Structural characterisation of amyloid-like fibrils formed by an amyloidogenic peptide segment of β-lactoglobulin

Abstract

Protein nanofibrils (PNFs) represent a promising class of biobased nanomaterials for biomedical and materials science applications. In the design of such materials, a fundamental understanding of the structure-function relationship at both molecular and nanoscale levels is essential. Here we report investigations of the nanoscale morphology and molecular arrangement of amyloid-like PNFs of a synthetic peptide fragment consisting of residues 11-20 of the protein β-lactoglobulin (β-LG_11-20), an important model system for PNF materials. Nanoscale fibril morphology was analysed by atomic force microscopy (AFM) that indicates the presence of polymorphic self-assembly of protofilaments. However, observation of a single set of ¹³C and ¹⁵N resonances in the solid-state NMR spectra for the β-LG_11-20 fibrils suggests that the observed polymorphism originates from the assembly of protofilaments at the nanoscale but not from the molecular structure. The secondary structure and inter-residue proximities in the β-LG_11-20 fibrils were probed using NMR experiments of the peptide with ¹³C- and ¹⁵N-labelled amino acid residues at selected positions. We can conclude that the peptides form parallel β-sheets, but the NMR data was inconclusive regarding inter-sheet packing. Molecular dynamics simulations confirm the stability of parallel β-sheets and suggest two preferred modes of packing. Comparison of molecular dynamics models with NMR data and calculated chemical shifts indicates that both packing models are possible.

Scheme 1. Schematic display of β-LG full sequence and peptides derived from β-LG identified in PNFs by mass spectrometry. Peptides fragments that differ by one residue are depicted using one stripped line. A and B represents bovine β-LG variants. The red-highlighted peptide segment is chosen to study here.

Fig. 1. ThT fluorescence spectra of β-LG_11–20 peptide solution (1 mg mL⁻¹) with 5% pure β-LG PNF seeds, incubated at 50 °C and pH 2 for 0, 24, 36, 48, and 96 hours, respectively.

Fig. 2. (A) AFM height images of β-LG_11–20 fibrils formed at 50 °C and pH 2 (after 96 hours). Images were collected in air. The right side shows the profiles with different periodicity that are highlighted in the left side image. (B and C) AFM height images of β-LG_11–20 displaying a mesh-like (B) and web-like (C) structure in areas with a high fibril density. (D and E) Statistical AFM analysis of fibril morphologies. The periodicity distribution showing that the majority of formed fibrils have periodicity of 35 ± 5 nm (D) and the correlation between periodicity and fibril heights (E).

Fig. 3. Secondary ¹³C′ (A), ¹³C^α (B), ¹³C^β (C), and ¹⁵N (D) chemical shifts of the β-LG_11–20 fibrils. The secondary shifts were calculated as Δδ = δ(exp) − δ(X), where δ(exp) is the experimentally observed chemical shift of either the monomer values in liquid, M(aq), monomer in solid state, M(s), or for the fibrils, F(s), whereas δ(X) represents the Wishart random coil values in ppm. The secondary ¹³C′ and ¹⁵N shifts for D1, Q3, K4, W9, Y10 could not be calculated as the residues were not labelled in any of the samples.

Fig. 4. (A) Ramachandran plot showing the ϕ and ψ values predicted by TALOS+ (red) and the values obtained from the two best MD models (blue). (B) A magnification with error bars (±1 STD) included.

Fig. 5. 2D ¹³C–¹³C solid-state DARR NMR spectra of the mixed (50 : 50%) fibrils IT-VAG. The lines indicate the assigned spin systems. The yellow arrows highlight inter-molecular cross peaks.

Fig. 6. (A) A representative structure of four peptides in parallel alignment. For simplicity, only the protein backbone is shown, and the tyrosine and tryptophane moieties are shown in green and grey spheres, respectively. The structure is (i) stabilised by the π–π stacking interactions involving Tyr and Trp moieties, (ii) stabilised by hydrogen bonding interactions within the neighbouring peptides, and (iii) the peptides form β-sheet secondary structure during our MD simulations (as represented by the arrows). (B) In a similar manner to panel A, a 20-peptide sheet was built to study a stability of this more complex structure. Even after ca. 13 ns of MD simulation, the end point of our simulation showed that the π–π stacking and H-bonding interactions are not preserved among all Tyr and Trp moieties (highlighted in green and grey), despite the ability to preserve most of the β-sheets (yellow). (C) MD simulation (30 ns) of 40 peptides in the anti-parallel B–B zipper mode packing (stabilised by 194 ± 9 H-bonding interactions). (D) MD simulation (30 ns) of 40 peptides in the parallel B–B zipper mode packing (stabilised by 192 ± 9 H-bonding interactions). Panels C and D show the most stable arrangements of a 40-peptide double sheets; the non-stable packing variants are shown in Fig. S7 in the ESI.

Fig. 7. Experimental vs. DFT/PBE0 calculated ¹³C C^α and C^β chemical shifts for monomer (A) in liquid-state, parallel BB-PZ (B), and anti-parallel BB-aPZ (C) molecular ensembles β-sheets. The calculated values for panel B and C were obtained from snapshots taken at the end point of our 30 ns of MD simulations, and the experimental values are taken from solid-state MAS NMR.