Calorimetric analysis of the Ca 2+-binding βγ-crystallin homolog protein S from Myxococcus xanthus: intrinsic stability and mutual stabilization of domains

Abstract

The βγ-crystallin superfamily consists of a class of homologous two-domain proteins with Greek-key fold. Protein S, a Ca 2+-binding spore-coat protein from the soil bacterium Myxococcus xanthus exhibits a high degree of sequential and structural homology with γB-crystallin from the vertebrate eye lens. In contrast to γB-crystallin, which undergoes irreversible aggregation upon thermal unfolding, protein S folds reversibly and may therefore serve as a model in the investigation of the thermodynamic stability of the eye-lens crystallins. The thermal denaturation of recombinant protein S (PS) and its isolated domains was studied by differential scanning calorimetry in the absence and in the presence of Ca 2+ at varying pH. Ca 2+-binding leads to a stabilization of PS and its domains and increases the cooperativity of their equilibrium unfolding transitions. The isolated N-terminal and C-terminal domains (NPS and CPS) obey the two-state model, independent of the pH and Ca 2+-binding; in the case of PS, under all conditions, an equilibrium intermediate is populated. The first transition of PS may be assigned to the denaturation of the C-terminal domain and the loss of domain interactions, whereas the second one coincides with the denaturation of the isolated N-terminal domain. At pH 7.0, in the presence of Ca 2+, where PS exhibits maximal stability, the domain interactions at 20 °C contribute 20 kJ/mol to the overall stability of the intact protein.