Conformational fluctuation of native-like and molten-globule-like cytochrome c observed by time-resolved hole burning

Yutaka Shibata, Hirokazu Takahashi, Rina Kaneko, Atusi Kurita, Takashi Kushida

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14 Citations (Scopus)

Abstract

Nanosecond to millisecond conformational fluctuations of Zn-substituted cytochrome c (ZnCytc) have been studied by the time-resolved transient hole- burning method. The investigation of low-temperature dynamics has been made on the ZnCytc solution sample in a water-glycerol mixture. The conformational fluctuations in the native-like and the molten-globule (MG)-like states have been compared for the aqueous solution samples at room temperature. ZnCytc in the MG-like state has been prepared by adding 200 mM NaClO4 to the protein solution with a pH of 2.1, and the formation of the MG-like stare has been confirmed by both the far-UV CD and the visible absorption spectra. The hole spectrum of ZnCytc has been found to consist of two nearly degenerate components, that is, the Q(x) and Q(y) bands. The temporal change in the Q(x) component hole spectrum has been extracted by fitting the observed hole spectrum to the three-Gaussian form. The experimental results for ZnCytc dissolved in a water-glycerol mixture have revealed that the conformational fluctuation of ZnCytc is suppressed around 200 K, which is nearly the same temperature as the glass-like transition point of Zn-substituted myoglobin (ZnMb) and also as the glass-transition point of the solvent. This supports the idea of the solvent-induced glass-like transition of a protein. It has been also found that at physiological temperatures the time scale of the conformational fluctuation of ZnCytc lies around a few tens of nanoseconds, which is 2-3 orders of magnitude faster than that of ZnMb. The experimental results for the aqueous solution samples have shown that the difference between the native-like and the MG-like states is not conspicuous. However, they are indicative of the appearance of the slower conformational fluctuation in the MG-like state.

Original languageEnglish
Pages (from-to)1802-1810
Number of pages9
JournalBiochemistry
Volume38
Issue number6
DOIs
Publication statusPublished - 1999 Feb 9

ASJC Scopus subject areas

  • Biochemistry

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