A thermodynamic study of mutant forms of Streptomyces subtilisin inhibitor. II. Replacements at the interface of dimer formation, Val13

Abstract

Amino acid replacements in a homodimeric protein, Streptomyces subtilisin inhibitor, at the position of Val13, which is located at the center of the β-sheet interface of the domain-domain interaction, changed both the overall stability and the denaturational scheme. All the mutant forms lost stability, losses in free energy at 82.21°C at pH 7.0 obtained by calorimetric measurements ranging from 10.3 kcal (mol dimer)-1 for the Gly mutant, which involved a loss in enthalpy without a compensating loss in entropy to 0.84 kcal (mol dimer)-1 for the Ile mutant, which involved comparable losses in enthalpy and entropy. A gain in enthalpy for the Ala mutant was overcome by a gain in entropy, resulting in a loss in free energy by 6.78 kcal (mol dimer)−1. It was found that decreases in enthalpy and entropy showed good correlation with increasing side-chain hydrophobicity, while no such correlation was present for free energy. Replacements with Gly, Ala, Met and Phe altered the thermal denaturational scheme from N2 ⇌ 2D which was the case for the Leu and Ile mutants as well as for the wild-type, to N2 ⇌ 2N ⇌ 2D, where the first step call be regarded as a dissociation of the native dimer followed by a major unfolding in the second step. The free energy acquired by forming the dimer was estimated to be 2.03 kcal mol−1 for the Ala mutant at 66.95°C at pH 9.5 and lower for the other three mutants. © 1995 Academic Press Limited.