title: Formation of oligomeric cytochrome c during folding by intermolecular hydrophobic interaction between N- and C-terminal α-helices.
abstract: We have previously shown that horse cytochrome c (cyt c) forms oligomers by domain swapping its C-terminal α-helix when interacting with ethanol. Although folding of cyt c has been studied extensively, formation of domain-swapped oligomers of cyt c during folding has never been reported. We found that domain-swapped oligomeric cyt c is produced during refolding from its guanidinium ion-induced unfolded state at high protein concentrations and low temperatures. The obtained dimer exhibited a domain-swapped structure exchanging the C-terminal α-helical region between molecules. The extent of dimer formation decreased significantly for the folding of C-terminal cyt c mutants with reduced hydrophobicity achieved by replacement of hydrophobic residues with Gly in the C-terminal region, whereas a large amount of heterodimers was generated for the folding of a mixture of N- and C-terminal mutants. These results show that cyt c oligomers are formed through intermolecular hydrophobic interaction between the N- and C-terminal α-helices during folding. A slow phase (4-5 s) was observed in addition to a 400-500 ms phase during folding of a high concentration of cyt c in the presence of 1.17 M guanidine hydrochloride. The fast phase is attributed to the intramolecular ligand exchange process, and we attribute the slow phase to the ligand exchange process in oligomers. These results show that it is important to consider formation of domain-swapped oligomeric proteins when folding at high protein concentrations.
authors.affiliation: Graduate School of Materials Science, Nara Institute of Science and Technology , 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
authors.forename: Partha Pratim
authors.initials: PP
authors.lastname: Parui
authors.forename: Megha Subhash
authors.initials: MS
authors.lastname: Deshpande
authors.forename: Satoshi
authors.initials: S
authors.lastname: Nagao
authors.forename: Hironari
authors.initials: H
authors.lastname: Kamikubo
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Komori
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Higuchi
authors.forename: Mikio
authors.initials: M
authors.lastname: Kataoka
authors.forename: Shun
authors.initials: S
authors.lastname: Hirota
mesh_terms: D000818:Animals; D018360:Crystallography, X-Ray; D045304:Cytochromes c / Q000737:chemistry*; D006736:Horses; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs / Q000502:physiology*; D055503:Protein Multimerization / Q000502:physiology*; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae
chemical_list: D045304:Cytochromes c