ELECTROSTATIC CALCULATIONS OF SIDE-CHAIN PK(A) VALUES IN MYOGLOBIN AND COMPARISON WITH NMR DATA FOR HISTIDINES
ELECTROSTATIC CALCULATIONS OF SIDE-CHAIN PK(A) VALUES IN MYOGLOBIN AND COMPARISON WITH NMR DATA FOR HISTIDINES
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DOI：
10.1021/bi00082a027
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发表时间：
1993-08-10
期刊：
影响因子：
2.9
通讯作者：
WRIGHT, PE
中科院分区：
文献类型：
--
作者：
BASHFORD, D;CASE, DA;WRIGHT, PE
通过二维双量子核磁共振实验测定了一氧化碳抹香鲸肌红蛋白(MbCO)中12个组氨酸残基的定点滴定曲线。观察到其中8个组氨酸残基在可达的pH范围内滴定，并测定了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的估计值有很大不同，但它们与最近测定的偏水肌红蛋白的值非常一致。这些实验值(加上之前确定的酪氨酸滴定)与使用数值泊松-玻尔兹曼模型和多点滴定的蒙特卡罗处理的肌红蛋白晶体结构的预测进行了比较。描述了现有模型的扩展，它解释了组氨酸的替代互变构体。为了评估结果对计算细节的敏感性，报告了使用半径和电荷以及五种晶体结构的几种选择进行计算。总体而言，计算的滴定行为与观察到的滴定行为之间的一致性表明，尽管该理论模型忽略了构象涨落和晶体和溶液之间可能存在的平均结构的差异，但它捕捉到了该体系中的大部分静电行为。滴定基团之间的相互作用通常很重要；通常，这些相互作用会导致更渐进的单点滴定(平均Hill系数约为0.8)，在某些情况下，相互作用非常强，需要将两个侧链视为一个单位，单个残基可能参与两步滴定。推测这种两步滴定中的组氨酸和天然构象中具有异常低pK(A)值的羧酸残基可能参与了酸诱导的MbCO的部分去折叠。
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.