Botelho M M, Valente-Mesquita V L, Oliveira K M, Polikarpov I, Ferreira S T
Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Brazil.
Eur J Biochem. 2000 Apr;267(8):2235-41. doi: 10.1046/j.1432-1327.2000.01226.x.
Beta-lactoglobulin, the main whey protein in bovine milk, exists in several isoforms of which the most abundant are isoforms A and B. We have previously reported the denaturation of beta-lactoglobulin A by hydrostatic pressure [Valente-Mesquita, V.L., Botelho, M.M. & Ferreira, S.T. (1998) Biophys. J. 75, 471-476]. Here, we compare the pressure stabilities of isoforms A and B. These isoforms differ by two amino-acid substitutions: Asp64 and Val118 in isoform A are replaced by glycine and alanine, respectively, in isoform B. Replacement of the buried Val118 residue by the smaller alanine side-chain is not accompanied by significant structural rearrangements of the neighbouring polypeptide chain and creates a cavity in the core of beta-lactoglobulin. Pressure denaturation experiments revealed different stabilities of the two isoforms. Standard volume changes (DeltaVunf) of - 49 +/- 8 mL.mol-1 and -75 +/- 3 mL.mol-1, and unfolding free energy changes (DeltaGunf) of 8.5 +/- 1.3 kJ.mol-1 and 11.3 +/- 0.4 kJ.mol-1 were obtained for isoforms A and B, respectively. The volume occupied by the two methyl groups of Val118 removed in the V118A substitution is approximately 40 A3 per monomer of beta-lactoglobulin, in excellent agreement with the experimentally measured difference in DeltaVunf for the two isoforms (DeltaDeltaVunf = 26 mL.mol-1, corresponding to approximately 43 A3 per monomer). Thus, the existence of a core cavity in beta-lactoglobulin B may explain its enhanced pressure sensitivity relative to beta-lactoglobulin A. beta-Lactoglobulin undergoes a reversible pH-induced conformational change around pH 7, known as the Tanford transition. We have compared the pressure denaturation of beta-lactoglobulin A at pH 7 and 8. Unfolding free energy changes of 8.5 +/- 1.3 and 8.3 +/- 0.3 kJ.mol-1 were obtained at pH 7 and 8, respectively, showing that the thermodynamic stability of beta-lactoglobulin is identical at these pH values. Interestingly, DeltaVunf was dependent on pH, and varied from -49 +/- 8 mL.mol-1 to -68 +/- 2 mL.mol-1 at pH 7 and 8, respectively. The large increase in DeltaVunf at pH 8 relative to pH 7 appears to be associated with an overall expansion of the protein structure and could explain the increased pressure sensitivity of beta-lactoglobulin at alkaline pH.
β-乳球蛋白是牛乳中的主要乳清蛋白，存在多种异构体，其中最丰富的是A和B两种异构体。我们之前曾报道过静水压对β-乳球蛋白A的变性作用[瓦伦特 - 梅斯基塔，V.L.，博特略，M.M. & 费雷拉，S.T.（1998年）《生物物理杂志》75卷，471 - 476页]。在此，我们比较了A和B两种异构体的压力稳定性。这两种异构体有两个氨基酸替换的差异：异构体A中的Asp64和Val118在异构体B中分别被甘氨酸和丙氨酸取代。较小的丙氨酸侧链取代了埋藏的Val118残基，并未伴随相邻多肽链的显著结构重排，却在β-乳球蛋白的核心形成了一个空腔。压力变性实验揭示了两种异构体具有不同的稳定性。异构体A和B的标准体积变化（ΔVunf）分别为 - 49 ± 8 mL·mol⁻¹ 和 - 75 ± 3 mL·mol⁻¹，展开自由能变化（ΔGunf）分别为8.5 ± 1.3 kJ·mol⁻¹ 和11.3 ± 0.4 kJ·mol⁻¹。在V118A替换中去除的Val118的两个甲基所占据的体积，对于β-乳球蛋白的每个单体约为40 ų，这与两种异构体实验测得的ΔVunf差异（ΔΔVunf = 26 mL·mol⁻¹，对应每个单体约43 ų）非常吻合。因此，β-乳球蛋白B中存在核心空腔可能解释了其相对于β-乳球蛋白A增强的压力敏感性。β-乳球蛋白在pH 7左右会发生可逆的pH诱导构象变化，即所谓的坦福德转变。我们比较了pH 7和8时β-乳球蛋白A的压力变性情况。在pH 7和8时获得的展开自由能变化分别为8.5 ± 1.3和8.3 ± 0.3 kJ·mol⁻¹，表明β-乳球蛋白在这些pH值下的热力学稳定性相同。有趣的是，ΔVunf依赖于pH，在pH 7和8时分别从 - 49 ± 8 mL·mol⁻¹ 变化到 - 68 ± 2 mL·mol⁻¹。相对于pH 7，pH 8时ΔVunf的大幅增加似乎与蛋白质结构的整体膨胀有关，这可以解释β-乳球蛋白在碱性pH下压力敏感性的增加。
