Figure 5.

Amyloid aggregation propensity of GLP-1 and the related Ex4-Tc5b analogues. The aggregation behavior of GLP-1 (a–c), Tc-truncated Δ^31–39Ex4-Tc5b (d–f), full-length Ex4 (g–i), Ex4-Tc5b (j–l), and Ex4-Tc5b_CC (m–o) was characterized by using the thioflavin-T binding assay, CD spectroscopy, and AFM techniques. The first row shows the emitted fluorescence of thioflavin-T assays at acidic and neutral pH, at concentrations of 1 and 5 mg mL^–1 (acidic conditions) collected over a 3-day-long period. The average fluorescence and standard deviation of three parallel measurements were plotted as error-band functions. Fluorescence of GLP-1 exceeded the limit of detection range (indicated by ⊥ in panel a). Long-term aggregation was also monitored by CD spectroscopy under acidic (second row) and neutral conditions (Figure S4) at a concentration of 1 mg mL^-1. The initial CD spectra exhibit characteristic CD properties of mainly α-helical folded structures. Over time, observable decreases in intensity associated with unfolding (b–k) occur, rather than the distinctive α-helical to β-sheet transition (Figure S4f). However, AFM micrographs (third row) of the 72 h agitated acidic ThT samples, at concentrations of 1 mg mL^–1 (c,f,i,o) and 5 mg mL^–1 (l) reveal fibril formation (c,f,l). Objects revealed by AFM of granulated or rectangular topology are NaCl salt crystals, crystallized during the vacuum drying process (i,o).