Thermodynamics of the Op18/stathmin-tubulin interaction
Thermodynamics of the Op18/stathmin-tubulin interaction
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DOI：
10.1074/jbc.m305546200
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发表时间：
2003-10-03
影响因子：
4.8
通讯作者：
Steinmetz, MO
中科院分区：
文献类型：
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作者：
Honnappa, S;Cutting, B;Steinmetz, MO
OP18/stathmin(Stathmin)是一种参与微管微丝系统调节的内在无序蛋白。Stathmin的一个功能是将微管蛋白二聚体隔离到组装不能的复合体中，最近的研究发现每个stathmin分子有两个微管蛋白结合位点。用高灵敏度等温滴定量热法测定了在10℃，80 mm管子，pH 6.8，1 mM EGTA，1 mM MgCl2，1 mM GTP条件下，这两个结合位点具有相同的亲和力，平衡结合常数K-0=6.0×10~(6)M~(-1)。获得了微管蛋白-Stathmin结合平衡的DeltaC(P)(0)=-860cal mol(-1)K-1的较大的负摩尔热容变化，表明疏水效应是结合反应的主要驱动力。用GDP代替β-微管蛋白上的GTP对微管蛋白-他汀类蛋白结合平衡的热力学参数没有显著影响。通过圆二色谱和核磁共振对所提出的Stathmin的pH敏感双功能进行了进一步的评价。在低温下，Stathmin被发现具有广泛的螺旋结构，但没有任何稳定的三级结构。然而，在含有两个微管蛋白亚基的复合体中，stathmin采用了稳定的构象。单独的蛋白质和复合体的稳定性和构象都不受pH的微小变化的显著影响。与pH 6.8相比，在pH 7.5时，Stathmin对微管蛋白的亲和力降低了4倍。这种下降可能是由于stathmin的C末端结合较弱所致。这些发现不支持Stathmin作为一种对pH敏感的蛋白质发挥作用的观点。
Op18/stathmin (stathmin) is an intrinsically disordered protein involved in the regulation of the microtubule filament system. One function of stathmin is to sequester tubulin dimers into assembly incompetent complexes, and recent studies revealed two tubulin binding sites per stathmin molecule. Using high sensitivity isothermal titration calorimetry, we document that at 10 degreesC and under the conditions of 80 mM PIPES, pH 6.8, 1 mM EGTA, 1 mM MgCl2, 1 mM GTP these two binding sites are of equal affinity with an equilibrium binding constant of K-0 = 6.0 x 10(6) M-1. The obtained large negative molar heat capacity change of DeltaC(p)(0) = -860 cal mol(-1) K-1 (referring to tubulin) for the tubulin-stathmin binding equilibrium suggests that the hydrophobic effect is the major driving force of the binding reaction. Replacing GTP by GDP on beta-tubulin had no significant effect on the thermodynamic parameters of the tubulin-stathmin binding equilibrium. The proposed pH-sensitive dual function of stathmin was further evaluated by circular dichroism spectroscopy and nuclear magnetic resonance. At low temperatures, stathmin was found to be extensively helical but devoid of any stable tertiary structure. However, in complex with two tubulin subunits stathmin adopts a stable conformation. Both the stability and conformation of the individual proteins and complexes were not significantly affected by small changes in pH. A 4-fold decrease in affinity of stathmin for tubulin was revealed at pH 7.5 compared with pH 6.8. This decrease could be attributed to a weaker binding of the C terminus of stathmin. These findings do not support the view that stathmin works as a pH-sensitive protein.