The position 68(E11) side chain in myoglobin regulates ligand capture, bond formation with heme iron, and internal movement into the xenon cavities
The position 68(E11) side chain in myoglobin regulates ligand capture, bond formation with heme iron, and internal movement into the xenon cavities
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DOI：
10.1074/jbc.m506333200
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发表时间：
2005-11-18
影响因子：
4.8
通讯作者：
Friedman, JM
中科院分区：
文献类型：
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作者：
Dantsker, D;Roche, C;Friedman, JM
光解离后，配体与肌红蛋白的重新结合表现出与蛋白质内发生的多个一级成对重组过程相关的复杂动力学模式，以及代表来自溶剂的二级配体重新结合的更简单的双分子相。通过使用溶胶-凝胶封装、添加甘油作为沐浴介质和温度调节（- 15 --> 65 摄氏度）的组合，可以获得从类低温到溶液相性质的平滑过渡。该方法应用于一系列双突变体肌红蛋白 CO（H64L/V68X，其中 X = Ala、Val、Leu、Asn 和 Phe），旨在检查在不存在与远端组氨酸的空间和极性相互作用的情况下，位置 68(E11) 侧链对内部重结合相的出现和消失的贡献。根据粘度、温度和 E11 侧链立体化学的影响，三个主要相 B --> A、C --> A 和 D-->A 可以分别从以下位置分配给配体重新结合：(i) 远端血红素口袋，(ii) 大幅侧链构象松弛之前的氙气腔，以及 (iii) 68(E11) 位置侧显着构象松弛后的氙气腔链。 B-->A 和 C-->A 相的相对振幅明显取决于 E11 侧链的大小和形状，E11 侧链在空间上调节配体返回血红素铁原子和配体迁移到氙气腔。内部氙气腔提供了一个短暂的对接位点，允许侧链松弛和水进入空出的远端口袋，这反过来又显着减慢配体重组。
After photodissociation, ligand rebinding to myoglobin exhibits complex kinetic patterns associated with multiple first-order geminate recombination processes occurring within the protein and a simpler bimolecular phase representing second-order ligand rebinding from the solvent. A smooth transition from cryogenic-like to solution phase properties can be obtained by using a combination of sol-gel encapsulation, addition of glycerol as a bathing medium, and temperature tuning (- 15 --> 65 degrees C). This approach was applied to a series of double mutants, myoglobin CO ( H64L/ V68X, where X = Ala, Val, Leu, Asn, and Phe), which were designed to examine the contributions of the position 68( E11) side chain to the appearance and disappearance of internal rebinding phases in the absence of steric and polar interactions with the distal histidine. Based on the effects of viscosity, temperature, and the stereochemistry of the E11 side chain, the three major phases, B --> A, C --> A, and D-->A, can be assigned, respectively, to ligand rebinding from the following: (i) the distal heme pocket, (ii) the xenon cavities prior to large amplitude side chain conformational relaxation, and (iii) the xenon cavities after significant conformational relaxation of the position 68( E11) side chain. The relative amplitudes of the B --> A and C-->A phases depend markedly on the size and shape of the E11 side chain, which regulates sterically both ligand return to the heme iron atom and ligand migration to the xenon cavities. The internal xenon cavities provide a transient docking site that allows side chain relaxations and the entry of water into the vacated distal pocket, which in turn slows ligand recombination markedly.