Sakai K, Sakurai K, Sakai M, Hoshino M, Goto Y
Institute for Protein Research, Osaka University, Suita, Japan.
Protein Sci. 2000 Sep;9(9):1719-29.
Bovine beta-lactoglobulin A assumes a dimeric native conformation at neutral pH, while the conformation at pH 2 is monomeric but still native. Beta-lactoglobulin A has a free thiol at Cys121, which is buried between the beta-barrel and the C-terminal major alpha-helix. This thiol group was specifically reacted with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the presence of 1.0 M Gdn-HCI at pH 7.5, producing a modified beta-lactoglobulin (TNB-bIg) containing a mixed disulfide bond with 5-thio-2-nitrobenzoic acid (TNB). The conformation and stability of TNB-bIg were studied by circular dichroism (CD), tryptophan fluorescence, analytical ultracentrifugation, and one-dimensional 1H-NMR. The CD spectra of TNB-bIg indicated disordering of the native secondary structure at pH 7.5, whereas a slight increase in the alpha-helical content was observed at pH 2.0. The tryptophan fluorescence of TNB-bIg was significantly quenched compared with that of the intact protein, probably by the energy transfer to TNB. Sedimentation equilibrium analysis indicated that, at neutral pH, TNB-bIg is monomeric while the intact protein is dimeric. In contrast, at pH 2.0, both the intact beta-lactoglobulin and TNB-bIg were monomeric. The unfolding transition of TNB-bIg induced by Gdn-HCl was cooperative in both pH regions, although the degree of cooperativity was less than that of the intact protein. The 1H-NMR spectrum for TNB-bIg at pH 3.0 was native-like, whereas the spectrum at pH 7.5 was similar to that of the unfolded proteins. These results suggest that modification of the buried thiol group destabilizes the rigid hydrophobic core and the dimer interface, producing a monomeric state that is native-like at pH 2.0 but is molten globule-like at pH 7.5. Upon reducing the mixed disulfide of TNB-bIg with dithiothreitol, the intact beta-lactoglobulin was regenerated. TNB-bIg will become a useful model to analyze the conformation and stability of the intermediate of protein folding.
牛β-乳球蛋白A在中性pH值下呈二聚体天然构象，而在pH 2时的构象为单体但仍为天然构象。β-乳球蛋白A在Cys121处有一个游离巯基，它埋藏在β-桶和C端主要α-螺旋之间。在pH 7.5的1.0 M盐酸胍存在下，该巯基与5,5'-二硫代双(2-硝基苯甲酸)(DTNB)发生特异性反应，生成一种含有与5-硫代-2-硝基苯甲酸(TNB)混合二硫键的修饰β-乳球蛋白(TNB-bIg)。通过圆二色性(CD)、色氨酸荧光、分析超速离心和一维1H-NMR研究了TNB-bIg的构象和稳定性。TNB-bIg的CD光谱表明在pH 7.5时天然二级结构发生紊乱，而在pH 2.0时观察到α-螺旋含量略有增加。与完整蛋白质相比，TNB-bIg的色氨酸荧光显著淬灭，可能是由于能量转移到TNB。沉降平衡分析表明，在中性pH值下，TNB-bIg是单体，而完整蛋白质是二聚体。相反，在pH 2.0时，完整的β-乳球蛋白和TNB-bIg都是单体。尽管协同程度低于完整蛋白质，但在两个pH区域中，盐酸胍诱导的TNB-bIg的去折叠转变都是协同的。pH 3.0时TNB-bIg的1H-NMR光谱类似天然光谱，而pH 7.5时的光谱与去折叠蛋白质的光谱相似。这些结果表明，埋藏巯基的修饰使刚性疏水核心和二聚体界面不稳定，产生一种在pH 2.0时类似天然状态但在pH 7.5时类似熔球状态的单体状态。用二硫苏糖醇还原TNB-bIg中的混合二硫键后，完整的β-乳球蛋白得以再生。TNB-bIg将成为分析蛋白质折叠中间体构象和稳定性的有用模型。
