Presence of a Slow Dimerization Equilibrium on the Thermal Unfolding of the 205−316 Thermolysin Fragment at Neutral pH

Abstract

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 a 2N a 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. Nevertheless, at pH 7.5 the dimerization equilibrium slows down below about 30°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°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.It is well established that the folding pathways of proteins can occur via one or more intermediates, which can range from relatively unordered, pre-molten globules and moltenglobule states to highly ordered, near-native intermediate states (Ptitsyn, 1995, and references therein). These intermediate states sometimes have been well characterized, showing the early-forming persistent structural elements in specific domains of the protein, which later lead to the complete folding of the rest of the chain (Radford et al., 1992;Dobson et al., 1994). Isolated fragments from globular proteins are being widely used as models to provide insight into the folding problem (Vita et al., 1979(Vita et al., , 1984(Vita et al., , 1989Fontana, 1990;Chaffotte et al., 1991;Peng & Kim, 1994;Rico et al., 1994;Conejero-Lara et al., 1994). These submolecular domains can provide information about local events which could well be of importance during the folding of the complete polypeptide chain.In previous studies Fontana and co-workers have isolated and characterized a set of thermolysin fragments, most of them from the C-terminal part of the protein, using mainly circular dich...

NMR Solution Structure of the 205−316 C-Terminal Fragment of Thermolysin. An Example of Dimerization Coupled to Partial Unfolding

“…The existence of a monomer-dimer equilibrium has been discussed elsewhere for 205-316 thermolysin fragment at low concentrations (Azuaga et al, 1995;Conejero-Lara & Mateo, 1996). Nevertheless, we expected this fragment to be mostly dimeric under the high protein concentration used in our NMR study.…”

“…In a previous paper (Conejero-Lara & Mateo, 1996), we have reported on the far-and near-UV-CD spectra of the monomeric and dimeric states of the 205-316 fragment. Far UV-CD spectra for monomeric and dimeric fragments are very similar suggesting that they do not differ in helical structure.…”

“…The aromatic Tyr, Trp, and (to a smaller extent) Phe contribute to the far UV-CD signal at 222 nm, indicative of helix content, but the dependences of this contribution on helix content and length, on the sequence position, and on the orientation of the aromatic chromophores remain to be determined (Chakrabartty et al, 1993). It is likely that this aromatic contribution in the monomer is different from that in the dimer since near-UV-CD spectra (characteristic of ordered tertiary structure around aromatic residues) suggest that the dimer has a higher content of fixed tertiary structure than the monomer (Conejero-Lara & Mateo, 1996). Therefore, aromatic contributions complicate the interpretation of the CD data so that no clear-cut evidence can be drawn about actual differences in helical structure between the monomer and dimer.…”

“…The folding and stability properties of fragments of thermolysin, a well-characterized metalloprotease isolated from Bacillus thermoproteolyticus (Titani et al, 1972;Holmes & Matthews, 1982), have been the subject of growing interest in recent years (Vita et al, 1979(Vita et al, , 1984(Vita et al, , 1989Vita & Fontana, 1982;Dalzoppo et al, 1985;Conejero-Lara et al, 1994;Azuaga et al, 1995;Conejero-Lara & Mateo, 1996). This was due to the finding that short amino acid sequences of the protein fold into native-like conformations and undergo cooperative unfolding transitions under the appropiate conditions.…”

Observation of a Pressure-Induced Unfolding Intermediate of Thermolysin by Using Pressure-Jump Method

“…3 These domains seem to have differential stability against physical and chemical perturbations and, in addition, certain partial sequences of these domains are known to fold autonomously under moderate conditions. [7][8][9][10] We have reported the effects of high pressure on TLN activity and spectroscopic properties over a range of temperature and pressure. [4][5][6] The pressure-induced spectroscopic changes of TLN were explained by a simple two-state transition model, accompanied with a large and negative reaction volume change, ∆V.…”