Fig. 2: Biophysical characterisation of the amyloid-like properties of PAM4.

a Concentration-dependent Th-T kinetic assays of the PAM4 peptide (n = 3 independent repeats), along with end-state Th-T fluorescence values shown in the right bar plot, with individual values shown as points. b Cross-β diffraction pattern produced by oriented fibres containing PAM4 peptide fibrils. Intensity quantification along the meridional and equatorial axis of the pattern indicate the presence of an intense 4.7 Å and 10.9 Å reflection, respectively. c FTIR spectrum produced from fibril deposits of the PAM4 peptide. The prominent 1631 cm⁻¹ and 1680 cm⁻¹ peaks are indicative of a dominant β-sheet secondary structure. d Polarised microscopy reveals an apple-green birefringence shown by PAM4 peptide deposits which typically signifies the presence of amyloid aggregates. (Scale Bar = 100 μM). e Atomic force microscopy imaging of PAM4 fibrils. Multiple helical morphologies can be observed in a single field of view. A single representative image is shown (n = 3 independent repeats). f Electron micrograph of fibrils formed by assembly of the PAM4 peptide. Higher magnifications of individual fibrils (lower images) showcase the presence of highly polymorphic amyloid fibrils. A single representative image is shown (n = 3 independent repeats). g–h Concentration-dependent seeding quantification performed by counting the percentage of expressing cells containing fluorescent puncta (n = 3 independent repeats) and representative images of the dose-dependent seeding of tauRD-YFP conjugate construct with PAM4. (Scale bar = 20 μm). i, j Representative images (Bar = 20 μm) and quantification of puncta-positive cells transiently expressing the tauRD-YFP construct, following transduction with 5 μM of peptide seeds. The individual points are colour-coded to match the regions highlighted in Fig. 1, with underlined segments representing the aggregation motifs predicted by Cordax and Waltz. Bar plots represent mean values ± SEM (n = 4 independent samples). The solid and dashed horizontal lines indicate the mean and SEM corresponding to the untreated biosensor condition. Statistical significance was determined using one-way ANOVA with Dunnett’s correction for multiple comparisons.