Investigations of the low-frequency spectral density of cytochrome c upon equilibrium unfolding.
Investigations of the low-frequency spectral density of cytochrome c upon equilibrium unfolding.
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DOI：
10.1021/jp404881k
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发表时间：
2013-08-22
期刊：
影响因子：
--
通讯作者：
Champion PM
中科院分区：
文献类型：
--
作者：
Sun Y;Karunakaran V;Champion PM
用紫外-可见吸收光谱、共振拉曼光谱和振动相干光谱研究了盐酸胍(GdHCl)诱导的马心细胞色素c(Cytc)的平衡去折叠过程。使用包括完全折叠(N)和未折叠(U)状态的三态模型以及分配给Lys结合的血红素的中间体(I)成功地拟合了去折叠过程。VCS谱首次揭示了几种对细胞色素c展开敏感的低频血红素模式：γa(~50 cm−1)，γb(~80 cm−1)，γc(~100 cm−1)和VS(His-Fe-His)，位于205cM−1。这些平面外模式具有潜在的功能相关性，并被蛋白质诱导的血红素扭曲激活。在pH 7.0和20°C时，N-I和I-U转变的自由能分别为4.6kcal/M和11.6kcal/M。同时引入咪唑取代蛋氨酸配体，使其展开过程可以模拟为两态体系。在展开过程中，γb~80 cm−1模的强度几乎保持不变，而其他低频模的幅度与在较高频率观察到的光谱变化相一致。这证实了血红素变形的变化与蛋白质在展开时发生的三级结构变化有关。这些研究还表明，相干振荡的衰减敏感地依赖于血红素与周围水溶剂之间的耦合。
The equilibrium unfolding process of ferric horse heart cytochrome c (cyt c), induced by guanidinium hydrochloride (GdHCl), was studied using UV-vis absorption spectroscopy, resonance Raman spectroscopy and vibrational coherence spectroscopy (VCS). The unfolding process was successfully fit using a three-state model which included the fully folded (N) and unfolded (U) states, along with an intermediate (I) assigned to a Lys bound heme. The VCS spectra revealed for the first time several low frequency heme modes that are sensitive to cytochrome c unfolding: γa (~50 cm−1), γb (~80cm−1), γc (~100cm−1), and vs(His-Fe-His) at 205 cm−1. These out-of-plane modes have potential functional relevance and are activated by protein-induced heme distortions. The free energies for the N-I and the I-U transitions at pH 7.0 and 20°C were found to be 4.6 kcal/M and 11.6 kcal/M, respectively. Imidazole was also introduced to replace the methionine ligand so the unfolding can be modeled as a two-state system. The intensity of the mode γb~80 cm−1 remains nearly constant during the unfolding process, while the amplitudes of the other low frequency modes track with spectral changes observed at higher frequency. This confirms that the heme deformation changes are coupled to the protein tertiary structural changes that take place upon unfolding. These studies also reveal that damping of the coherent oscillations depends sensitively on the coupling between heme and the surrounding water solvent.
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