Wenk M, Herbst R, Hoeger D, Kretschmar M, Lubsen N H, Jaenicke R
Institute of Biophysics and Physical Biochemistry, University of Regensburg, Germany.
Biophys Chem. 2000 Aug 30;86(2-3):95-108. doi: 10.1016/s0301-4622(00)00161-7.
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.
人源和牛源γS-晶体蛋白（HγS和BγS）及其分离的N端和C端结构域被克隆，并作为重组蛋白在大肠杆菌中表达。根据其光谱和流体动力学性质，发现HγS和BγS是真实的。全长蛋白和分离的结构域均为单体，具有高热稳定性和pH稳定性。热力学表征利用了在不同pH值下化学诱导和热诱导的平衡去折叠转变。尽管γS-晶体蛋白具有典型的双结构域结构，但它并未表现出双峰去折叠特征。对于BγS，在pH 7.0时，C端结构域比N端结构域稳定性低，而对于HγS则相反。差示扫描量热法证实了化学诱导平衡去折叠转变的结果。在2.0至11.5的整个pH范围内，HγS-晶体蛋白及其分离的结构域（HγS-N和HγS-C）遵循两态模型。完整双结构域蛋白的两态去折叠表明组成结构域的稳定性非常相似。显然，结构域之间的相互作用对γS-晶体蛋白的整体稳定性贡献不大。相比之下，结构密切相关的γB-晶体蛋白的极端稳定性很大程度上归因于结构域间的相互作用。
