Fig. 3: IM30* dimerizes via its unstructured C-terminus.

Limited proteolysis of IM30*: IM30* was incubated with the endoproteinase GluC at different enzyme-to-substrate ratios for 30 or 60 min, respectively. The peptide patterns were analyzed via SDS-PAGE. The band highlighted with a black box was analyzed by mass spectrometry. Below, the predicted secondary structure of IM30 is shown, whereby each box represents an α-helical segment. After treatment with endoproteinase GluC, a stable helix 2/3 fragment was identified (with a sequence coverage of ~60%). For more details, see Supplementary Fig. 7. b The difference of relative HDX between IM30* and IM30 WT after 10 s mapped on the predicted IM30 monomer structure¹⁹ revealed an increased flexibility of helix 1 and helices 3a-5/6 of IM30* compared to the WT. A large part of helix 7 and the linker region between helix 7 and 6 is highly flexible in both variants. Dark gray regions mark sites where no peptides were detected in the HDX experiment, and thus no data is available. (For more details see Supplementary Fig. 8). c SDS-PAGE analysis of A227C mutants of IM30 WT and IM30* in absence of reducing agents. d IM30 monomer models generated based on the SAXS data were aligned at helix 2 and 3a to visualize the flexibility of the C-terminal region. Helices 2 and 3a (amino acids 26–144) are depicted in gray, the C-terminal amino acids 145–267 (H3b-7) are colored in green, and amino acids 1–25 (H1) in red. The N-terminal region (red) fills a small volume, starting from the rim between helix 2 and 3 with an only small overlap between the N-terminal and the C-terminal regions. However, the C-terminal region (green) nearly fills the entire conformational space at the end of the structural core, with higher accumulation at the very end of helix 3a.