Folding Of Cyt Research Articles

Formation of Oligomeric Cytochrome c during Folding by Intermolecular Hydrophobic Interaction between N- and C-Terminal α-Helices

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.

A common folding mechanism in the cytochrome c family

Of the globular proteins, cytochrome c (cyt c) has been used extensively as a model system for folding studies. Here we analyse the folding pathway of different cyt c proteins from prokaryotes and eukaryotes, and attempt to single out general correlations between structural determinants and folding mechanisms. Recent studies provide evidence that the folding pathway of several cyt c proteins involves the formation of a partially structured intermediate. Using state-of-the-art kinetic analysis on published data, we show that such a folding intermediate is an obligatory on-pathway species that might represent either a defined local minimum in the reaction coordinate or an unstable high-energy state. Available data also indicate that some essential structural features of the folding intermediate and transition states are highly conserved across this protein family. Thus, cyt c proteins share a consensus folding mechanism in spite of large differences in physico-chemical properties and thermodynamic stability. This novel outlook on the folding of cyt c can shed light on much published data and might offer a general scheme by which to plan new experiments.

A model for the misfolded bis-His intermediate of cytochrome c: the 1?56 N-fragment

We have characterized the ferric and ferrous forms of the heme-containing (1–56 residues) N-fragment of horse heart cytochrome c (cyt c) at different pH values and low ionic strength by UV–visible absorption and resonance Raman (RR) scattering. The results are compared with native cyt c in the same experimental conditions as this may provide a deeper insight into the cyt c unfolding-folding process. Folding of cyt c leads to a state having the heme iron coordinated to a histidine (His18) and a methionine (Met80) as axial ligands. At neutral pH the N-fragment (which lacks Met80) shows absorption and RR spectra that are consistent with the presence of a bis-His low spin heme, like several non-native forms of the parental protein. In particular, the optical spectra are identical to those of cyt c in the presence of a high concentration of denaturants; this renders the N-fragment a suitable model to study the heme pocket microenvironment of the misfolded (His–His) intermediate formed during folding of cyt c. Acid pH affects the ligation state in both cyt c and the N-fragment. Data obtained as a function of pH allow a correlation between the structural properties in the heme pocket of the N-fragment and those of non-native forms of cyt c. The results underline that the (57–104 residues) segment under native-like conditions imparts structural stability to the protein by impeding solvent access into the heme pocket.

A model for the misfolded bis-His intermediate of cytochrome c: the 1–56 N-fragment

We have characterized the ferric and ferrous forms of the heme-containing (1–56 residues) N-fragment of horse heart cytochrome c (cyt c) at different pH values and low ionic strength by UV–visible absorption and resonance Raman (RR) scattering. The results are compared with native cyt c in the same experimental conditions as this may provide a deeper insight into the cyt c unfolding-folding process. Folding of cyt c leads to a state having the heme iron coordinated to a histidine (His18) and a methionine (Met80) as axial ligands. At neutral pH the N-fragment (which lacks Met80) shows absorption and RR spectra that are consistent with the presence of a bis-His low spin heme, like several non-native forms of the parental protein. In particular, the optical spectra are identical to those of cyt c in the presence of a high concentration of denaturants; this renders the N-fragment a suitable model to study the heme pocket microenvironment of the misfolded (His–His) intermediate formed during folding of cyt c. Acid pH affects the ligation state in both cyt c and the N-fragment. Data obtained as a function of pH allow a correlation between the structural properties in the heme pocket of the N-fragment and those of non-native forms of cyt c. The results underline that the (57–104 residues) segment under native-like conditions imparts structural stability to the protein by impeding solvent access into the heme pocket.

Hydrophobic effect of trityrosine on heme ligand exchange during folding of cytochrome c

Effect of a hydrophobic peptide on folding of oxidized cytochrome c (cyt c) is studied with trityrosine. Folding of cyt c was initiated by pH jump from 2.3 (acid-unfolded) to 4.2 (folded). The Soret band of the 2-ms transient absorption spectrum during folding decreased its intensity and red-shifted from 397 to 400 nm by interaction with trityrosine, whereas tyrosinol caused no significant effect. The change in the transient absorption spectrum by interaction with trityrosine was similar to that obtained with 100 mM imidazole, which showed that the population of the intermediate His/His coordinated species increased during folding of cyt c by interaction with trityrosine. The absorption change was biphasic, the fast phase (82 ± 9 s −1) corresponding to the transition from the His/H 2O to the His/Met coordinated species, whereas the slow phase (24 ± 3 s −1) from His/His to His/Met. By addition of trityrosine, the relative ratio of the slow phase increased, due to increase of the His/His species at the initial stage of folding. According to the resonance Raman spectra of cyt c, the high-spin 6-coordinate and low-spin 6-coordinate species were dominated at pH 2.3 and 4.2, respectively, and these species were not affected by addition of trityrosine. These results demonstrated that the His/His species increased by interaction with trityrosine at the initial stage of cyt c folding, whereas the heme coordination structure was not affected by trityrosine when the protein was completely unfolded or folded. Hydrophobic peptides thus may be useful to study the effects of hydrophobic interactions on protein folding.

Folding Properties of Cytochrome c Studied by Photocleavable o-Nitrobenzyl Modification of Methionine 65 and 80

Abstract Folding properties of cytochrome c (cyt c) were studied by specific o-nitrobenzyl modification of its methionines and subsequent UV light irradiation. Absorption changes with time scales of about 50 µs, 10 ms, 30–100 ms, and 250–500 ms were observed during folding of cyt c.