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) Study holds ProTherm entries: 3645, 3646 Extra Details: NaN3(0.01%) was added in the experiment solute perturbation analysis; Structural stability;,partially folded state

Submission Details

ID: Rib9udm23

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:22 p.m.

Version: 1

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