The role of a disulfide bridge in the stability and folding kinetics of Arabidopsis thaliana cytochrome c6A |
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Authors: | Jody M. MasonDerek S. Bendall Christopher J. HoweJonathan A.R. Worrall |
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Affiliation: | a Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, CO4 3SQ, UKb Department of Biochemistry, University of Cambridge, Downing Site, Tennis Court Road, Cambridge CB2 1QW, UK |
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Abstract: | Cytochrome c6A is a eukaryotic member of the Class I cytochrome c family possessing a high structural homology with photosynthetic cytochrome c6 from cyanobacteria, but structurally and functionally distinct through the presence of a disulfide bond and a heme mid-point redox potential of + 71 mV (vs normal hydrogen electrode). The disulfide bond is part of a loop insertion peptide that forms a cap-like structure on top of the core α-helical fold. We have investigated the contribution of the disulfide bond to thermodynamic stability and (un)folding kinetics in cytochrome c6A from Arabidopsis thaliana by making comparison with a photosynthetic cytochrome c6 from Phormidium laminosum and through a mutant in which the Cys residues have been replaced with Ser residues (C67/73S). We find that the disulfide bond makes a significant contribution to overall stability in both the ferric and ferrous heme states. Both cytochromes c6A and c6 fold rapidly at neutral pH through an on-pathway intermediate. The unfolding rate for the C67/73S variant is significantly increased indicating that the formation of this region occurs late in the folding pathway. We conclude that the disulfide bridge in cytochrome c6A acts as a conformational restraint in both the folding intermediate and native state of the protein and that it likely serves a structural rather than a previously proposed catalytic role. |
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Keywords: | Cyt, cytochrome At, Arabidopsis thaliana Pl, Phormidium laminosum Em, heme mid-point redox potential LIP, loop insertion peptide wt, wild-type Amp, ampicillin Cam, chloramphenicol LB, lysogeny-broth IPTG, isopropyl β-D-1-thiogalactopyranoside GuHCl, guanidine hydrochloride CD, Circular dichroism NHE, normal hydrogen electrode DTT, Dithiothreitol DTNB, 5,5&prime -dithiobis(2-nitrobenzoic acid) |
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