Figure 3.

SDS-PAGE gel of purified heterododecamers of mtDod(WT) (WT, light blue in cartoon) and mtDod-PAS-Strep (S, orange with red encircled “S”). L: Ladder. For analysis of heterododecamer composition, the Tris–glycine (Lämmli) SDS-PAGE system was used, and samples were prepared without heat treatment and SDS (for full length gel images see Supplementary Fig. S6). For mtDod(WT) two bands at about 40 kDa and slightly above 50 kDa are observable (both below the weight of the dodecamer, about 90 kDa). The band at about 40 kDa represents likely the dodecamer and the band above seems to be caused by the mild denaturation conditions during sample preparation. The appearance of additional bands at higher molecular weight is also observable in other lanes. We want to note that these bands don’t depict the hexamer and the dodecamer, as all protein–protein interactions, which would stabilize the hexamer, are present in higher numbers in the dodecamer with also other additional stabilizing interactions. The origin of the mtDod dodecamer migration behaviour and the molecular mechanism behind the bands at higher molecular weight is not clear. (a) SDS-PAGE gel of heterododecamers obtained by refolding mtDod(WT) and mtDod-PAS-Strep at different ratios. Each band represents a heterododecamer with a defined composition of mtDod-PAS-Strep and mtDod(WT) (13 bands for 13 possible compositions, higher molecular weight bands above about 160 kDa excluded). (b) SDS-PAGE gel of purified heterododecamers formed during polycistronic expression of mtDod(WT) and mtDod-PAS-Strep. Bicis(WT:S): Bicistronic expression vector design: mtDod(WT) encoding gene first and mtDod-PAS-Strep encoding gene second. Bicis(S:WT): Bicistronic expression vector design: mtDod-PAS-Strep encoding gene first and mtDod(WT) encoding gene second. Tricis(WT:WT:S): Tricistronic expression vector design: mtDod(WT) encoding gene first and second and mtDod-PAS-Strep encoding gene third.