Extended Data Fig. 2: Aggregation properties of the Agp and Bab BASS11 amyloid motifs.
From: Characterization of an amyloid-based antiphage defence system in Escherichia coli
a, Time course of BASS11 synthetic peptide amyloid aggregation followed by ThT fluorescence. Agp(2-25) (upper panel) or Bab(86-108) (lower panel) peptides were incubated at the given concentration with 50 µM ThT. Results are given in triplicates in different shades of red, magenta and blue for peptide concentrations of 30, 15 and 7.5 µM respectively. The buffer alone controls are given in shades of grey. b, Bathochromic shift in the Congo Red absorbance spectrum in the presence of Agp(2-25) (upper panel) or Bab(86-108) (lower panel) measured at 25 µM Congo Red and 90 µM of synthetic peptide, absorbance are normalized to the absorbance value at the maximal absorbance wavelength. Results are given as means +/−s.d. for triplicates. The absorbance spectra were recorded 15 sec, 10 min and 50 min after diluting the peptides (dissolved in 8 M GuHCl 250 mM acetic acid 1 mM DTT) into 100 mM Tris-HCl pH 8 150 mM NaCl 25 µM Congo Red. The absorbance spectrum of the buffer alone control is given in grey. A picture of the Agp(2-25), Bab(86-108) samples and buffer alone control (-) is given at the right. c, Time course of aggregation of recombinant full-length Bab-6his followed by ThT fluorescence at 7.5 µM (in triplicates) is given in different shades of red. The time course of Bab(86-108) synthetic peptide at the same concentration (in triplicates) is given in shades of blue. The buffer alone controls (in triplicates) is given in shades of grey. d, Representative example of electron micrograph of full length Bab-6his fibrils. Scale bar is 200 nm. e, The graph gives the proportion of cells containing GFP foci at different time points after transformation for the listed strains expressing wild-type or mutant versions of the BASS11 motif of Bab or Agp fused to GFP (BASS11-GFP). Results are given as means +/− s.d. of three technical replicates. P-values are calculated from one-way ANOVA and are Tukey’s multiple comparison tests comparing the values for the mutants (N12P or N100P) to the corresponding wild-type. f, Insolubility of BASS11-GFP in cells with foci. Anti-GFP western-blot of crude and fractionated E. coli extracts. Cells were harvested 5h or 24h after transformation and crude extracts (tot.) were centrifuged for 1h at 17,000g to yield the supernatant (sup.) and pellet (pel.) fractions. Size of molecular weight markers is given in kDa.
