(A) Primary sequence of h-IAPP, r-IAPP and I26P-IAPP. Mature polypeptides contain a disulfide between Cys2 and Cys7, indicated by brackets, and an amidated C-terminus. Amino acid positions that differ from h-IAPP are indicated in red. (B) Representative SDS-PAGE of photochemically cross-linked non-toxic r-IAPP and I26P-IAPP show the presence of low order oligomers. Peptide solutions were incubated at 25°C until time points corresponding to h-IAPP toxicity and ultracentrifuged for 20,000 g for 20 min. Aliquots of the supernatants were removed and irradiated for 10 s for cross-linking, and analyzed using silver staining. Lane-1, markers (molecular weight: KDaltons); lane-2, h-IAPP; lane 3, r-IAPP; and lane 4, I26P-IAPP. (C) Quantitative analysis of the gels shown in panel B reveal a distribution of low order oligomers ranging up to hexamers: h-IAPP (red); r-IAPP (green); and I26P-IAPP (blue). (D) Thioflavin-T binding assays of aliquots of buffer (gold), r-IAPP (green) or I26P-IAPP (blue) at different time points over the course of aggregation. (E) TEM image of r-IAPP oligomers after 14 h incubation (Scale bars: 200 nm). (F) Alamar Blue reduction assays of β-cells treated with buffer (gold), r-IAPP (green) or I26P-IAPP (blue) at different time points over the course of aggregation. (G) Light microscopy image of viable β-cells after treatment with r-IAPP aggregates shown in panel E. (H) CD spectra of I26P-IAPP (blue), r-IAPP (green) and h-IAPP (red). Data is plotted as mean residue ellipticity. (I) TEM image of I26P-IAPP oligomers after 14 h incubation. (J) Light microscopy image of viable β-cells treated with oligomers shown in panel I. IAPP solutions contained 20 µM peptide. The final peptide concentration after dilution into β-cell assays was 14 µM. Data represent mean ± SD of three to six replicate wells per condition and three replicate experiments per group (NSD: no significant difference; Scale bars: 200 nm). Figure 5—figure supplements 1–7 provide additional biochemical information, biophysical characterization and toxicity experiments for the non-toxic, non-amyloidogenic r-IAPP and h-IAPP mutants.