Thermodynamic analysis of helix-engineered forms of the activation domain of human procarboxypeptidase A2.

TitleThermodynamic analysis of helix-engineered forms of the activation domain of human procarboxypeptidase A2.
Publication TypeJournal Article
Year of Publication2000
AuthorsFernández, A. M., V. Villegas, J. C. Martínez, N. A. J. van Nuland, F. Conejero-Lara, F. X. Avilés, L. Serrano, V. V. Filimonov, and P. L. Mateo
JournalEur J Biochem
Volume267
Issue19
Pagination5891-9
Date Published2000 Oct
ISSN0014-2956
KeywordsAmino Acid Sequence, Amino Acid Substitution, Calorimetry, Differential Scanning, Carboxypeptidases, Carboxypeptidases A, Circular Dichroism, Enzyme Activation, Enzyme Precursors, Humans, Hydrogen-Ion Concentration, Magnetic Resonance Spectroscopy, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Protein Conformation, Protein Denaturation, Protein Folding, Protein Renaturation, Protein Structure, Tertiary, Sequence Alignment, Sequence Homology, Amino Acid, Thermodynamics
Abstract

Thermodynamic characterization of the activation domain of human procarboxypeptidase A2, ADA2h, and its helix-engineered mutants was carried out by differential scanning calorimetry. The mutants were engineered by changing residues in the exposed face of the two alpha helices in order to increase their stability. At neutral and alkaline pH the three mutants, alpha-helix 1 (M1), alpha-helix 2 (M2) and alpha-helix 1 and alpha-helix 2 (DM), were more stable than the wild-type domain, in the order DM, M2, M1 and wild-type. Under these conditions the CD and NMR spectra of all the variants are very similar, indicating that this increase in stability is not the result of gross structural changes. Calorimetric analysis shows that the stabilizing effect of mutating the water-exposed surfaces of the helices seems to be mainly entropic, because the mutations do not change the enthalpy or the increase in heat capacity of denaturation. The unfolding behavior of all variants changes under acidic conditions: whereas wild-type and M1 have a strong tendency to aggregate, giving rise to a beta conformation upon unfolding, M2 and DM unfold reversibly, M2 being more stable than DM. CD and NMR experiments at pH 3.0 suggest that a region involving residues of the second and third beta strands as well as part of alpha-helix 1 changes its conformation. It seems that the enhanced stability of the altered conformation of M2 and DM reduces the aggregation tendency of ADA2h at acidic pH.

Alternate JournalEur. J. Biochem.
PubMed ID10998048