Human and bovine gammaS-crystallin (HgammaS and BgammaS) and their isolated N- and C-terminal domains were cloned and expressed as recombinant proteins in E. coli. HgammaS and BgammaS 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, gammaS-crystallin does not show bimodal unfolding characteristics. In the case of BgammaS, at pH 7.0, the C-terminal domain is less stable than the N-terminal one, whereas for HgammaS 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, HgammaS-crystallin and its isolated domains (HgammaS-N and HgammaS-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 gammaS-crystallin. In contrast, the structurally closely related gammaB-crystallin owes much of its extreme stability to domain interactions. Study holds ProTherm entries: 9750, 9751, 9752, 9753, 9754, 9755, 9756, 9757, 9758, 9759, 9760, 9761, 9762, 9763, 9764 Extra Details: additive : DTT(5 mM), crystallins; differential scanning calorimetry; protein folding;,protein stability; domain interaction
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:38 p.m.
|Number of data points||21|
|Proteins||Beta-crystallin S ; Beta-crystallin S ; Beta-crystallin S ; Beta-crystallin S|
|Assays/Quantities/Protocols||Experimental Assay: dCp ; Experimental Assay: dHcal ; Experimental Assay: Tm ; Experimental Assay: Cm ; Experimental Assay: Cm ; Experimental Assay: Cm ; Experimental Assay: Cm|
|Libraries||Mutations for sequence MYKIQIFEKGDFNGQMHETTEDCPSIMEQFHMREVHSCKVLEGAWIFYELPNYRGRQYLLDKKEYRKPVDWGAASPAVQSFRRIVE ; Mutations for sequence GQYKIQIFEKGDFSGQMYETTEDCPSIMEQFHMREIHSCKVLEGVWIFYELPNYRGRQYLLDKKEYRKPIDWGAASPAVQSFRRIVE|