Kinetic mechanism of folding and unfolding of Rhodobacter capsulatus cytochrome c2.
Kinetic mechanism of folding and unfolding of Rhodobacter capsulatus cytochrome c2.
复制标题
荚膜红细菌细胞色素c2折叠和展开的动力学机制。
DOI：
10.1021/bi961976k
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Roder,H
中科院分区：
文献类型：
--
作者：
Sauder,JM;MacKenzie,NE;Roder,H
尽管存在边缘序列同源性，来自光合细菌的细胞色素c2和线粒体细胞色素c表现出一些惊人的结构相似性，包括三个主要螺旋的三级排列。为了比较这两个关系较远的蛋白质组的折叠机制，在存在和不存在稳定盐硫酸钠的情况下，对来自荚膜红杆菌的细胞色素c2的折叠/解折叠反应进行平衡和动力学测量，将其作为盐酸胍(GuHCl)浓度的函数。血红素对 Trp67 荧光的猝灭被用作构象探针。避免了由于非天然组氨酸连接引起的动力学复杂性，因为细胞色素c2仅含有一种组氨酸His17，它在天然和变性条件下形成轴向血红素配体。定量动力学模型表明平衡和动力学结果与具有两个连续中间体的最小四态机制一致。在低 GuHCl 浓度下停流测量（爆发相）的 2 ms 死区时间内观察到荧光大幅下降，随后初始振幅向展开过渡区域呈 S 形恢复，这归因于与展开分子快速交换的密集折叠中间体。低 GuHCl 浓度下几乎不依赖变性剂的过程反映了紧凑中间体向天然状态的限速转化。在高 GuHCl 浓度下，一个几乎不依赖变性剂的过程归因于解折叠过程中限速的 Met96−铁去连接过程，这一过程得到了咪唑结合动力学的支持。平衡转变中点附近折叠和展开速率对 GuHCl 的强烈依赖性归因于每个中间体及其在折叠和展开过程中的过渡态的不稳定。硫酸钠的添加将速率曲线转变为更高的变性剂浓度，这可以从盐对部分和完全折叠状态的相对稳定作用来理解。
In spite of marginal sequence homology, cytochromec2from photosynthetic bacteria and the mitochondrial cytochromescexhibit some striking structural similarities, including the tertiary arrangement of the three main helices. To compare the folding mechanisms for these two distantly related groups of proteins, equilibrium and kinetic measurements of the folding/unfolding reaction of cytochromec2fromRhodobacter capsulatuswere performed as a function of guanidine hydrochloride (GuHCl) concentration in the absence and presence of a stabilizing salt, sodium sulfate. Quenching of the fluorescence of Trp67 by the heme was used as a conformational probe. Kinetic complexities due to non-native histidine ligation are avoided, since cytochromec2contains only one histidine, His17, which forms the axial heme ligand under native and denaturing conditions. Quantitative kinetic modeling showed that both equilibrium and kinetic results are consistent with a minimal four-state mechanism with two sequential intermediates. The observation of a large decrease in fluorescence during the 2-ms dead-time of the stopped-flow measurement (burst phase) at low GuHCl concentration, followed by a sigmoidal recovery of the initial amplitude toward the unfolding transition region, is attributed to a well-populated compact folding intermediate in rapid exchange with unfolded molecules. A nearly denaturant-independent process atlowGuHCl concentrations reflects the rate-limiting conversion of a compact intermediate to the native state. AthighGuHCl concentrations, a process with little denaturant dependence is attributed to the rate-limiting Met96−iron deligation process during unfolding, which is supported by the kinetics of imidazole binding. The strong GuHCl-dependence of folding and unfolding rates near the midpoint of the equilibrium transition is attributed to destabilization of each intermediate and their transition states in folding and unfolding. Addition of sodium sulfate shifts the rate profile to higher denaturant concentration, which can be understood in terms of the relative stabilizing effect of the salt on partially and fully folded states.