Presence of a slow dimerization equilibrium on the thermal unfolding of the 205-316 thermolysin fragment at neutral pH

Differential scanning calorimetry and size-exclusion chromatography have been used to characterize the dimerization and unfolding of the 205-316 C-terminal fragment of thermolysin at pH 7.5. We show that the folded fragment dimerizes at low temperature with a moderate affinity and undergoes thermal unfolding according to a N-2 reversible arrow 2N reversible arrow 2U model. This behavior has already been observed at acid pH, where a similar dissociation equilibrium has been found [Azuaga, A., Conejero-Lara, F., Rivas G., De Filippis, V., Fontana, A., & Mateo, P. L. (1995) Biochim. Biophys. Acta 1252, 95-102]. Nevertheless, at pH 7.5 the dimerization equilibrium slows down below about 30 degrees C, with virtually no interconversion between the monomeric and the dimeric states of the fragment. We have studied the kinetics of interconversion between monomer and dimer by size-exclusion chromatography experiments and have shown that a very high energy barrier (83.8 kJ/mol at 26.5 degrees C) exists between either state. A mathematical analysis of the DSC thermograms on the basis of the proposed model has allowed us to obtain the thermodynamic characterization of the dimerization and the unfolding processes of the fragment and confirms the kinetic parameters obtained in the chromatographic experiments. The thermodynamic functions for the unfolding of the fragment are compatible with some degree of disorder in the structures of both the monomer and the dimer. According to circular dichroism measurements, the dimerization of the fragment seems to be linked to some conformational change in the subunits, most probably due to a rearrangement of the existing secondary-structure elements. This fragment displays several features already observed in folding intermediates, such as the partial disorder of the polypeptidic chain, association processes, and kinetic barriers between different regions in the conformational space.