Ligand and proton exchange dynamics in recombinant human myoglobin mutants.
Ligand and proton exchange dynamics in recombinant human myoglobin mutants.
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DOI：
10.1016/0022-2836(89)90456-7
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发表时间：
1989-05
影响因子：
5.6
通讯作者：
D. Lambright;S. Balasubramanian;S. Boxer
中科院分区：
文献类型：
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作者：
D. Lambright;S. Balasubramanian;S. Boxer
已经制备了人肌红蛋白的位点特异性突变体，其中赖氨酸45被精氨酸（K45R）取代，天冬氨酸60被谷氨酸（D60E）取代，以检查这些残基及其相互作用对蛋白质动力学的影响。这些蛋白质通过多种方法进行了研究，包括一维和二维质子核磁共振波谱，远端和近端组氨酸NH质子的交换动力学（作为pH值的函数），CO形式的闪光光解和配体替代动力学。这些蛋白质的CO形式的电子吸收和质子核磁共振谱几乎相同，表明血红素口袋的结构没有被这些突变改变。然而，突变体K45R的CO结合和质子交换动力学都发生了实质性的变化，而突变体D60E的行为与参考的人肌红蛋白没有区别。相对于参比物，K45R具有较快的CO双分子重组速率和较慢的CO脱配速率。CO结合动力学与pH（6.5 ~ 10）和离子强度（0 ~ 1m-NaCl）无关。在K45R中，远端组氨酸NH的交换率明显低于参比物，而近端组氨酸NH的交换率没有改变。人类蛋白质的交换行为类似于在45号位置有赖氨酸的肌红蛋白与在这个位置有精氨酸的抹香鲸肌红蛋白交换率的比较。这表明，汇率差异在很大程度上反映了赖氨酸→精氨酸的替代作用。缺乏CO动力学与这种取代的简单相关性意味着它们对其他因素也很敏感。具体的动力学模型，其中精氨酸取代赖氨酸在位置45可以影响配体结合动力学，概述。这些实验表明，表面残基的相对保守变化可以以一种不易从静态结构预测的方式实质性地扰乱配体和质子交换动力学。
Site-specific mutants of human myoglobin have been prepared in which lysine 45 is replaced by arginine (K45R) and aspartate 60 by glutamate (D60E), in order to examine the influence of these residues and their interaction on the dynamics of the protein. These proteins were studied by a variety of methods, including one and two-dimensional proton nuclear magnetic resonance spectroscopy, exchange kinetics for the distal and proximal histidine NH protons as a function of pH in the met cyano forms, flash photolysis of the CO forms, and ligand replacement kinetics.The electronic absorption and proton nuclear magnetic resonance spectra of the CO forms of these proteins are virtually identical, indicating that the structure of the heme pocket is unaltered by these mutations. There are, however, substantial changes in the dynamics of both CO binding and proton exchange for the mutant K45R, whereas the mutant D60E exhibits behavior indistinguishable from the reference human myoglobin. K45R has a faster CO bimolecular recombination rate and slower CO off-rate relative to the reference. The kinetics for CO binding are independent of pH (6.5 to 10) as well as ionic strength (0 to 1m-NaCl). The exchange rate for the distal histidine NH is substantially lower for K45R than the reference, whereas the proximal histidine NH exchange rate is unaltered. The exchange behavior of the human proteins is similar to that reported for a comparison of the exchange rates for myoglobins having lysine at position 45 with sperm whale myoglobin, which has arginine at this position. This indicates that the differences in exchange rates reflects largely the Lys → Arg substitution. The lack of a simple correlation for the CO kinetics with this substitution means that these are sensitive to other factors as well. Specific kinetic models, whereby substitution of arginine for lysine at position 45 can affect ligand binding dynamics, are outlined. These experiments demonstrate that a relatively conservative change of a surface residue can substantially perturb ligand and proton exchange dynamics in a manner that is not readily predicted from the static structures.