Figure 5.

(A) In vitro aggregation analysis of AC-GSKLVFFAEGS-NH₂ (i), (ii) TEM characterization of AC-GSKLVFFAEGS-NH₂ over 15 min showing the initial formation of narrow lamellar aggregates, which tend to wider lamellar aggregates over 5 h of incubation. (iii), (iv) Number (%) of the internal contour length (L, nm) values over 15 min and 5 h for n=80 full-length analyzed lamellar aggregates. (v), (vi) Number (%) fibril width (W, nm) analysis (n=300) indicates a significant 23.05 nm increase over time. (B) In vitro aggregation analysis of Ac-GSGSKLVFFAEGSGS-NH₂ reveals the spontaneous formation of micellar morphologies over 15 min of incubation followed by their morphological evolution into short protofibrilar species accompanied by 10-40 nm spherical aggregates over 30 min of incubation. Mature rigid fibrils were finally formed over 5 h of incubation. (i-iii, v) TEM images of the 15 min, 30 min and 5 h incubated Ac-GSGSKLVFFAEGSGS-NH₂ peptide depicting the morphology transition from micelles to rigid fibrils. (iv) Small (~ 20 nm) and large nanoscale aggregates (50-80 nm) in diameter after 30 min of incubation. Scale bars: 25 nm, except for bottom right-100 nm. (vi) Internal contour length (L, nm) of the micelles determined by number (%) count. (vii) Number (%) determination of the internal contour fibril length (L, nm) upon 30 min of incubation (n=390). (viii) Number (%) determination of the internal contour fibril length (L, nm) upon 5 h of incubation (n=80). (ix) Fibril width (W, nm) analysis (n=300) of the respective protofibrils and mature fibrils analyzed in panels vii and viii. L (nm), W (nm) values were estimated similarly as for the other peptides. (C) Persistence length (λ, nm) bar chart indicating the bending rigidity of the four respective peptides (Ac-KLVFFAE-NH₂, Ac-GSKLVFFAE-NH₂, Ac-GSKLVFFAEGS-NH₂, Ac-GSGSKLVFFAEGSGS-NH₂) over time (< 1 h-orange and 5 h-green). Data points shown are the mean values (n=3) ± (SEM). Statistical analysis performed through unpaired t-test determining two tailed p-values (APA): < 0.12 (ns), < 0.033 (*), < 0.002 (**), < 0.001 (***). (D), (E) Number (%) vs local curvature (1/R_c, nm) graphs of the four respective peptides over time. Peptides’ concentration: 1 mM in MilliQ H₂O, incubation temperature: 37 °C. Internal contour length and fibril width values were calculated through FiberApp. λ values were estimated using the bond correlation function (BCF), mean squared end-to-end distance (MSED), and mean-squared midpoint displacement (MSMD) methods and are presented as mean values determined by the three methods. λ and 1/R_c values were obtained through FiberApp from the corresponding contour length fibril analysis.