Burova T V, Choiset Y, Tran V, Haertlé T
Institute of Biochemical Physics, Russian Academy of Sciences, Moscow.
Protein Eng. 1998 Nov;11(11):1065-73. doi: 10.1093/protein/11.11.1065.
Mixed disulfide derivatives of bovine beta-lactoglobulin (BLG) were studied by circular dichroism (CD), gel-permeation HPLC and high-sensitivity differential scanning calorimetry (HS-DSC). It was shown that modification of Cys121 with mercaptopropionic acid and mercaptoethanol does not affect the secondary structure of BLG, but results instead in tertiary and quaternary structure changes. At neutral pH, the equilibrium dimer<==>monomer of modified beta-lactoglobulin is shifted towards monomeric form. In contrast to native BLG, thermal denaturation of modified beta-lactoglobulin is fully reversible in neutral and acidic pH as demonstrated by CD and HS-DSC measurements. Modification of Cys121 results in a significant decrease of transition temperature (-6 degrees C) and enthalpy (-106 kJ/mol) at pH 2.05 while unfolding heat capacity increment remains unchanged. Thermal unfolding transitions of native and modified beta-lactoglobulin at pH 2.05 are well approximated by a two-state model suggesting that no intermediate states appear after modification. The difference in Gibbs energy of denaturation between native and modified beta-lactoglobulin, 8.5 kJ/mol at 37 degrees C and pH 2.05, does not depend on the nature of the introduced group (charged or neutral). Computer analysis of possible interactions involving Cys121 in a three-dimensional structure of beta-lactoglobulin revealed that the thiol group is too far away from neighboring residues to form side-chain hydrogen bonds. This suggests that the sulfhydryl group of Cys121 may contribute to the maintenance of BLG tertiary structure via water mediated H-bonding.
采用圆二色光谱（CD）、凝胶渗透高效液相色谱法（HPLC）和高灵敏度差示扫描量热法（HS-DSC）对牛β-乳球蛋白（BLG）的混合二硫键衍生物进行了研究。结果表明，用巯基丙酸和巯基乙醇对Cys121进行修饰不会影响BLG的二级结构，反而会导致三级和四级结构的变化。在中性pH条件下，修饰后的β-乳球蛋白的平衡二聚体⇌单体向单体形式移动。与天然BLG不同，CD和HS-DSC测量结果表明，修饰后的β-乳球蛋白在中性和酸性pH条件下的热变性是完全可逆的。在pH 2.05时，Cys121的修饰导致转变温度显著降低（-6℃）和焓显著降低（-106 kJ/mol），而展开热容量增量保持不变。天然和修饰后的β-乳球蛋白在pH 2.05时的热展开转变可以很好地用两态模型来近似，这表明修饰后没有出现中间态。天然和修饰后的β-乳球蛋白在37℃和pH 2.05时的变性吉布斯自由能差异为8.5 kJ/mol，不依赖于引入基团的性质（带电荷或中性）。对β-乳球蛋白三维结构中涉及Cys121的可能相互作用进行计算机分析发现，硫醇基团与相邻残基距离太远，无法形成侧链氢键。这表明Cys121的巯基可能通过水介导的氢键有助于维持BLG的三级结构。
