Structural characterization of an equilibrium unfolding intermediate in cytochrome c
Structural characterization of an equilibrium unfolding intermediate in cytochrome c
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DOI：
10.1016/j.jmb.2006.01.055
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发表时间：
2006-03-31
影响因子：
5.6
通讯作者：
Roder, H
中科院分区：
文献类型：
--
作者：
Latypov, RF;Cheng, H;Roder, H
虽然变性剂诱导的细胞色素c的去折叠转变最初被认为是一个合作的过程，最近的光谱研究表明，从两个状态的行为与平衡中间体的积累一致的偏差。然而，很少有人知道这种状态的结构和热力学性质，以及它是否是稳定的非天然血红素配体的存在下。我们使用各种光谱探针，包括荧光，近紫外和远紫外CD，血红素吸收带的Soret可见和近红外光谱区域，以及二维NMR监测可逆变性剂诱导的氧化马细胞色素c的展开平衡。全局拟合技术被用于定量解释的结果在一个三态模型，这使我们能够确定的内在光谱性质的中间体。在pH 5的平衡实验中，使用盐酸胍或尿素作为变性剂，观察到一个良好的填充中间体，无论是野生型细胞色素c以及H33 N突变体选择，以防止形成非天然的组氨酸-血红素连接。对于中间体的更详细的结构表征，我们使用2D H-1-N-15相关光谱来跟踪各个骨架酰胺基团的峰强度变化。在我们的光学和NMR研究中观察到的平衡态包含许多天然的结构特征，包括结构良好的α-螺旋子域，短的Trp 59-血红素距离和溶剂屏蔽的血红素环境，但缺乏天然的Met 80硫-铁连接，并显示在侧链包装和其他三级相互作用的主要扰动。这些结构性质让人想起细胞色素c的A-状态，在酸性高盐条件下发现的紧凑的变性形式，以及在折叠后期填充的动力学中间体。变性剂诱导的中间体也类似于改变血红素连接的碱性形式的细胞色素c，这表明天然甲硫氨酸配体的破坏有利于在存在和不存在非天然配体的情况下积累结构类似的状态。(c)2006爱思唯尔有限公司保留所有权利。
Although the denaturant-induced unfolding transition of cytochrome c was initially thought to be a cooperative process, recent spectroscopic studies have shown deviations from two-state behavior consistent with accumulation of an equilibrium intermediate. However, little is known about the structural and thermodynamic properties of this state, and whether it is stabilized by the presence of non-native heme ligands. We monitored the reversible denaturant-induced unfolding equilibrium of oxidized horse cytochrome c using various spectroscopic probes, including fluorescence, near and far-UV CD, heme absorbance bands in the Soret visible and near-IR regions of the spectrum, as well as 2D NMR. Global fitting techniques were used for a quantitative interpretation of the results in terms of a three-state model, which enabled us to determine the intrinsic spectroscopic properties of the intermediate. A well-populated intermediate was observed in equilibrium experiments at pH 5 using either guanidine-HCl or urea as a denaturant, both for wild-type cytochrome c as well as an H33N mutant chosen to prevent formation of non-native His-heme ligation. For a more detailed structural characterization of the intermediate, we used 2D H-1-N-15 correlation spectroscopy to follow the changes in peak intensity for individual backbone amide groups. The equilibrium state observed in our optical and NMR studies contains many native-like structural features, including a well-structured alpha-helical subdomain, a short Trp59-heme distance and solvent-shielded heme environment, but lacks the native Met80 sulfur-iron linkage and shows major perturbations in side-chain packing and other tertiary interactions. These structural properties are reminiscent of the A-state of cytochrome c, a compact denatured form found under acidic high-salt conditions, as well as a kinetic intermediate populated at a late stage of folding. The denaturant-induced intermediate also resembles alkaline forms of cytochrome c with altered heme ligation, suggesting that disruption of the native methionine ligand favors accumulation of structurally analogous states both in the presence and absence of non-native ligands. (c) 2006 Elsevier Ltd. All rights reserved.