Vanderheeren G, Hanssens I, Noyelle K, Van Dael H, Joniau M
Interdisciplinary Research Center, Katholieke Universiteit Leuven Campus Kortrijk, Kortrijk, Belgium.
Biophys J. 1998 Nov;75(5):2195-204. doi: 10.1016/S0006-3495(98)77663-5.
In this work we have studied the interaction of the hydrophobic fluorescent probe 1,1'-bis(4-anilino-5-naphthalenesulfonate) (bis-ANS), with the native state of apo- and Ca2+-bound goat alpha-lactalbumin (GLA). In 10 mM Tris-HCl, pH 7.5, at 4 degrees C in 2 mM EGTA as well as at 37 degrees C in 2 mM Ca2+, the native protein is close to its thermal transition. Therefore, it can be expected that in both conditions the protein is equally susceptible to interaction with bis-ANS. Nevertheless, we have observed a number of interesting differences in the interaction of the dye with the apo and Ca2+ form. Native apo-GLA binds two bis-ANS molecules and in the complex with bis-ANS, the far-UV circular dichroism (CD) spectrum of apo-GLA becomes similar to that of the protein in the molten globule state. In contrast, native Ca2+-GLA binds five bis-ANS molecules and the far-UV CD spectrum of native Ca2+-GLA is conserved for the complex. In both cases, the high activation energies observed in kinetic experiments indicate that upon binding, large parts of the protein structure have to be reorganized. The reduced perturbation of the protein structure in the presence of Ca2+ can be attributed to local stabilization effects.
在这项工作中，我们研究了疏水荧光探针1,1'-双（4-苯胺基-5-萘磺酸）（双-ANS）与脱辅基和结合Ca2+的山羊α-乳白蛋白（GLA）天然状态的相互作用。在10 mM Tris-HCl，pH 7.5，4℃下2 mM EGTA中以及37℃下2 mM Ca2+中，天然蛋白质接近其热转变温度。因此，可以预期在这两种条件下蛋白质与双-ANS相互作用的敏感性相同。然而，我们观察到染料与脱辅基形式和Ca2+形式相互作用存在一些有趣的差异。天然脱辅基-GLA结合两个双-ANS分子，在与双-ANS形成的复合物中，脱辅基-GLA的远紫外圆二色性（CD）光谱变得类似于处于熔球态的蛋白质的光谱。相反，天然Ca2+-GLA结合五个双-ANS分子，并且天然Ca2+-GLA与复合物的远紫外CD光谱保持不变。在这两种情况下，动力学实验中观察到的高活化能表明，结合时蛋白质结构的大部分必须重新组织。Ca2+存在下蛋白质结构扰动的减少可归因于局部稳定作用。
