Investigations of the Low-Frequency Spectral Density of Cytochrome c upon Equilibrium Unfolding

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• 作者：Yuhan Sun ; Venugopal Karunakaran ; Paul M. Champion
• 刊名：The Journal of Physical Chemistry B
• 出版年：2013
• 出版时间：August 22, 2013
• 年：2013
• 卷：117
• 期：33
• 页码：9615-9625
• 全文大小：458K
• 年卷期：v.117,no.33(August 22, 2013)
• ISSN：1520-5207

文摘

The equilibrium unfolding process of ferric horse heart cytochrome c (cyt c), induced by guanidinium hydrochloride (GdHCl), was studied using UV鈥搗is 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 cyt c unfolding: 纬_a (50 cm^鈥?), 纬_b (80 cm^鈥?), 纬_c (100 cm^鈥?), and 谓_s(His-Fe-His) at 205 cm^鈥?. These out-of-plane modes have potential functional relevance and are activated by protein-induced heme distortions. The free energies for the N鈥揑 and the I鈥揢 transitions at pH 7.0 and 20 掳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 纬_b80 cm^鈥? 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.