title: Unfolding dynamics of cytochrome c revealed by single-molecule and ensemble-averaged spectroscopy.
abstract: Denaturant-induced conformational change of yeast iso-1-cytochrome c (Cytc) has been comprehensively investigated in the single-molecule and bulk phases. By fluorescence-quenching experiments with dye-labelled heme-protein (Alexa 488-labelled Cytc, Cytc-A488), we clearly show that the fluorescence quenching observed from folded Cytc-A488 is due mainly to photoinduced electron transfer (PET) between electron-donating amino acids such as tryptophan and the dye attached to the protein. In addition, the unfolding process of Cytc-A488 observed in the single-molecule and bulk phases can be explained well in terms of a three-state model: Cytc unfolds through an intermediate with a native-like compactness. By quantitative analysis of fluorescence correlation spectroscopy (FCS) data, we were able to observe a relaxation time of ∼1.5 μs corresponding to segmental motion and fast folding dynamics of 55 μs in the unfolded state of Cytc. The results presented here also suggest that a combination of single-molecule and ensemble-averaged spectroscopy is necessary to provide convincing and comprehensive assignments of protein kinetics.
authors.affiliation: The Institute of Scientific and Industrial Research (SANKEN), Osaka University, Ibaraki, Osaka, Japan.
authors.forename: Jungkweon
authors.initials: J
authors.lastname: Choi
authors.forename: Sooyeon
authors.initials: S
authors.lastname: Kim
authors.forename: Takashi
authors.initials: T
authors.lastname: Tachikawa
authors.forename: Mamoru
authors.initials: M
authors.lastname: Fujitsuka
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Majima
mesh_terms: D045304:Cytochromes c / Q000737:chemistry*; D006834:Hydrazines / Q000737:chemistry*; D008958:Models, Molecular; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence
chemical_list: C417561:Alexa 488 hydrazide; D006834:Hydrazines; D045304:Cytochromes c