title: Maintenance of the secondary structure of horse cytochrome c during the conversion process of monomers to oligomers by addition of ethanol.
abstract: We have previously shown that polymerization of cytochrome c (cyt c) occurs by successively domain swapping its C-terminal α-helix in the presence of ethanol. However, the factors that govern the conversion process of monomers to domain-swapped oligomers remain unknown. We found that oligomeric cyt c is produced in the presence of ethanol and the oligomers precipitate due to low solubility. The optical absorption spectra revealed that in the presence of 30-40% ethanol, the Met-heme coordination in cyt c is dissociated. However, according to circular dichroism and small-angle X-ray scattering measurements, the α-helical structure of cyt c is maintained in solution with a little perturbation and the radius of gyration increases slightly but without dissociation of the C-terminal α-helix from the rest of the protein by the addition of ethanol. Solid-state nuclear magnetic resonance spectra showed that oligomeric cyt c in the precipitate also retains most of its α-helical structure. In the transmission electron microscopic image of the precipitate obtained by the addition of ethanol to cyt c, spherical particles with diameters of about 3 nm were detected. These results indicate that oligomeric cyt c forms through a state with the Met80 region locally unfolded, while maintaining the secondary structure, possibly an open monomer.
authors.affiliation: Graduate School of Materials Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan. hirota@ms.naist.jp
authors.forename: Shun
authors.initials: S
authors.lastname: Hirota
authors.forename: Mariko
authors.initials: M
authors.lastname: Ueda
authors.forename: Yugo
authors.initials: Y
authors.lastname: Hayashi
authors.forename: Satoshi
authors.initials: S
authors.lastname: Nagao
authors.forename: Hironari
authors.initials: H
authors.lastname: Kamikubo
authors.forename: Mikio
authors.initials: M
authors.lastname: Kataoka
mesh_terms: D000818:Animals; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D002942:Circular Dichroism; D045304:Cytochromes c / Q000737:chemistry* / Q000302:isolation & purification / Q000648:ultrastructure; D000431:Ethanol / Q000737:chemistry*; D006418:Heme / Q000737:chemistry; D006736:Horses; D009682:Magnetic Resonance Spectroscopy; D046529:Microscopy, Electron, Transmission; D010084:Oxidation-Reduction; D010316:Particle Size; D011485:Protein Binding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D012995:Solubility
chemical_list: D000431:Ethanol; D006418:Heme; D045304:Cytochromes c