Fig 1.
Far-UV CD spectra of pEAβ(3–40) and Aβ(1–40).
Peptides were dissolved in buffer (20–25% TFE in 50 mM potassium phosphate, pH 2.8). CD spectra were recorded at 20°C from 260 to 187 nm, accumulated 10 times and background corrected. (a) CD spectra of 25 μM pEAβ(3–40) in 25%, 23%, 22%, 21% and 20% TFE showed a shift from α-helical structure towards β-sheets with decreasing TFE concentrations. (b) The CD spectrum of 25 μM Aβ(1–40) indicated α-helices in 20% TFE while the spectrum of 25 μM pEAβ(3–40) in 20% TFE showed mainly β-sheet rich structures.
Fig 2.
Aggregation kinetics of pEAβ(3–40) and Aβ(1–40) in TFE.
(a) 25 μM of monomerized pEAβ(3–40) were dissolved in buffer with various TFE contents (25%, 23%, 22%, 21% and 20% TFE in 50 mM potassium phosphate, pH 2.8) including 10 μM ThT. pEAβ(3–40) aggregated in 20% and 21% TFE but was significantly decreased in aqueous solution with higher TFE concentration. (b) 25 μM of monomerized pEAβ(3–40) and Aβ(1–40) were dissolved in buffer (20% TFE in 50 mM potassium phosphate, pH 2.8) including 10 μM ThT. An increase in ThT fluorescence was observed for pEAβ(3–40) but not for Aβ(1–40) within 72 h.
Fig 3.
TEM image of pEAβ(3–40) in 50 mM potassium phosphate pH 2.8 containing 20% TFE.
Monomerized pEAβ(3–40) (25 μM) was incubated for fibrillation at 20°C for five days and grids were prepared by negative staining. pEAβ(3–40) incubated in aqueous TFE solution formed large twisted fibrils up to several hundred nm in size which accumulate into large aggregates ranging from 1–5 μm in diameter.
Fig 4.
1H,15N-HSQC of pEAβ(3–40) and Aβ(1–40).
25 μM of the monomerized peptides were dissolved in aqueous solution (50 mM potassium phosphate, pH 2.8) containing 20% TFE. Spectra were recorded on a 600 MHz Bruker spectrometer at 5°C. Overlay of the spectrum of pEAβ(3–40) (red) and Aβ(1–40) (blue) indicate that the pyroglutamate modification affects the N-terminal signals significantly towards E11 as well as V40.
Fig 5.
Chemical shift changes Δδ(1H,15N) = ((10*Δδ1HN)2 + (Δδ15N)2)1/2 between pEAβ(3–40) and Aβ(1–40) plotted as a function of the pEAβ(3–40) sequence.
Pyroglutamate formation affects the N-terminal amino acids with decreasing effect towards the C-terminus and, interestingly, also the C-terminal V40.