Latypov Ramil F, Cheng Hong, Roder Navid A, Zhang Jiaru, Roder Heinrich
Basic Science Division, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA.
J Mol Biol. 2006 Mar 31;357(3):1009-25. doi: 10.1016/j.jmb.2006.01.055. Epub 2006 Feb 3.
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 1H-15N 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 sub-domain, 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.
虽然最初认为细胞色素c的变性剂诱导的去折叠转变是一个协同过程，但最近的光谱研究表明其偏离了二态行为，这与平衡中间体的积累一致。然而，对于该状态的结构和热力学性质以及它是否由非天然血红素配体的存在而稳定化知之甚少。我们使用各种光谱探针监测了氧化马细胞色素c的可逆变性剂诱导的去折叠平衡，这些探针包括荧光、近紫外和远紫外圆二色性、光谱的Soret、可见和近红外区域中的血红素吸收带以及二维核磁共振。全局拟合技术用于根据三态模型对结果进行定量解释，这使我们能够确定中间体的固有光谱性质。在pH 5的平衡实验中，使用盐酸胍或尿素作为变性剂，野生型细胞色素c以及选择用于防止形成非天然组氨酸-血红素连接的H33N突变体均观察到了一个丰度较高的中间体。为了对中间体进行更详细的结构表征，我们使用二维1H-15N相关光谱来跟踪各个主链酰胺基团峰强度的变化。我们在光学和核磁共振研究中观察到的平衡状态包含许多类似天然的结构特征，包括结构良好的α-螺旋亚结构域、较短的色氨酸59-血红素距离和溶剂屏蔽的血红素环境，但缺乏天然的甲硫氨酸80硫-铁连接，并且在侧链堆积和其他三级相互作用中表现出主要扰动。这些结构性质让人联想到细胞色素c的A状态，这是一种在酸性高盐条件下发现的紧密变性形式，也是折叠后期出现的动力学中间体。变性剂诱导的中间体也类似于血红素连接改变的细胞色素c的碱性形式，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。
