1. Volume 112, Issue 8, Pages 1621-1633 (April 2017)
Pyroglutamate-Modified Amyloid-β(3–42) Shows α-Helical Intermediates before Amyloid Formation 
Christina Dammers, Kerstin Reiss, Lothar Gremer, Justin Lecher, Tamar Ziehm, Matthias Stoldt, Melanie Schwarten, Dieter Willbold 
Biophysical Journal 
Volume 112, Issue 8, Pages (April 2017)
DOI: /j.bpj
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2. Figure 1
CD spectra and aggregation kinetics of pEAβ (3–42) and Aβ (1–42). Far-UV CD spectra were recorded on a Jasco J-1100 spectropolarimeter at 20°C from 260 to 190 nm, accumulated 12 times, and corrected for the buffer. (A) CD spectra of 25 μM pEAβ (3–42) in 50 mM potassium phosphate (pH 2.8) containing 30%5, 40%, or 50% TFE, respectively (black, red, and blue lines). With decreasing TFE concentration the content of α-helices was reduced and β-sheet-rich structures appeared. (B) CD spectra of 25 μM Aβ (1–42) in 50 mM potassium phosphate (pH 2.8) containing 30%, 40%, or 50% TFE (green, purple, and orange lines), respectively. Although the spectrum of pEAβ (3–42) showed β-sheet structural elements in 50% and 40% TFE, Aβ (1–42) is primarily α-helix rich but forms β-sheet structures in 30% TFE comparable to that of pEAβ (3–42) at higher TFE concentrations, i.e., 40%. (C) Aggregation kinetics measured by ThT assay, where 25 μM pEAβ (3–42) was dissolved in 50 mM potassium phosphate (pH 2.8) containing 30%, 40%, or 50% TFE (black, red, and blue lines, respectively) including 10 μM ThT. (D) Kinetics of 25 μM Aβ (1–42) were measured in 50 mM potassium phosphate (pH 2.8) containing 30%, 40%, and 50% TFE (green, purple, and orange lines) including 10 μM ThT (pH 2.8). The fluorescence emission at 492 nm was monitored on a fluorescence spectrometer (excitation at 440 nm), recorded every 15 min for 62 h. The measurement was performed fivefold and averaged, and the buffer was subtracted for background correction. An increase of ThT fluorescence and faster aggregation kinetics of pEAβ (3–42) were observed with decreasing TFE concentration. No fibril formation of Aβ (1–42) could be detected within 62 h. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
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3. Figure 2
TEM images of pEAβ (3–42) and Aβ (1–42). pEAβ (3–42) (upper) and Aβ (1–42) (lower) peptides were dissolved in 50 mM potassium phosphate (pH 2.8) containing 40% TFE and incubated at 20°C for 5 days. Grids were prepared by negative staining. Aggregated pEAβ (3–42) formed large twisted fibrils, whereas Aβ (1–42) formed non-fibrillary branched networks. The average fibril length of pEAβ (3–42) was 9.7 ± 1.5 nm. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
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4. Figure 3
(1H-15N)-HSQC of pEAβ (3–42) and Aβ (1–42). (A) pEAβ (3–42) (100 μM) was dissolved in aqueous solution containing 30%, 40%, or 50% TFE in 50 mM potassium phosphate (pH 2.8) (black, red, and blue lines, respectively). Spectra were recorded on a 600 MHz Bruker spectrometer at 20°C. The spectra of pEAβ (3–42) in 50% TFE differs slightly from that in 40% TFE due to the change in TFE concentration. Notably, some peak intensities, such as those of H13, E22, M35, and G37, are drastically decreased. The spectrum of pEAβ (3–42) in 30% TFE revealed that only the crosspeaks of the very N-terminal aa residues pE3, F4, and R5 were left. (B) Overlay of 100 μM pEAβ (3–42) (red) and Aβ (1–42) (purple) in aqueous solution (50 mM potassium phosphate (pH 2.8)) containing 40% TFE. Peaks with prominent changes in chemical shifts are marked with assignments. Spectra were recorded on a 600 MHz Bruker spectrometer at 20°C. Pyroglutamate modification affects the N-terminal crosspeaks toward H14 (assigned in black). Crosspeaks exclusively for Aβ (1–42) were labeled in purple and in red for pEAβ (3–42). (C) Changes in chemical shifts of pEAβ (3–42) compared to those of Aβ (1–42) were obtained from (1H-15N)-HSQC of pEAβ (3–42) and Aβ (1–42) in 40% TFE and calculated according to the formula Δδ(1H,15N) = ((Δδ1HN)2 + (1/5∗Δδ15N)2)1/2. Chemical-shift changes are plotted as a function of the primary sequence of pEAβ (3–42). Pyroglutamate formation affects the N-terminal aa residues up to G9 with decreasing effect toward the C-terminus. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
