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) <==> 2N <==> 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. Study holds ProTherm entries: 5010 Extra Details:

Submission Details

ID: 7CKf48fN4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:27 p.m.

Version: 1

Publication Details
Conejero-Lara F;Mateo PL,Biochemistry (1996) Presence of a slow dimerization equilibrium on the thermal unfolding of the 205-316 thermolysin fragment at neutral pH. PMID:8639498
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
5DLH 2015-09-04T00:00:00+0000 2.25 SFX structure of thermolysin
5ONP 2017-08-04T00:00:00+0000 1.34 Alzheimer's Amyloid-Beta Peptide Fragment 1-40 in Complex with Cd-substituted Thermolysin
5ONQ 2017-08-04T00:00:00+0000 1.17 Alzheimer's Amyloid-Beta Peptide Fragment 29-40 in Complex with Cd-substituted Thermolysin
5WR2 2016-11-29T00:00:00+0000 2.0 Thermolysin, SFX liganded form with oil-based carrier
5WR3 2016-11-29T00:00:00+0000 2.1 Thermolysin, SFX liganded form with water-based carrier
5WR4 2016-11-29T00:00:00+0000 2.1 Thermolysin, SFX unliganded form with oil-based carrier
5WR5 2016-11-29T00:00:00+0000 1.9 Thermolysin, liganded form with cryo condition 1
5WR6 2016-11-29T00:00:00+0000 2.3 Thermolysin, liganded form with cryo condition 2
6FHP 2018-01-15T00:00:00+0000 1.7 DAIP in complex with a C-terminal fragment of thermolysin
6FSM 2018-02-19T00:00:00+0000 1.39 Crystal structure of TCE-treated Thermolysin

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
99.4 Thermolysin P43133 THER_GEOSE
100.0 Thermolysin P00800 THER_BACTH