Multistep nucleus formation and a separate subunit contribution of the amyloidgenesis of heat-denatured monellin

Abstract

Monellin (MN) is a sweet-tasting plant protein known to form fibrous aggregates in the heat-denatured state. Here the amyloid-type aggregation process of MN is extensively characterized. The amyloidgenesis was initiated in a highly denatured state of MN. A seeding effect of skipping a lag phase of the amyloid formation kinetics established a nucleation-dependent aggregation mechanism. A finely controlled experimental protocol revealed an additional prenucleus stage preceding the maturation of the nucleus, indicating that the initial lag phase is composed of multiple conformational events. The results obtained for the aggregation properties of the separate A and B subunit chains of MN and a recombinant single-chain MN suggest that the B chain exclusively contributed to the amyloid-type aggregation. These findings suggest a scheme for the amyloidgenesis of MN and their subunits, and provide a unique model of amyloidgenesis that is regulated by the subunit composition of protein.

Fig. 1.

(A) Heat denaturation curves of MN in 10 mM glycine (pH 2.5) and 0.3 M NaCl monitored by ellipticity at 215 nm (solid line) or 280 nm (○). The measurements were started at 25°C, and the temperature was increased by 1°C/min. The concentration of MN was 67 μM. (B) Far-UV CD spectra of MN in 10 mM glycine (pH 2.5). The concentration of MN was 45 μM. Broken line: at 25°C. Solid line: at 70°C with no cosolvent. (○): at 70°C in the presence of 0.3 M NaCl. (*): at 70°C in the presence of 0.5 M urea.

Fig. 2.

(A) Kratky profiles of SAXS of MN in 10 mM glycine (pH 2.5) at 25°C (bold line) and 75°C (thin line). The concentration of MN was 450 μM. (B) Guinier plots of SAXS of MN in 10 mM glycine (pH 2.5) measured at 25°C. The concentration of MN was 270 μM. (○): no cosolvent, no heating. (•): no cosolvent, heat-treated. (*): 0.1 M NaCl, heat-treated. The heat treatment was done at 75°C for 20 min, and then recovered at 25°C for 6 h.

Fig. 3.

Negatively-stained TEM images of MN aggregates formed in 0.3 M NaCl (A) and 0.5 M urea (B).

Fig. 4.

(A) Thioflavin T (ThT) fluorescence and (B) IR spectra of MN. Thin solid lines: no heating. Bold solid lines: heat-treated in 0.3 M NaCl. Broken lines: heat-treated without NaCl. The concentration of MN was 270 μM for (B). The measurements for (A) were done after 40× dilution in 10 mM Tris (pH 7.5) containing 10 μM ThT.

Fig. 5.

(A) Amyloid formation kinetics of MN monitored by ThT fluorescence at various concentrations of MN and NaCl. The MN samples were incubated at 75°C in 10 mM glycine (pH 2.5). (○) 90 μM MN in 0.3 M NaCl. (|*) 45 μM MN in 0.3 M NaCl. (•) 45 μM MN in 0.35 M NaCl. F₄₈₃: fluorescence intensity of ThT emitted at 483 nm wavelength. (B) Amyloid formation kinetics of MN monitored by ThT fluorescence at 55°C (○), 60°C (•), 67.5°C (▵) and 75°C (|*). The MN concentration was 67 μM in 10 mM glycine (pH 2.5) and 0.3 M NaCl. Fluorescence changes for (A) and (B) were normalized with their maximum changes (achieved typically at 600 min). (C) ThT binding of MN measured after heat treatment for 45 min in 10 mM glycine (pH 2.5) and 0.3 M NaCl, plotted against the incubation temperature. The concentration of MN was 67 μM.

Fig. 6.

Amyloid formation kinetics of MN in 10 mM glycine (pH 2.5) and 0.3 M NaCl at 67.5°C monitored by ThT binding (□), intrinsic Trp fluorescence (•) and turbidity (*). The concentration of MN was 67 μM. Each datum was normalized with its maximum change (achieved typically at 600 min).

Fig. 7.

Amyloid formation kinetics of MN in 10 mM glycine (pH 2.5) and 0.3 M NaCl at 56°C monitored by the CD spectroscopy. (A) The far-UV CD spectra at the incubation times of 5 (bold broken line), 30 (thin broken line) and 120 min (bold solid line). (B) The kinetic changes of [θ]₂₀₉ (○), [θ]₂₂₀ (•) and ThT binding (*). F₄₈₃: fluorescence intensity of ThT emitted at 483 nm wavelength. The concentration of MN was 67 μM.

Fig. 8.

A seeding effect on the amyloid formation kinetics of MN (67 μM) in 10 mM glycine (pH 2.5) and 0.3 M NaCl at 67.5°C monitored by ThT binding. (○) no seeding. (•) seeded with 10 μM of fully-matured MN amyloid. The plot of (*) was made as follows. The sample at various incubation times at 67.5°C was added as a seed in one-tenth volume to a fresh sample. This seeded sample was then incubated at 67.5°C for 30 min, and its ThT binding was monitored. The ThT fluorescence intensity was plotted against the incubation period of the seed preparation. F₄₈₃: fluorescence intensity of ThT emitted at 483 nm wavelength.

Fig. 9.

Effects of the preheating treatment on the amyloid formation kinetics of MN (67 μM) in 10 mM glycine (pH 2.5) and 0.3 M NaCl at 67.5°C, monitored by ThT binding (A) and intrinsic Trp fluorescence (B). The sample was preheated for 0 (○), 15 (•), and 30 min (*) at 67.5°C, and left for 24 h at 25°C before the second increase in the temperature. Arrows in (B): see the text. The traces of (•) and (*) start at 15 and 30 min because the preheating time is added to the second heat incubation time. F₄₈₃: fluorescence intensity of ThT emitted at 483 nm wavelength.

Fig. 10.

TEM images of the aggregates of the B chain of MN (A) and SMN (B) formed in 10 mM glycine (pH 2.5) and 0.3 M NaCl at 67.5°C.

Fig. 11.

An aggregation scheme of heat-denatured monellin.