Fluorenylmethyloxycarbonyl (Fmoc)-protected amino acids, and N,N,N′,N′- tetramethyl-O-(1H-benzotriazol-1-yl) uronium hexafluorophosphate (HBTU) were procured from Novabiochem (Darmstadt, Germany). Rink amide resin, N,N-diisopropylethylamine (DIPEA), piperidine, trifluoroacetic acid, ethanedithiol, thioanisole, acetic anhydride, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), and Thioflavin T (ThT) were from Sigma-Aldrich Chemicals Pvt. Ltd.
N,N-dimethylformamide, diethyl ether, and m-cresol was obtained from Merck, India. 1-hydroxybenzotriazole hydrate (HOBt) was obtained from Sisco Research Laboratory, India. All other reagents were of the highest grade available.
2.2.2 Peptide synthesis and characterization
Peptides were synthesized on solid support using Fmoc chemistry by employing HBTU/HOBt/DIPEA activation strategy. For 14-residue peptides, HATU activation was used for coupling the turn-inducing amino acids (8th and 9th amino acids). On-resin acetylation of the N-terminus was carried out using five equivalents of acetic anhydride and ten equivalents of DIPEA. The peptides were cleaved from the resin using a cocktail mixture containing TFA:m-
cresol:thioanisole:ethanedithiol (20:2:2:1) and precipitated in ice-cold diethyl ether. The crude peptides were dried, dissolved in DMSO, and purified on a reversed-phase C-18 column on a Shimadzu Prominence Modular HPLC instrument (Shimadzu, Kyoto, Japan) using a linear gradient of acetonitrile containing 0.1% TFA (2.25% acetonitrile/min). The purified peptides were characterized by MALDI-TOF (Bruker Autoflex Speed MALDI-TOF-TOF, USA) mass spectrometry.
2.2.3 Peptide aggregation
Peptides were dissolved in HFIP and their concentrations estimated by measuring absorption at 280 nm for peptides containing tyrosine or tryptophan and at 254 nm for peptides lacking these amino acids. Molar absorption coefficients of peptides were calculated by adding the molar absorption coefficients of the aromatic amino acids at corresponding wavelengths i.e Tyr (ε280 = 1280 M-1cm-1), Trp (ε280 = 5690 M-1cm-1), and Phe (ε254 = 143 M-1cm-1). The concentrations of the peptides were adjusted to 1.25 mM in HFIP. Subsequently, the peptides were diluted in water to obtain 250 µM concentration having 20%
HFIP, and their aggregation studied.
2.2.4 Circular dichroism (CD) spectroscopy
Far-UV CD spectra were recorded on a Chirascan CD spectropolarimeter (Applied Photophysics, UK) for the 24 h old samples with suitable dilution in 20% HFIP. The spectra were recorded in a 1 mm path length cell with a step size of 0.5 nm, corrected by subtracting the blank spectrum, i.e. the spectrum for 20%
HFIP, and smoothed. The data was converted to mean residue ellipticity, [θ]MRE, using the formula: [θ]MRE = (Mr × θobs)/(100× l × c), where Mr is the mean residue weight (peptide molecular weight/number of amino acid residues), θobs is the observed ellipticity in millidegrees, l is the path length in decimeters, and c is the peptide concentration in mg/ml [27].
2.2.5 Fourier transform infrared (FTIR) spectroscopy
FTIR spectra were recorded on a Bruker Alpha-E spectrometer (Bruker Optik GmbH, Germany) with Eco attenuated total reflection (ATR) single reflection ATR sampling module equipped with ZnSe ATR crystal. Peptides (10 μl) were spread out and dried as films on ZnSe crystal, and ATR-FTIR spectra were recorded at a resolution of 4 cm-1.
2.2.6 Molecular dynamics [MD] simulations
Peptides in a fully extended conformation (φ = ψ =180°) were constructed using Ribosome.[24] The dihedral angles φ and ψ for DPro, wherever applicable, were fixed to 70° and -45°, respectively. The amino-terminus was acetylated while C- terminus was amidated for each of the peptides. The lysine and glutamate side- chains were electrically charged, rendering the peptide electrically neutral; no counter-ion was added separately. Energy minimization and MD simulation were performed using the Gromacs software package (v 5.0.4) [28] with the GROMOS96 54a7 forcefield [29] at the CDAC-IIT Guwahati Supercomputing facility. The Ribosome-constructed structures were energy minimized in vacuum using the Steepest Descent algorithm. The vacuum energy-minimised peptide structures were solvated with water using simple point charge [30] water model in a cubic box with a distance of 1.5 nm between the peptide and the edge of the box. The system comprising of one peptide molecule and 25,148 water molecules was energy-minimised. A small position restrained dynamics was performed before the final production of the MD run. MD simulation was carried out for 100 ns at 300 K and 1 bar by using leap-frog integration method [31]. Temperature and pressure couplings were done with v-rescale [32] and Berendsen [33]
methods, respectively. Long-range electrostatics was calculated with fast, smooth Particle mesh Ewald (PME) method with cubic interpolation and Fourier grid spacing of 0.1 nm [34, 35]. The Verlet cut off-scheme was followed for short- range electrostatics and van der Waals interactions, with the cut-off value set to 1.0 nm for both rcoulomb and rvdw. Bond lengths were constrained using the LINCS algorithm [36]. All the simulations were carried out using periodic
boundary conditions in all the directions. The results were analysed using the built-in Gromacs utilities. Discovery Studio Visualizer was used for visualization [37].
2.2.7 Thioflavin T (ThT) fluorescence assay
ThT fluorescence spectra for the 24 h-old peptide samples in 20% HFIP were recorded on a Jasco FP8500 spectrofluorometer (Jasco, Japan). The peptides were diluted to 20 µM concentration in 5 mM phosphate buffer, pH 7.0, containing 10 µM ThT. The samples were excited at 440 nm, and fluorescence emission recorded from 460 to 550 nm. Excitation and emission slit widths were 2.5 and 5 nm, respectively. Spectra of the peptides without ThT were recorded as blanks and subtracted. Fluorescence intensity at 482 nm is reported in the results.
2.2.8 Transmission electron microscopy (TEM)
The peptide samples (72 h-old) were diluted 2.5-fold in deionized water and drop-casted on Formvar/Carbon coated 200-mesh copper grids. After 2 minutes, the solvent was blotted out from the periphery of the grid by Whatman I filter paper. The grids were stained with saturated uranyl acetate negative stain solution, which was blotted out after 30 seconds. The grids were then dried and images recorded on JAM-2100 LaB6 (JEOL, Japan) transmission electron microscope at 120 kV.