Evidence for kinetic intermediate states during the refolding of GdnHCl-denatured MM-creatine kinase. Characterization of a trapped monomeric species
Evidence for kinetic intermediate states during the refolding of GdnHCl-denatured MM-creatine kinase. Characterization of a trapped monomeric species
复制标题
DOI：
10.1021/bi981828p
复制
发表时间：
1998-12-15
期刊：
影响因子：
2.9
通讯作者：
Vial, C
中科院分区：
文献类型：
--
作者：
Leydier, C;Clottes, E;Vial, C
研究了氯化胍变性兔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，而不是蛋白质或DTT的浓度。即使经过几个小时的重折叠，也只有45%的原始活性二聚体形式被恢复。复活率受到高度稳定但非生产性单体物种的积累的限制。“正确”和“不正确”形式的比率取决于暴露在变性剂中的时间，这可能与关于Pro异构化的未折叠状态的异质性群体的存在一致。在复性过程中观察到的第一个快速反应导致出现坍塌的单体状态，显示出预熔化球状状态的特征。这些爆裂物种迅速转化为更具结构的单体，类似于熔融的球状状态，具有部分折叠的C-末端结构域。这些瞬时中间体中的一部分(45%)结合成活性二聚体，而其余的(55%)通过重新洗牌被捕获在单体的死端产物中。我们的结果有力地表明：(I)二聚体状态是表达催化活性的先决条件，(Ii)复性的动力学中间体与平衡去折叠时观察到的非常相似，以及(Iii)在这些条件下肌酸激酶的复性受到非活性错误折叠的非二聚单体积累的限制。
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.