Fig. 3
Myricetin exhibits high-affinity binding to the functional region of the PAP248–286 monomer. a The electrostatic interaction of myricetin with PAP248–286. PAP248–286 (100 μg/ml) was incubated with serially diluted myricetin (12.5, 6.25, 3.13 and 1.56 μg/ml) at 37 °C for 30 min. The samples were then centrifuged at 5000 rpm for 15 min; the peptides remaining in the supernatant were electrophoresed through 10% acidic native polyacrylamide gels and detected by WB using a polyclonal antibody against PAP248–286. b Binding affinity of myricetin to PAP248–286. PAP248–286 (50 μg/ml) was immobilized on a sensor chip. Subsequently, twofold serial dilutions of recombinant myricetin were injected as the analyte. The affinity constant KD is the ratio of the dissociation constant Kd to the association constant Ka (KD= Kd/Ka). c Binding affinity of myricetin to an irrelevant N36 peptide (50 μg/ml). d Binding affinity of an irrelevant flavonoid (phlorizin) to PAP248–286 (50 μg/ml). e Presumed binding sites of myricetin to PAP248–286. According to the computational docking results, myricetin formed hydrogen bonds with Leu258, GIn259, Met271 and Arg273, and it bound to Val264 and Leu268 by hydrophobic interactions. f Amyloid fibril formation by wild-type PAP257–273 and six mutants. Wild-type and mutant peptides (3 mg/ml) were agitated at 1400 rpm at 37 °C for 48 h; the presence of fibrils was then monitored using ThT assays. g Myricetin inhibition of fibril formation by wild-type and mutants PAP257–273. Wild-type and mutants PAP257–273 were mixed with myricetin (200, 100, 50 and 25 μg/ml), and the mixtures were agitated at 1400 rpm at 37 °C. The samples were then collected and monitored using ThT assays. Percent inhibition was calculated from the following equation: (1-ThT fluorescence of fibrils with Myr/ThT fluorescence of fibrils without Myr) × 100. Average values (± SD) were calculated from triplicate measurements; the data shown represent one representative trial of three independent experiments