Engineering amyloid-like assemblies from unstructured peptides via site-specific lipid conjugation

Abstract

Aggregation of amyloid beta (Aβ) into oligomers and fibrils is believed to play an important role in the development of Alzheimer's disease (AD). To gain further insight into the principles of aggregation, we have investigated the induction of β-sheet secondary conformation from disordered native peptide sequences through lipidation, in 1-2% hexafluoroisopropanol (HFIP) in phosphate buffered saline (PBS). Several parameters, such as type and number of lipid chains, peptide sequence, peptide length and net charge, were explored keeping the ratio peptide/HFIP constant. The resulting lipoconjugates were characterized by several physico-chemical techniques: Circular Dichroism (CD), Attenuated Total Reflection InfraRed (ATR-IR), Thioflavin T (ThT) fluorescence, Dynamic Light Scattering (DLS), solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy and Electron Microscopy (EM). Our data demonstrate the generation of β-sheet aggregates from numerous unstructured peptides under physiological pH, independent of the amino acid sequence. The amphiphilicity pattern and hydrophobicity of the scaffold were found to be key factors for their assembly into amyloid-like structures.

Figure 1. Characterization of β-sheet amyloid-like aggregates of Palm1–15.

A) CD spectra at 30 µM peptide Palm1–15 (green) and controls Acetyl1–15 (red) and Aβ1–15 (orange) in 2% HFIP/PBS (v/v). B) Profile of ThT emission with different peptide concentrations in the presence of 24 µM of dye. Fluorescence was measured at 485 nm with excitation at 440 nm. C, D, E) ssNMR of Palm1–15 uniformly labeled at Ala2, Ser8 and Gly9: (¹³C-¹³C) 2D PDSD correlation spectra with mixing times of 20 ms (C) and 150 ms (D); ¹H-¹³C FSLG-HETCOR spectra (E) together with chemical shift predictions based on secondary structure (red for random coil, green for β-sheet, blue for α-helix). F) Electron micrographs of negatively stained Palm1–15 aggregates formed over 24 hour incubation in 2% HFIP/PBS (v/v). Samples were negatively stained with 2% Uranyl Acetate in water. Scale bar 0.1 µm. Magnification 22000 x.

Figure 2. Parameters influencing scaffold conformation.

A) CD spectra and B) ThT fluorescence of tetrapalmitoylated peptides with shortened length, 9 or 5 amino acids (Palm1–9 in blue and Palm1–5 in orange respectively) or different order of amino acids, reverse or scrambled (Palm15-1 in green and scPalm15 in red, respectively) to model sequence Palm1–15. C) CD and D) ThT of tetrapalmitoylated peptides with different isoelectric point with pI (and net charge) value indicated on top of each column (Palm1–15(D7K) in green, Palm1–15(E3A, D7K) in blue, Palm1–15(E3K, D7K) in orange, Palm1–15(E3K, D7K, E11K) in red and Palm1–15 in black). E) CD and F) ThT of peptides with different number/position of palmitic chains (Palm1–15(1C) in orange, Palm1–15(2C) in red, Palm1–15(1N1C) in green, Palm1–15(4C) in blue and Palm1–15 in black). G) CD and H) ThT of peptides acylated with different lipid chain length (Acetyl1–15 in orange, Butyl1–15 in blue, Octyl1–15 in green, Dodecyl1–15 in red and Palm1–15 in black). Peptides were 30 µM in 2% HFIP/PBS (v/v) (CD) and 15 µM in 1% HFIP/PBS (v/v) (ThT, 24 µM). Fluorescence was measured at 485 nm with excitation at 440 nm. Values are the average of 3 replicates, normalized to the Palm1–15 emission (taken as 100%).