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首页> 外文期刊>Journal of Molecular Biology >The Natively Helical Chain Segment 169-188 of Escherichia coli Adenylate Kinase is Formed in the Latest Phase of the Refolding Transition.
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The Natively Helical Chain Segment 169-188 of Escherichia coli Adenylate Kinase is Formed in the Latest Phase of the Refolding Transition.

机译：大肠杆菌腺苷酸激酶的天然螺旋链节169-188形成于重折叠转变的最新阶段。

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摘要

The refolding transition of Escherichia coli adenylate kinase (AK) was investigated by monitoring the refolding kinetics of a selected 20 residue helical segment in the CORE domain of the protein. Residues 169 and 188 were labeled by 1-acetamido-methyl-pyrene, and by bimane, respectively. The experiment combines double-jump stopped-flow fast mixing initiation of refolding and time-resolved Forster energy transfer spectroscopy for monitoring the conformational transitions (double-kinetics experiment). Two kinetic phases were found in the denaturant-induced unfolding of AK. In the first phase, the fluorescence quantum yields of both probes decreased. The distribution of the distances between them transformed from the native state's narrow distribution with the mean distance corresponding to the distance in the crystal structure, to a distribution compatible with an unordered structure. In the second, slow step of denaturation, neither the fluorescence parameters of the probes nor the distance distribution between them changed. This step appeared to be a transformation of the fast-folding species formed in the first phase, to the slow-folding species. Refolding of the fast-folding species of the denatured state of AK was also a two-phase process. During the first fast phase, within less than 5ms, the fluorescence emission of both probes increased, but the distance distribution between the labeled sites was unchanged. Only during the second slow refolding step did the intramolecular distance distribution change from the characteristic of the denatured state to the narrow distribution of the native state. This experiment shows that for the case of the CORE domain of AK, the large helical segment of residues 169-188 was not formed in the first compaction step of refolding. The helical conformation of this segment is established only in the second, much slower, refolding phase, simultaneously with the completion of the native structure.
机译：通过监测蛋白质的CORE域中选定的20个残基螺旋片段的重折叠动力学，研究了大肠杆菌腺苷酸激酶（AK）的重折叠过渡。残基169和188分别用1-乙酰氨基甲基甲基py和Bimane标记。该实验结合了重折叠的双跳停流快速混合起始和时间分辨的Forster能量转移光谱，用于监测构象转变（双动力学实验）。在变性剂诱导的AK展开中发现了两个动力学相。在第一阶段，两种探针的荧光量子产率均降低。它们之间的距离分布从原始状态的窄分布（其平均距离对应于晶体结构中的距离）转变为与无序结构兼容的分布。在第二个缓慢的变性步骤中，探针的荧光参数和探针之间的距离分布都没有改变。此步骤似乎是将第一阶段形成的快速折叠物种转变为缓慢折叠物种。 AK变性状态的快速折叠物种的重折叠也是一个两阶段过程。在第一个快速阶段中，在不到5毫秒内，两个探针的荧光发射都增加了，但标记位点之间的距离分布没有变化。仅在第二缓慢重折叠步骤中，分子内距离分布才从变性状态的特征变为天然状态的窄分布。该实验表明，对于AK的CORE域，在重折叠的第一个压实步骤中未形成残基169-188的大螺旋段。该片段的螺旋构象仅在第二个慢得多的重折叠阶段建立，同时完成天然结构。

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