Thioflavine T interaction with synthetic Alzheimer's disease β‐amyloid peptides: Detection of amyloid aggregation in solution

Abstract

Thioflavine T (ThT) associates rapidly with aggregated fibrils of the synthetic PjACderived peptides p( 1-28) and p(1-40), giving rise to a new excitation (ex) (absorption) maximum at 450 nm and enhanced emission (em) at 482 nm, as opposed to the 385 nm (ex) and 445 nrn (em) of the free dye. This change is dependent on the aggregated state as monomeric or dimeric peptides do not react, and guanidine dissociation of aggregates destroys the signal. There was no effect of high salt concentrations. Binding to the p(1-40) is of lower affinity, Kd 2 p M , while it saturates with a Kd of 0.54 pM for p(1-28). Insulin fibrils converted to a P-sheet conformation fluoresce intensely with ThT. A variety of polyhydroxy, polyanionic, or polycationic materials fail to interact or impede interaction with the amyloid peptides. This fluorometric technique should allow the kinetic elucidation of the amyloid fibril assembly process as well as the testing of agents that might modulate their assembly or disassembly.Keywords: P/A4; dye fluorescence; pH dependence Amyloid was defined by Virchow (1855) on the basis of a blue staining reaction with iodine (starch-like) followed by treatment with acid. ThS and ThT, (structure in Fig. 1A) characteristically stain amyloid-like deposits in a number of pathophysiological conditions (Vassar and Culling, 1959;KelCnyi, 1967). Along with Congo red they are believed to specifically interact in some unknown way with the crossed-0-sheet structure common to amyloid structures comprised of different materials. Naiki et al. (1989Naiki et al. ( , 1990Naiki et al. ( , 1991 have described the interaction of these dyes with amyloid proteins derived from serum amyloid A protein and apoAII to give enhanced fluorescence emission. The work described in this manuscript documents a similar phenomenon for interactions with aggregated species of synthetic peptides (Fig. 1B) whose sequences are derived from a protein (0/A4) isolated from deposits

Myricetin Prevents Fibrillogenesis of Hen Egg White Lysozyme

“…30 By monitoring the kinetics of HEWL fibril formation by fluorescence spectroscopy, we found that the relative fluorescence of HEWL increased with extended incubation times (Figure 2A), which is in agreement with previous studies. 31 The consistent increase in ThT fluorescence intensity observed when HEWL was incubated alone, demonstrates that formation of amyloid fibrils proceeded rapidly and without a lag phase ( Figure 2A To determine an effective concentration range of myricetin for inhibiting HEWL fibril formation, varying aliquots of myricetin were mixed with HEWL to final concentrations of 1, 10, and 100 μM; at these concentrations we did not observe significant toxic effects of myricetin on cell survival (data not shown) and the ThT fluorescence intensity measured ( Figure 2B). Myricetin (and quercitin control) was observed to exhibit a dosedependent effect on HEWL fibril formation ( Figure 2B).…”

In silico and in vitro studies to elucidate the role of Cu2+ and galanthamine as the limiting step in the amyloid beta (1–42) fibrillation process

“…The formation of Ab fibrils and oligomers requires a conformational change from an a-helix to a bsheet conformation, which is encouraged by the formation of a salt bridge between Asp 23 or Glu 22 and Lys 28. Recently, Cu 21 and various drugs used for AD treatment, such as galanthamine (Reminyl V R ), have been reported to inhibit the formation of Ab fibrils. However, the mechanism of this inhibition remains unclear.…”

“…However, the mechanism of this inhibition remains unclear. Therefore, the aim of this work was to explore how Cu 21 and galanthamine prevent the formation of Ab 1-42 fibrils using molecular dynamics (MD) simulations (20 ns) and in vitro studies using fluorescence and circular dichroism (CD) spectroscopies. The MD simulations revealed that Ab 1-42 acquires a characteristic U-shape before the a-helix to b-sheet conformational change.…”

“…The formation of a salt bridge between Asp 23 and Lys 28 was also observed beginning at 5 ns. However, the MD simulations of Ab 1242 in the presence of Cu 21 or galanthamine demonstrated that both ligands prevent the formation of the salt bridge by either binding to Glu 22 and Asp 23 (Cu 21 ) or to Lys 28 (galanthamine), which prevents Ab 1242 from adopting the Ucharacteristic conformation that allows the amino acids to transition to a b-sheet conformation. The docking results revealed that the conformation obtained by the MD simulation of a monomer from the 1Z0Q structure can form similar interactions to those obtained from the 2BGE structure in the oligomers.…”

“…The docking results revealed that the conformation obtained by the MD simulation of a monomer from the 1Z0Q structure can form similar interactions to those obtained from the 2BGE structure in the oligomers. The in vitro studies demonstrated that Ab remains in an unfolded conformation when Cu 21 and galanthamine are used. Then, ligands that bind Asp 23 or Glu 22 and Lys 28 could therefore be used to prevent b turn formation and, consequently, the formation of Ab fibrils.…”