Schobert B, Lanyi J K
Biochemistry. 1986 Jul 15;25(14):4163-7. doi: 10.1021/bi00362a026.
The influence of different anions on the deprotonation of the retinal Schiff base of halorhodopsin in the dark was investigated. We find that a large number of anions cause a significant increase of the pKa of the Schiff base, an effect attributed to binding to "site I" on the protein. The concentration dependencies of the spectroscopic shifts associated with the changes of the pKa yielded dissociation constants (and thus binding energies) for the anions, which were related to the Stokes radii. The data fit the predictions of electrostatic interaction between the anions and the positive charge associated with site I, if the latter is located within a few angstroms from the surface of the protein. The specificity of site I toward various anions is quantitatively explained by the differences in the change of Born energy upon transfer of the anions from water to the binding site. The changes in the deprotonation energy of the Schiff base upon the binding of anions, delta delta Gdeprot, could be calculated from the delta pKa at infinite anion concentration. Unexpectedly, the delta delta Gdeprot values were remarkably close to the energies of binding to site I. Thus, site I and the Schiff base are strongly electrostatically coupled, either because of close proximity or because of the possibility of allosteric energy transfer between them.
研究了不同阴离子在黑暗中对嗜盐菌视紫红质视网膜席夫碱去质子化的影响。我们发现大量阴离子会导致席夫碱的pKa显著升高，这种效应归因于与蛋白质上“位点I”的结合。与pKa变化相关的光谱位移的浓度依赖性产生了阴离子的解离常数（从而得到结合能），这些常数与斯托克斯半径有关。如果位点I位于距蛋白质表面几埃的范围内，这些数据符合阴离子与位点I相关正电荷之间静电相互作用的预测。位点I对各种阴离子的特异性可以通过阴离子从水转移到结合位点时玻恩能量变化的差异来定量解释。阴离子结合时席夫碱去质子化能量的变化，即ΔΔGdeprot，可以从无限阴离子浓度下的ΔpKa计算得出。出乎意料的是，ΔΔGdeprot值与结合到位点I的能量非常接近。因此，位点I和席夫碱通过紧密相邻或它们之间变构能量转移的可能性而强烈静电耦合。
