Protein surface charge effect on 3D domain swapping in cells for c-type cytochromes

Many c-type cytochromes (cyts) can form domain-swapped oligomers. The positively charged Hydrogenobacter thermophilus (HT) cytochrome (cyt) c(552) forms domain-swapped oligomers during expression in the Escherichia coli (E. coli) expression system, but the factors influencing the oligomerization remain unrevealed. Here, we found that the dimer of the negatively charged Shewanella violacea (SV) cyt c(5) exhibits a domain-swapped structure, in which the N-terminal helix is exchanged between protomers, similar to the structures of the HT cyt c(552) and Pseudomonas aeruginosa (PA) cyt c(551) domain-swapped dimers. Positively charged horse cyt c and HT cyt c(552) domain swapped during expression in E. coli, whereas negatively charged PA cyt c(551) and SV cyt c(5) did not. Oligomers were formed during expression in E. coli for HT cyt c(552) attached to either a co- or post-translational signal peptide for transportation through the cytoplasm membrane, but not for PA cyt c(551) attached to either signal peptide. HT cyt c(552) formed oligomers in E. coli in the presence and absence of rare codons. More oligomers were obtained from the in vitro folding of horse cyt c and HT cyt c(552) by the addition of negatively charged liposomes during folding, whereas the amount of oligomers for the in vitro folding of PA cyt c(551) and SV cyt c(5) did not change significantly by the addition. These results indicate that the protein surface charge affects the oligomerization of c-type cyts in cells; positively charged c-type cyts assemble on a negatively charged membrane, inducing formation of domain-swapped oligomers during folding.