Extended Data Figure 7. CNTNW cysteine cross-linking mutants, except for the negative control (CNTNWS109C,A373C), spontaneously cross-link to form covalent trimers.
a, Western blot of MBP-CNTNW cross-linking mutants in crude membrane preparations. Each cross-linking pair, except for the negative control, cross-links spontaneously to form covalently linked dimers and trimers, and this reaction is reversible by addition of reducing reagent. Data is representative of three independent experiments. b, Cross-linked CNTNW mutants have a similar elution volume in size exclusion chromatography as Cys-less CNT, as shown by size-exclusion chromatography and SDS-PAGE analysis. c, The purified cysteine cross-linking mutants were analyzed by Coomassie-stained SDS-PAGE before and after PreScission protease treatment to remove the MBP-His tag. After PPX treatment, monomeric, dimeric, and trimeric CNTNW can be seen in the SDS-PAGE gel, indicated by the blue, green, and purple arrows, respectively. Air-oxidized protein in the peak fraction (*) was reconstituted into proteoliposomes for the flux assay shown in Fig. 3.