Real-time monitoring of the aggregation of Alzheimer's amyloid-β via1H magic angle spinning NMR spectroscopy

Abstract

Proton magic-angle-spinning NMR used for real-time analysis of amyloid aggregation reveals that mechanical rotation of Aβ_1-40 monomers increases the rate of formation of aggregates, and that the increasing lag-time with peptide concentration suggests the formation of growth-incompetent species. EGCG's ability to shift off-pathway aggregation is also demonstrated.

Figure 1

Real-time monitoring of Aβ_1–40 monomer depletion under MAS. Experimentally measured ¹H NMR signal intensities for selected aliphatic and aromatic resonances of freshly prepared 50 μM Aβ₄₀ as a function of time under 5 kHz MAS and 298 K. Additional experimental results showing the decay of monomer peaks are shown in Figure S4. Time=0 was the starting time of NMR data acquisition, which is <10 minutes from the fresh sample preparation.

Figure 2. Aβ_1–40 monomer depletion promoted by EGCG

(A) 1D ¹H NMR spectra of 50 μM Aβ_1–40 in presence of 50 μM EGCG obtained at the indicated times under 5 kHz MAS and 298 K. (B, C, D) Change in ¹H signal intensities for selected aliphatic and aromatic resonances of Aβ_1–40 (B), EGCG (C), and the newly appeared peaks (indicated as O1 and O2) (D). Additional experimental results on the changes in peak intensities are shown in Figures S6 and S7.

Figure 3

TEM images of Aβ_1–40 aggregates. TEM images of 50 μM Aβ_1–40 after spinning for 68 hours in the absence (A) and presence of 50 μM EGCG (1:1 Aβ_1–40:EGCG molar ratio) after spinning for 42 hours (B). TEM images of 5 μM Aβ_1–40 after spinning for 80 hours (C) and 150 μM Aβ_1–40 under static condition after 48 hours (D).

Figure 4

Concentration dependent monomer decay for Aβ₄₀. (A) Aggregation kinetics of Aβ_1–40 measured from ¹H NMR signal intensity of methyl resonance (0.78 ppm) under 5 kHz MAS for various peptide concentrations. The monomer decay curve was fitted using the equation, y=(1−A)*exp(−b*x)+A. The parameters used for the monomer decay curves are given in (B). Additional experimental results on the changes in the peak intensities are shown in Figures S8 and S9 and Table S1.