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首页> 外文期刊>Biochemistry >Evidence for kinetic intermediate states during the refolding of GdnHCl-denatured MM-creatine kinase. Characterization of a trapped monomeric species
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Evidence for kinetic intermediate states during the refolding of GdnHCl-denatured MM-creatine kinase. Characterization of a trapped monomeric species

机译：GdnHCl变性的MM-肌酸激酶重折叠过程中的动力学中间状态的证据。捕获单体种类的表征

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摘要

The kinetics of refolding of guanidinium chloride-denatured rabbit MM-creatine kinase was investigated. Recovery of enzymatic activity is biphasic, depending on the temperature but not on the protein or DTT concentration. Only 45% of the original, active dimeric form is recovered even after several hours of refolding. The reactivation yield is limited by the accumulation of a highly stable but nonproductive monomeric species. The ratio of "correct" to "incorrect" forms depends on the duration of exposure to the denaturant, which may be consistent with the existence of a heterogeneous population of unfolded states with regard to proline isomerization. The first fast reaction observed during renaturation results in the appearance of collapsed monomeric states, displaying features of a pre-molten globule state. These burst species are rapidly transformed into more structured monomers resembling a molten globule state possessing a partially folded C-terminal domain. A proportion of these latter transient intermediates (45%) associates into an active dimer, while the remainder (55%) is trapped by reshuffling in a monomeric dead-end product. Our results strongly indicate that (i) the dimeric state is a prerequisite for the expression of catalytic activity, (ii) the kinetic intermediates of refolding are very similar to those observed during equilibrium unfolding, and (iii) refolding of creatine kinase in these conditions is limited by the accumulation of inactive misfolded nondimerizable monomer. [References: 57]
机译：研究了氯化胍变性的兔MM-肌酸激酶的复性动力学。酶活性的恢复是双相的，取决于温度，而不取决于蛋白质或DTT的浓度。即使重新折叠数小时，也只能回收到原始活性二聚体形式的45％。再活化产率受到高度稳定但非生产性单体物种的积累的限制。 “正确的”与“错误的”形式的比例取决于暴露于变性剂的持续时间，这与脯氨酸异构化的未折叠状态的异质群体的存在相一致。复性过程中观察到的第一个快速反应导致出现塌陷的单体态，显示出熔融前球状态的特征。这些破裂的物质迅速转变为具有熔融的球状状态，具有部分折叠的C-末端结构域的结构更好的单体。后者中的一部分过渡中间体（45％）缔合为活性二聚体，而其余（55％）通过改组进入单体的最终产物而被捕集。我们的结果强烈表明（i）二聚体状态是表达催化活性的先决条件;（ii）重折叠的动力学中间体与平衡解折叠过程中观察到的非常相似;（iii）在这些条件下肌酸激酶的重折叠受非活性错折叠的不可二聚单体的积累限制。 [参考：57]

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