Bashford D, Case D A, Dalvit C, Tennant L, Wright P E
Department of Molecular Biology, Scripps Research Institute, La Jolla, California 92037.
Biochemistry. 1993 Aug 10;32(31):8045-56. doi: 10.1021/bi00082a027.
Site-specific titration curves for 12 histidine residues in carbon monoxy sperm whale myoglobin (MbCO) have been determined from two-dimensional (2D) double quantum NMR experiments. Eight of these histidine residues are observed to titrate over the accessible pH range, and pK(a) values have been determined; bounds on the titration midpoints of the remaining four histidines are also reported. Results for residues 48, 81, and 119 differ significantly from those estimated from earlier, one-dimensional studies, but they are in good agreement with values recently determined for metaquomyoglobin. These experimental values (plus those determined earlier for tyrosine titrations) are compared to predictions from crystal structures of myoglobin using a numerical Poisson-Boltzmann model and a Monte Carlo treatment of the multiple-site titration. An extension of existing models is described that accounts for alternate tautomers for histidines. Calculations are reported using several choices for radii and charges, and for five crystal structures, in order to assess the sensitivity of the results to details of the calculations. In general, the agreement between calculated and observed titration behavior suggests that this theoretical model captures much of the electrostatic behavior in this system, even though it ignores conformational fluctuations and the differences in mean structures that may exist between crystal and solution. Interactions among titrating groups are often important; in general, these interactions lead to more gradual individual site titrations (the mean Hill coefficient is about 0.8), and in several cases the interactions are so strong that two side chains need to be considered as a unit and single residues may participate in two-step titrations. It is suggested that histidines involved in such two-step titrations and carboxylic acid residues with abnormally low pK(a) values in the native conformation may be involved in the acid-induced partial unfolding of MbCO.
通过二维（2D）双量子核磁共振实验，已确定了一氧化碳抹香鲸肌红蛋白（MbCO）中12个组氨酸残基的位点特异性滴定曲线。观察到其中8个组氨酸残基在可及的pH范围内发生滴定，并已确定了pK(a)值；还报告了其余4个组氨酸滴定中点的界限。残基48、81和119的结果与早期一维研究估计的结果有显著差异，但与最近测定的间位肌红蛋白的值非常一致。使用数值泊松-玻尔兹曼模型和多位点滴定的蒙特卡罗处理方法，将这些实验值（加上早期酪氨酸滴定确定的值）与肌红蛋白晶体结构的预测值进行了比较。描述了现有模型的扩展，该扩展考虑了组氨酸的交替互变异构体。为了评估结果对计算细节的敏感性，报告了使用几种半径和电荷选择以及五种晶体结构的计算结果。总体而言，计算和观察到的滴定行为之间的一致性表明该理论模型捕捉了该系统中的许多静电行为，尽管它忽略了构象波动以及晶体和溶液之间可能存在的平均结构差异。滴定基团之间的相互作用通常很重要；一般来说，这些相互作用导致单个位点的滴定更加平缓（平均希尔系数约为0.8），并且在几种情况下，相互作用非常强，以至于两个侧链需要作为一个单元来考虑，单个残基可能参与两步滴定。有人认为，参与这种两步滴定的组氨酸以及天然构象中pK(a)值异常低的羧酸残基可能参与了酸诱导的MbCO部分展开。
