant thereafter. At low GuHCl concentrations (0-0.3 M), the thermal unfolding of diphtheria toxin is characterized by the presence of two transitions corresponding to the A and B domains of the protein. At higher GuHCl concentrations (0.3-1 M), the A domain is unfolded at all temperatures, and only one transition corresponding to the B domain is observed. Under these conditions, the most stable protein conformation at low temperatures is a partially folded state in which the A domain is unfolded and the B domain folded. A general model that explicitly considers the energetics of domain interactions has been developed in order to account for the stability and cooperative behavior of diphtheria toxin. It is shown that this cooperative domain interaction model correctly accounts for the temperature location as well as the shape and area of the calorimetric curves. Under physiological conditions, domain-domain interactions account for most of the structural stability of the A domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutational analysis of hydrophobic domain interactions in γB‐crystallin from bovine eye lens

“…Missiakas et al, 1990;Markovic-Housley et al, 1994); and (ii) if cooperative interactions between domains are essential for the intrinsic stability of the domains, folding and unfolding are strictly coupled so that the complete protein behaves as a single cooperative unit (cf. Ramsay & Freire, 1990). Considering the present study, yB-crystallin can be classified between these two extremes, as each domain folds independently with further stability gained from domain pairing for one of the two domains.…”

The domains in γb-crystallin: identical fold-different stabilities

“…diphteria toxin. Its isolated A domain has a stability of À9.2 kJ/mol, whereas domain interactions between the A and B domains contribute an additional À10.5 kJ/mol to the stabilization of the A domain (Ramsey & Freire, 1990).…”

Role of the N-terminal region for the conformational stability of esterase 2 from Alicyclobacillus acidocaldarius

“…Following the original approaches developed by Brandts and co-workers [32], and Freire and co-workers [33], we have devised a model of two cooperative and coupled domains able to reproduce the main qualitative features of the DSC curves shown by the three investigated proteins.…”