Transition state of the rate-limiting step of heat denaturation of Cry3A delta-endotoxin

Heat denaturation of Cry3A delta-endotoxin from Bacillus thuringiensis var. tenebrionis and its 55 kDa fragment was studied by differential scanning m icrocalorimetry at low pH. Analysis of the calorimetric data has shown that denaturation of Cry3A delta-endotoxin is a nonequilibrium process at heati ng fates from 0.125 to 2 K/min. This means that the stability of delta-endo toxin (the apparent temperature of denaturation T-m) under these conditions is under kinetic control rather than under thermodynamic control. It has b een shown that heat denaturation of this protein is a one-step kinetic proc ess, The enthalpy of the process and its activation energy were measured as functions of temperature. The data obtained allow confirmation of the fact that the conformation of delta-endotoxin at the transition state only slig htly differs from its native conformation with respect to compactness and e xtent of hydration. The comparison of the activation energy for intact delt a-endotoxin and the 55 kDa fragment showed that the transition of the molec ule to a transition state does not cause any changes in the conformation of three N-terminal alpha-helices. Complete removal of the N-terminal domain of delta-endotoxin and 40 amino acids from the C-terminus beta-sheet domain III causes an irreversible loss of the tertiary structure. Thus, during pr otein folding the nucleation core determining protein stability does not in volve its three initial alpha-helices but may include the remaining alpha-h elices of the N-terminal domain. The functional significance of peculiariti es of structure arrangement of the delta-endotoxin molecule is discussed.