Fig. 2.

PAN’s coiled-coils do not adopt symmetrical conformations. a Cartoon representation of the PAN-M87C mutant CC-OB domains (based on PDB: 3H43 [10.2210/pdb3H43/pdb]). The PAN-M87C mutant contains a cysteine in place of methionine at the CC domains most proximal hydrophobic residue. These cysteines are expected to form a disulfide crosslink based on measured β-carbon distances in its crystal structure (PDB: 3H43 [10.2210/pdb3H43/pdb]). b Representative non-reducing SDS-PAGE and Coomassie staining of WT-PAN and PAN-M87C under reducing (1 mM DTT) and oxidizing (1 mM tetrathionate) conditions followed by desalting (See Methods for details). The mean percentage of dimer with standard deviations is indicated at the top of the gel (WT-PAN-red- n = 3; WT-PAN-ox- n = 16; PAN-M87C-red- n = 3; PAN-M87C-ox- n = 18). See Supplementary Fig. 1 for full-length lanes of oxidized samples used for this quantification. c Experiment with PAN-M87C similar to (b) but with a dose response with oxidizing reagent (tetrathionate) prior to desalting and SDS-PAGE. The quantification of percentage of crosslinked vs. the concentration of tetrathionate is also shown on the right. Error bars are shown (n = 3) but are smaller than the data points. d Representative gel showing the amount of PAN-M87C crosslinking after 5 min or 72 h of incubation with tetrothionate (n = 3). e PAN-M87C was incubated in 1 mM tetrathionate, and increasing amounts of denaturant (SDS: 0.00006, 0.0006, 0.003, 0.006, 0.03, 0.06, 0.3, and 0.6%) were added prior to desalting and SDS-PAGE. At higher levels of SDS, >95% crosslinking was observed. A representative gel is shown (n = 3). See Supplementary Fig. 8 for validation of quantitative SDS-PAGE analysis of PAN