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5. Figure 4
Structural characterization of pEAβ (3–42). Structural analysis was based on NMR chemical shift data for 100 μM pEAβ (3–42) in buffer (40% TFE in 50 mM potassium phosphate (pH 2.8)) at 20°C. (A) C′, Cα, Cβ, and Hα chemical shifts calculated as the difference between measured and random-coil chemical shifts, described by Zhang et al. (33), and sequence corrected for neighbor effects (34). (B) SSP score. (C) Secondary-structure determination by TALOS-N (35). The probability for an α-helical conformation is plotted for each aa residue. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
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6. Figure 5
Normalized crosspeak intensities of pEAβ (3–42) compared to Aβ (1–42). Crosspeak intensities of pEAβ (3–42) and Aβ (1–42) obtained from BT-HNCA+ (A) and BT-HNCO (B) spectra. The peak intensity of pEAβ (3–42) in 40% TFE was normalized to pEAβ (3–42) in 50% TFE (red) and to Aβ (1–42) in 40% TFE (black). The normalized crosspeak intensity is plotted against the primary aa sequence of pEAβ (3–42). Stars indicate overlapping crosspeaks and therefore no analysis of peak height. The Cα and C′ intensities of pEAβ (3–42) are decreased by an average of 15–20% compared to those of Aβ (1–42). Cα and C′ intensities of pEAβ (3–42) in 40% TFE were only half those of pEAβ (3–42) in 50% TFE, but this effect was smaller at the N-terminus and residues G25–A30. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
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7. Figure 6
Crosspeak intensities over time. (A) The (1H-15N)-HSQC crosspeak intensities were averaged over all residues in Aβ (1–42) (black) and pEAβ (3–42) (red) and reported relative to their initial value at the first measurement. The monomer abundance in pEAβ (3–42) decreased more rapidly than that in Aβ (1–42). (B) Crosspeak intensities over time for 100 μM pEAβ (3–42) (40% TFE in 50 mM potassium phosphate (pH 2.8)) at 20°C. A time series of 48 (1H-15N)-HSQCs (each 90 min) was recorded on an 800 MHz Agilent spectrometer. The total acquisition time was ∼72 h. The amide crosspeak intensity for selected aa residues is plotted against time. Within 15 h, most of the crosspeaks were near the background, but the N-terminal aa residues pE3, F4, and R5 (dark blue) were more stable. Amide crosspeak intensities for all aa residues are shown in Fig. S3. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
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8. Figure 7
Normalized crosspeak intensities in the presence of a 10-fold molar excess of non-isotopically enriched peptide. (1H-15N)-HSQC peak height of 25 μM [U-15N]-pEAβ (3–42) with an excess of 250 μM [U-14N]-pEAβ (3–42) was normalized to 25 μM [U-15N]-pEAβ (3–42) in 40% TFE. The decrease in crosspeak intensity is plotted against the primary aa sequence of pEAβ (3–42). On average, the peak densities are decreased to ∼20% of the original intensities.
Biophysical Journal  , DOI: ( /j.bpj )
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9. Figure 8
Schematic diagram of the transition of an α-helical intermediate to β-sheets. α-helices are depicted as cylinders and β-strands as zigzag lines. Unstructured pEAβ (3–42) monomers form TFE induced α-helices. These helices bundle together and generate a high local concentration of an aggregation-prone sequence that is likely to form β-strands. The formation of β-strands disrupts the α-helices and leads to the formation of β-sheet-rich assemblies.
Biophysical Journal  , DOI: ( /j.bpj )
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