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

Y. Shibata et al., Conformational fluctuation of native-like and molten-globule-like cytochrome c observed by time-resolved hole burning, BIOCHEM, 38(6), 1999, pp. 1802-1810
Citations number
Categorie Soggetti
Biochemistry & Biophysics
Journal title
ISSN journal
0006-2960 → ACNP
Year of publication
1802 - 1810
SICI code
Nanosecond to millisecond conformational fluctuations of Zn-substituted cyt ochrome c (ZnCytc) have been studied by the rime-resolved transient hole-bu rning method. The investigation of low-temperature dynamics has been made o n the ZnCytc solution sample in a water-glycerol mixture. The conformationa l fluctuations in the native-like and the molten-globule (MG)-like states h ave been compared for the aqueous solution samples at room temperature. ZnC ytc in the MG-like state has been prepared by adding 200 mM NaClO4 to the p rotein solution with a pH of 2.1, and the formation of the MG-like state ha s 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 degener ate components, that is, the Q(x) and Q(y) bands. The temporal change in th e Q(x) component hole spectrum has been extracted by fitting the observed h ole spectrum to the three-Gaussian form. The experimental results for ZnCyt c dissolved in a water-glycerol mixture have revealed that the conformation al fluctuation of ZnCytc is suppressed around 200 K, which is nearly the sa me temperature as the glass-like transition point of Zn-substituted myoglob in (ZnMb)and also as the glass-transition point of the solvent. This suppor ts the idea of the solvent-induced glass-like transition of a protein. It h as 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 experimen tal 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 fluctu ation in the MG-like state.