NMR Reveals Two-Step Association of Congo Red to Amyloid β in Low-Molecular-Weight Aggregates

Abstract

Aggregation of the Amyloid β peptide into amyloid fibrils is closely related to development of Alzheimer's disease. Many small aromatic compounds have been found to act as inhibitors of fibril formation, and have inspired the search for new drug candidates. However, the detailed mechanisms of inhibition are largely unknown. In this study, we have examined in detail the binding of the fibril-formation inhibitor Congo Red (CR) to monomeric Aβ(1-40) using a combination of 1D, 2D, saturation transfer difference, and diffusion NMR, as well as dynamic light scattering experiments. Our results show that CR binds to the fibril forming stretches of Aβ(1-40) monomers, and that complex formation occurs in two steps: An initial 1:1 CR:Aβ(1-40) complex is formed by a relatively strong interaction (K(d) ≈ 5 μM), and a 2:1 complex is formed by binding another CR molecule in a subsequent weaker binding step (K(d) ≈ 300 μM). The size of these complexes is comparable to that of Aβ(1-40) alone. The existence of two different complexes might explain the contradictory reports regarding the inhibitory effects of CR on the fibril-formation process.

Resolution of Oligomeric Species during the Aggregation of Aβ1–40 Using 19F NMR

“…Both spectra show a single intense resonance at 35.85 ppm (Figure 1A and B), which DOSY experiments show corresponds to a species with a hydrodynamic radius of 1.28 nm (Figure S2). This value is nearly identical to previous values obtained for non-aggregated Aβ 1-40 in buffer (1.29 nm) and in NaOH (1.32 nm)(37) and similar to single molecule measurements of monomeric Aβ 1-40 (0.9 nm). (38) The main resonance in the 19 F spectra at the initial time point is therefore very likely to correspond to the monomeric peptide in both samples.…”

Influence of the Aqueous Environment on Protein Structure—A Plausible Hypothesis Concerning the Mechanism of Amyloidogenesis

“…The B chain is further subdivided into three parts: the N-terminal fragment, the AmL section and the C-terminal fragment. As mentioned above and highlighted in numerous publications [36][37][38] AmL exhibits an amyloid-like structural form [50][51][52][53][54][55]. Results listed in Table 5 describe the SufC-SufD complex involved in iron-sulfur cluster biosynthesis as a whole (single common 3D Gaussian) and individually for each chain.…”

“…This fragment resembles typical amyloid structures, as presented in numerous research papers discussing the conformation of amyloids [36][37][38]. For this reason the fragment in question (151-365 in the A-B complex) is singled out and labeled "AmL", while the remaining components of each chain (the N-and C-terminal fragments) are analyzed separately.…”

A Small Molecule Inhibitor of Pot1 Binding to Telomeric DNA

“…Therefore, equivalent dry aliquots were resuspended in water or binding buffer (50 mM KPhos, pH 8.0, 50 mM NaCl, 3 mM BME). The concentration of CR in solution was determined by absorbance spectroscopy in water using a molar extinction coefficient of 45000 M -1 cm -1 at 495 nm (81). Thioflavin T concentration was determined using a molar extinction coefficient of 36000 M -1 cm -1 at 412 nm, which is the same in water and phosphate buffer (82, 83).…”