Human and bovine gamma S-crystallin (H gamma S and B gamma S) and their isolated N- and C-terminal domains were cloned and expressed as recombinant proteins in E. coli. H gamma S and B gamma S are found to be authentic according to their spectral and hydrodynamic properties. Both full-length proteins and isolated domains are monomeric and exhibit high thermal and pH stabilities. The thermodynamic characterization made use of chemically and thermally-induced equilibrium unfolding transitions at varying pH. In spite of its exemplary two-domain structure, gamma S-crystallin does not show bimodal unfolding characteristics. In the case of B gamma S, at pH 7.0, the C-terminal domain is less stable than the N-terminal one, whereas for H gamma S the opposite holds true. Differential scanning calorimetry confirms the results of chemically-induced equilibrium unfolding transitions. Over the whole pH range between 2.0 and 11.5, H gamma S-crystallin and its isolated domains (H gamma S-N and H gamma S-C) follow the two-state model. The two-state unfolding of the intact two-domain protein points to the close similarity of the stabilities of the constituent domains. Obviously, interactions between the domains do not contribute significantly to the overall stability of gamma S-crystallin. In contrast, the structurally closely related gamma B-crystallin owes much of its extreme stability to domain interactions. (C) 2000 Elsevier Science B.V. All rights reserved.
