Helms V, Wade R C
European Molecular Biology Laboratory, Heidelberg, Germany.
Proteins. 1998 Aug 15;32(3):381-96.
Hydration of protein cavities influences protein stability, dynamics, and function. Protein active sites usually contain water molecules that, upon ligand binding, are either displaced into bulk solvent or retained to mediate protein-ligand interactions. The contribution of water molecules to ligand binding must be accounted for to compute accurate values of binding affinities. This requires estimation of the extent of hydration of the binding site. However, it is often difficult to identify the water molecules involved in the binding process when ligands bind on the surface of a protein. Cytochrome P450cam is, therefore, an ideal model system because its substrate binds in a buried active site, displacing partially disordered solvent, and the protein is well characterized experimentally. We calculated the free energy differences for having five to eight water molecules in the active site cavity of the unliganded enzyme from molecular dynamics simulations by thermodynamic integration employing a three-stage perturbation scheme. The computed free energy differences between the hydration states are small (within 12 kJ mol-1) but distinct. Consistent with the crystallographic determination and studies employing hydrostatic pressure, we calculated that, although ten water molecules could in principle occupy the volume of the active site, occupation by five to six water molecules is thermodynamically most favorable.
蛋白质腔的水合作用会影响蛋白质的稳定性、动力学和功能。蛋白质活性位点通常含有水分子，在配体结合时，这些水分子要么被置换到本体溶剂中，要么被保留下来以介导蛋白质与配体的相互作用。为了计算准确的结合亲和力值，必须考虑水分子对配体结合的贡献。这需要估计结合位点的水合程度。然而，当配体结合在蛋白质表面时，往往很难确定参与结合过程的水分子。因此，细胞色素P450cam是一个理想的模型系统，因为其底物结合在一个埋藏的活性位点中，置换了部分无序的溶剂，并且该蛋白质在实验上有很好的表征。我们通过采用三阶段微扰方案的热力学积分，从分子动力学模拟中计算了未结合配体的酶的活性位点腔中含有五到八个水分子时的自由能差。计算得到的水合状态之间的自由能差很小（在12 kJ mol-1以内）但很明显。与晶体学测定和采用静水压力的研究一致，我们计算得出，虽然原则上十个水分子可以占据活性位点的体积，但五到六个水分子的占据在热力学上是最有利的。
