The aggregation kinetics of Alzheimer's beta-amyloid peptide is controlled by stochastic nucleation

Abstract

We report here a recombinant expression system that allows production of large quantities of Alzheimer's Abeta(1-40) peptide. The material is competent to dissolve in water solutions with "random-coil properties," although its conformation and factual oligomerization state are determined by the physico-chemical solution conditions. When dissolved in 50 mM sodium phosphate buffer (pH 7.4) at 37 degrees C, the peptide is able to undergo a nucleated polymerization reaction. The aggregation profile is characteristically bipartite, consisting of lag and growth phase. From these curves we determined the lag time as well as the rate of aggregation. Both values were found to depend on peptide concentration and addition or formation of seeds. Moreover, they can vary considerably between apparently identical samples. These data imply that the nucleation event is under influence of a stochastic factor that can manifest itself in profound macroscopic differences in the aggregation kinetics of otherwise indistinguishable samples.

Figure 1.

Purification of Aβ(1–40). Coomassie-stained gel of purified Aβ(1–40); (lanes A, F) marker; (lane B) cell lysate; (lane C) crude fusion protein after NiNTA chromatography; (lane D) cleaved fusion protein; (lane E) pure Aβ(1–40).

Figure 2.

Secondary structure of recombinant Aβ(1–40). (A) Far-UV CD spectra of Aβ(1–40) dissolved at different concentrations: 4.3 μM: filled circles; 21.3 μM: open circles; 42.6 μM: filled diamonds; 85.2 μM: open diamonds; 127.8 μM: filled squares; 213 μM: open squares. (Insert) Dependence of the mean residue weight ellipticity at 217 nm on peptide concentration. (B) ATR-FTIR spectrum of freshly dissolved Aβ(1–40) at 2 mg/mL concentration (457 μM). All spectra were recorded in 50 mM sodium phosphate (pH 7.4) at room temperature.

Figure 3.

Influence of pH and aggregation on the solubility of Aβ(1–40). Filled diamonds: total peptide concentration in solution. Open diamonds: peptide in the supernatant after centrifugation of a freshly dissolved solution. Filled circles: peptide in the supernatant after centrifugation of a solution after incubation for 24 h. Open circles: peptide in the supernatant after centrifugation of a solution after addition of 5% seeds and incubation for 24 h.

Figure 4.

Kinetic evaluation of the aggregation reaction. (A) Kinetics of Aβ(1–40) aggregation. t^l was determined by fitting the straight lines a to the baseline of the lag phase and b as a tangent to the steepest region of the growth phase curve (normally occurring at ~30% of the fluorescence increase reached at the end of the experiment). t^l is defined as the time point where the two lines a and b intersect. To obtain k, the growth phase was fitted the function y=A+B*exp (−kx) (curve c). (B) Families of 10 kinetic traces each of solutions containing 1.0 mg/mL, 0.5 mg/mL, and 0.01 mg/mL Aβ (1–40).

Figure 5.

Effect of concentration and seeding on k and t^l. (A) Concentration dependence of t^l and t_av ^l. t^l values at 1 mg/mL solutions are approximated with 0.1 h. (B) Concentration dependence of k and k_av. Dependence of t^l (C) and k (D) on the seed concentration. (E) t^l (crosses) and k (diamonds) of freshly dissolved recombinant Aβ (1–40) and after pre-treatment using DMSO, NaOH, or TFA/HFIP. Gray symbols: t^l and k. Black symbols: t_av^l and k_av. The panels show data from 10 (A, B, E) or 20 (C, D) individual experiments.