Table 1.
Solubility and secondary structure of binary alternating peptides.
Table 2.
Peptides used in this study.
Fig 1.
Activity screens of peptide amyloids in various conditions.
The hydrolysis rates of 4NPA measured for each of the peptide amyloids (in columns) are shown as a ratio to the negative control (buffer) with color-codes as indicated at the bottom of the Figure. A color code from black (rate ratio of 1.0) to light blue (1.5) to red (19) is used. Activities below the negative control are indicated with smaller disks and color-coded from light grey to black. Occasionally observed negative reaction rates are indicated by small blue disks. The peptide amyloids were tested in a total of 46 conditions of which 19 conditions are shown here (in rows). The peptides are numbered according to Table 2, including a short nomenclature highlighting only the variable residues (e.g. the peptide Ac-YVDVHVSV-CONH2 is abbreviated DHS). The conditions are sorted top-down with the highest observed activity at the top. A detailed list of the conditions tested are shown in Table B in S1 File, while below a compressed introduction to the different conditions is listed: (2) pH 7.3 with ZnCl2 (control for 33 without heating), (33) condition 2 and heated to 95°C for 1 h prior to screening, (46) condition 2 and screened in 50% DMSO, (39) condition 2 and screened with 4.75 M NaCl, (40) condition 2 and screened with 0.2 M MgCl2, (45) condition 2 and screened in 90% EtOH, (38) condition 2 and screened with 0.2 M NaCl, (30) pH 7.3 with ZnCl2 and 1 M Na2SO4, (35) pH 7.3 and screened in 4.75 M NaCl, (34) pH 7.3 and screened in 0.5 M NaCl, (37) pH 7.3 and screened in 2 M MgCl2, (27) pH 7.3 with ZnCl2 and 0.2 M MgCl2, (6) pH 7.3 with NiCl2, (4) pH 7.3 with CoCl2, (1) pH 7.3 without other additives, (41) condition 2 and screened in 2 M MgCl2, (5) pH 7.3 with CuCl2, (36) pH 7.3 and screened in 0.2 M MgCl2 and (8) pH 4.0 without other additives.
Fig 2.
Esterase activity of selected peptides at pH 7.3.
Esterase activity for a selection of peptides is shown. Plots show the initial hydrolysis rate of 4NPA as a function of initial substrate concentration together with the fit of the data to the Michaelis-Menten equation. The symbols for the individual peptides are in Table 3. In (a) all of the peptides that were measured are shown (the less active peptides greyed out), while in (b) the same data is plotted on a smaller scale to show the less active peptide amyloids.
Table 3.
Esterase activity of selected peptides at pH 7.3.
Fig 3.
Parallel kinetics of fibrillization and hydrolysis activity.
Kinetics of fibrillization measured by CD (c) matches the appearance of the 4NPA hydrolysis activity (a). a) A stock of 35 at 200 μM in 10 mM HCl was diluted to a final concentration of 50 μM into 50 mM HEPES pH 7.3 with 0.5 mM ZnCl2 and 1 mM 4NPA. After ca. 10 s mixing time, the hydrolysis of 4NPA was recorded every 1 s for 20 min at 400 nm and the first derivative of this signal (the instantaneous rate) is plotted as a function of time. The black lines are four independent measurements of these and the grey lines are two measurements of the background reaction (i.e. without peptide). b) Aggregates of 35 were prepared as in (a) except in the absence of substrate. After 1 h, 1 mM 4NPA was added and the hydrolysis rate was monitored. Once again, the black curves are 4 separate measurements and the grey lines 2 background measurements. c) Peptide 35 was prepared as in (a) and the CD signal at 218 nm was measured every 2 s starting ca. 12 s after mixing. The inset plot shows the CD spectrum of the sample after 1 h and the detector voltage (scale on the right).
Fig 4.
Esterase activity at pH 4.3 measured by NMR.
The relative concentration of substrate measured by 1D-1H NMR peak intensities is shown as a function of reaction time for: (◇) 40 μM of 21; (□) 40 μM of 12 and; (○) co-aggregate of 20 μM of 12 with 20 μM of 21; (×) buffer control (50 mM d3-NaOAc at pH 4.3 and 2% d6-DMSO). Only 1 in 12 data points are shown to avoid overcrowded plot markers.
Table 4.
Esterase activity of selected peptides at pH 4.3.