Gamma S-crystallin of bovine and human eye lens: solution structure, stability and folding of the intact two-domain protein and its separate domains

Human and bovine gamma S-crystallin (H gamma S and B gamma S) and their iso lated N- and C-terminal domains were cloned and expressed as recombinant pr oteins in E. coli. H gamma S and B gamma S are found to be authentic accord ing to their spectral and hydrodynamic properties. Both full-length protein s and isolated domains are monomeric and exhibit high thermal and pH stabil ities. The thermodynamic characterization made use of chemically and therma lly-induced equilibrium unfolding transitions at varying pH. In spite of it s 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-termi nal domain is less stable than the N-terminal one, whereas for H gamma S th e opposite holds true. Differential scanning calorimetry confirms the resul ts of chemically-induced equilibrium unfolding transitions. Over the whole pH range between 2.0 and 11.5, H gamma S-crystallin and its isolated domain s (H gamma S-N and H gamma S-C) follow the two-state model. The two-state u nfolding of the intact two-domain protein points to the close similarity of the stabilities of the constituent domains. Obviously, interactions betwee n the domains do not contribute significantly to the overall stability of g amma S-crystallin. In contrast, the structurally closely related gamma B-cr ystallin owes much of its extreme stability to domain interactions. (C) 200 0 Elsevier Science B.V. All rights reserved.