Characterization of kinetic folding intermediates of recombinant canine milk lysozyme by stopped-flow circular dichroism.
Characterization of kinetic folding intermediates of recombinant canine milk lysozyme by stopped-flow circular dichroism.
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DOI：
10.1021/bi050082
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发表时间：
2005-05
期刊：
影响因子：
2.9
通讯作者：
Masaharu Nakao;K. Maki;M. Arai;T. Koshiba;K. Nitta;K. Kuwajima
中科院分区：
文献类型：
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作者：
Masaharu Nakao;K. Maki;M. Arai;T. Koshiba;K. Nitta;K. Kuwajima
由变性剂诱导的犬乳溶菌酶平衡去折叠的中间体非常稳定，具有熔融球状状态的特征。此外，该蛋白质在复性过程中至少存在两个动力学中间体：一个是在停流测量的死区时间内形成的爆发期(第一)中间体，另一个是累积的速率常数为22的S(-)(1)中间体。为了弄清这些中间体与平衡中间体的关系，也为了表征蛋白质在复性过程中的结构变化，我们用10个不同波长的停流圆二色谱研究了这些中间体的动力学复性反应，得到了中间体的圆二色谱。比较了中间体的圆二色谱，以及从完全展开状态到平衡中间体的折叠过程中没有观察到的动力学，证明了爆裂相中间体与平衡中间体是等价的。代表从动力学中间体到天然状态变化的差异圆二色谱具有激子耦合带的特征，表明该蛋白质中的色氨酸残基在这一阶段发生了特异的堆积。根据这些发现，我们提出了一个与蛋白质折叠的分级机制相一致的犬乳溶菌酶的复性模型。
The intermediate in the equilibrium unfolding of canine milk lysozyme induced by a denaturant is known to be very stable with characteristics of the molten globule state. Furthermore, there are at least two kinetic intermediates during refolding of this protein: a burst-phase (first) intermediate formed within the dead time of stopped-flow measurements and a second intermediate that accumulates with a rate constant of 22 s(-)(1). To clarify the relationships of these intermediates with the equilibrium intermediate, and also to characterize the structural changes of the protein during refolding, here we studied the kinetic refolding reactions using stopped-flow circular dichroism at 10 different wavelengths and obtained the circular dichroism spectra of the intermediates. Comparison of the circular dichroism spectra of the intermediates, as well as the absence of observed kinetics in the refolding from the fully unfolded state to the equilibrium intermediate, has demonstrated that the burst-phase intermediate is equivalent to the equilibrium intermediate. The difference circular dichroism spectrum that represented changes from the kinetic intermediate to the native state had characteristics of an exciton coupling band, indicating that specific packing of tryptophan residues in this protein occurred in this phase. From these findings, we propose a schematic model of the refolding of canine milk lysozyme that is consistent with the hierarchical mechanism of protein folding.