Sauder J M, MacKenzie N E, Roder H
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
Biochemistry. 1996 Dec 24;35(51):16852-62. doi: 10.1021/bi961976k.
In spite of marginal sequence homology, cytochrome c2 from photosynthetic bacteria and the mitochondrial cytochromes c exhibit 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 cytochrome c2 from Rhodobacter capsulatus were 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 cytochrome c2 contains 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 at low GuHCl concentrations reflects the rate-limiting conversion of a compact intermediate to the native state. At high GuHCl 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.
尽管光合细菌的细胞色素c2与线粒体细胞色素c的序列同源性很低，但它们在结构上表现出一些显著的相似性，包括三个主要螺旋的三级排列。为了比较这两组远缘相关蛋白质的折叠机制，我们在有无稳定盐硫酸钠的情况下，对来自荚膜红细菌的细胞色素c2的折叠/去折叠反应进行了平衡和动力学测量，测量结果是盐酸胍（GuHCl）浓度的函数。利用血红素对色氨酸67荧光的猝灭作为构象探针。由于细胞色素c2仅含有一个组氨酸His17，且在天然和变性条件下均形成轴向血红素配体，因此避免了非天然组氨酸连接引起的动力学复杂性。定量动力学建模表明，平衡和动力学结果均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，停流测量的2毫秒死时间（爆发相）内荧光大幅下降，随后初始振幅向去折叠转变区域呈S形恢复，这归因于与未折叠分子快速交换的大量存在的紧密折叠中间体。低GuHCl浓度下几乎与变性剂无关的过程反映了紧密中间体向天然状态的限速转化。在高GuHCl浓度下，几乎不依赖变性剂的过程归因于去折叠过程中限速的甲硫氨酸96-铁解连接过程，咪唑结合动力学支持了这一点。平衡转变中点附近折叠和去折叠速率对GuHCl的强烈依赖性归因于折叠和去折叠过程中每个中间体及其过渡态的不稳定。添加硫酸钠将速率曲线移至更高的变性剂浓度，这可以从盐对部分折叠和完全折叠状态的相对稳定作用来理解。
