title: Recombinant expression, biophysical characterization, and cardiolipin-induced changes of two Caenorhabditis elegans cytochrome c proteins.
abstract: Cytochrome c (cyt c) is one of the most widely studied biomolecules, but not much is known about this protein from nematodes. Recombinant expression of Caenorhabditis elegans CYC-2.1 and CYC-2.2 allowed for detailed characterization of their structural features, redox properties, stabilities, and interactions with cardiolipin (CL)-containing liposomes. Using a variety of spectroscopic tools, we show that CYC-2.1 and CYC-2.2 adopt a globular α-helical fold with His/Met heme ligation. The longer CYC-2.2 has a lower thermodynamic stability than CYC-2.1 and lacks His residues to misligate to the heme in the protein's denatured state. Both C. elegans proteins bind to CL-containing liposomes, and these interactions promote the proteins' peroxidase activity but to a much greater degree for CYC-2.2. Dye-to-heme distance distributions from time-resolved fluorescence resonance energy transfer in bimane-labeled CYC-2.1 and CYC-2.2 revealed similar populations of extended and compact conformers for CL-bound proteins, suggesting that their distinct peroxidase activities in the presence of CL arise from differences in the local heme environments for the two polypeptide ensembles. Without inhibition from His misligation, a less stable and more prone to unfolding CYC-2.2 allows for better access of substrates to the heme and thus exhibits higher peroxidase activity. Similar features of the conformational ensembles of CYC-2.1 and CYC-2.2 to those of mammalian cyt c suggest that C. elegans proteins, particularly the former, could serve as useful models for examining the mechanism of cyt c-CL interactions in live organisms.
authors.affiliation: Department of Chemistry, Dartmouth College, Hanover, NH 03755, USA.
authors.forename: Amber J
authors.initials: AJ
authors.lastname: Vincelli
authors.forename: Danielle S
authors.initials: DS
authors.lastname: Pottinger
authors.forename: Fangfang
authors.initials: F
authors.lastname: Zhong
authors.forename: Jonas
authors.initials: J
authors.lastname: Hanske
authors.forename: Stéphane G
authors.initials: SG
authors.lastname: Rolland
authors.forename: Barbara
authors.initials: B
authors.lastname: Conradt
authors.forename: Ekaterina V
authors.initials: EV
authors.lastname: Pletneva
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D029742:Caenorhabditis elegans Proteins / Q000096:biosynthesis / Q000737:chemistry*; D002308:Cardiolipins / Q000737:chemistry*; D017124:Conserved Sequence; D045304:Cytochromes c / Q000096:biosynthesis / Q000737:chemistry*; D004926:Escherichia coli; D031541:Fluorescence Resonance Energy Transfer; D006139:Guaiacol / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D006736:Horses; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000096:biosynthesis / Q000737:chemistry*; D011485:Protein Binding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D020539:Sequence Analysis, Protein; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
chemical_list: D029742:Caenorhabditis elegans Proteins; D002308:Cardiolipins; D008081:Liposomes; D011994:Recombinant Proteins; D006418:Heme; D006139:Guaiacol; D045304:Cytochromes c; D010544:Peroxidases