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首页> 外文期刊>Biochemistry >Mechanism of folding of the dimeric core domain of Escherichia coli Trp repressor: A nearly diffusion-limited reaction leads to the formation of an on-pathway dimeric intermediate
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Mechanism of folding of the dimeric core domain of Escherichia coli Trp repressor: A nearly diffusion-limited reaction leads to the formation of an on-pathway dimeric intermediate

机译：大肠杆菌Trp阻遏物的二聚体核心结构域折叠的机制：几乎受扩散限制的反应导致路径上的二聚体中间体的形成

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摘要

A polypeptide corresponding to the core/dimerization domain of E. coli Trp repressor (TR), [2-66](2) TR, was constructed by insertion of a pair of stop codons into the trpR gene. The kinetic properties of the urea-induced folding of this core fragment were examined by intrinsic tryptophan fluorescence (FL) and circular dichroism (CD) spectroscopy. The kinetic response of wild-type TR (WT TR) is very complicated and has been interpreted to involve three parallel channels with multiple folding and isomerization reactions (Mann et al. (1995) Biochemistry 34, 14573-14580). The refolding of [2-66]2 TR can be described by a much simpler mechanism, involving an association reaction followed by a urea-dependent first-order folding reaction. The second-order rate constant for the association reaction approaches that of the diffusion limit, 3 x 10(8) M-1 s(-1) in 1 M urea at 15 degrees C. Double-jump experiments demonstrate that greater than or equal to 93% of the unfolded monomers proceed to the native dimer via the dimeric intermediate; several lines of evidence demonstrate that this dimeric species is an on-pathway intermediate. The subsequent first-order folding reaction of the dimeric intermediate to the native species involves development of additional secondary structure and tertiary structure. The kinetic folding mechanism of [2-66](2) TR suggests that: (1) the complexity of the folding kinetics of full-length WT TR arises from alternative interactions of the DNA reading heads with the dimerization core domain-not from the intertwined nature of the dimerization interface; (2) residues 2-66 contain all of the sequence information necessary to direct the near-diffusion-limited association reaction in a TR folding reaction; and (3) the formation of secondary and tertiary structure is concurrent with or precedes dimerization, and further development certainly follows the formation of quaternary structure. [References: 55]
机译：通过将一对终止密码子插入trpR基因，构建了对应于大肠杆菌Trp阻遏物（TR）核心/二聚体结构域的多肽[2-66]（2）TR。通过固有的色氨酸荧光（FL）和圆二色性（CD）光谱检查了尿素诱导的该核心片段折叠的动力学特性。野生型TR（WT TR）的动力学响应非常复杂，并且已经被解释为涉及具有多个折叠和异构化反应的三个平行通道（Mann等人（1995）Biochemistry 34，14573-14580）。 [2-66] 2 TR的重折叠可以用一种更简单的机制来描述，包括缔合反应，然后是尿素依赖性的一级折叠反应。缔合反应的二级速率常数接近于15°C在1 M尿素中的扩散极限3 x 10（8）M-1 s（-1）的常数。两次跳跃实验表明，大于或等于93％的未折叠单体通过二聚中间体进入天然二聚体;几条证据表明，该二聚体物质是一种途中中间体。二聚体中间体与天然物种的随后的一级折叠反应涉及另外的二级结构和三级结构的发展。 [2-66]（2）TR的动力学折叠机制表明：（1）全长WT TR的折叠动力学的复杂性是由DNA阅读头与二聚化核心结构域的交替相互作用引起的，而不是由二聚化界面的交织性质; （2）残基2-66包含在TR折叠反应中指导近扩散限制缔合反应所必需的所有序列信息; （3）二级和三级结构的形成与二聚化同时发生或先于二聚化，并且肯定会随着四级结构的形成而进一步发展。 [参考：55]

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