Fig. 4
From: A viral-fusion-peptide-like molecular switch drives membrane insertion of botulinum neurotoxin A1
Single-molecule analysis of the distribution of BoNT/A1i on individual liposomes. a Representative TIRF microscope images showing co-localization of Alexa 647-labeled BoNT/A1i-D12* complexes (right) with rhodamine-labeled liposomes (left). In these experiments, < 10% of liposomes were occupied by protein to minimize co-localization. The circled spots on the right were used for photobleaching analysis only when co-localized with rhodamine fluorescence. b Representative time traces of fluorescence emission from spots used to assess the oligomerization state. The bar above the panel indicates the excitation with red laser used to first bleach the Alexa 647 followed by green laser to probe for rhodamine emission. Examples are shown for 1-, 2-, and 3-step photobleaching events. Dashed lines highlight individual bleaching steps. (c) Distribution of bleaching steps for BoNT/A1i-D12* at pH 4.6 (n = 935) and pH 7.4 (n = 482). The increase in 1-step events and decrease in 3-step events upon raising the pH to 7.4 were statistically significant (asterisk) with p = 0.0001 and 0.0065, respectively. The decrease in 2-step events was just above significance with p = 0.077. d Distribution of photobleaching steps for oxidized BoNT/A1iDS-D12* at pH 4.6 (n = 589) and pH 7.4 (n = 535). No significant differences were observed when the BoNT-switch was locked by a disulfide bond. Error bars indicate standard error of measurement (SEM) from replicate analysis
