Sound-mediated nucleation and growth of amyloid fibrils

Abstract

Mechanical energy, specifically in the form of ultrasound, can induce pressure variations and temperature fluctuations when applied to an aqueous media. These conditions can both positively and negatively affect protein complexes, consequently altering their stability, folding patterns, and self-assembling behavior. Despite much scientific progress, our current understanding of the effects of ultrasound on the self-assembly of amyloidogenic proteins remains limited. In the present study, we demonstrate that when the amplitude of the delivered ultrasonic energy is sufficiently low, it can induce refolding of specific motifs in protein monomers, which is sufficient for primary nucleation; this has been revealed by MD. These ultrasound-induced structural changes are initiated by pressure perturbations and are accelerated by a temperature factor. Furthermore, the prolonged action of low-amplitude ultrasound enables the elongation of amyloid protein nanofibrils directly from natively folded monomeric lysozyme protein, in a controlled manner, until it reaches a critical length. Using solution X-ray scattering, we determined that nanofibrillar assemblies, formed either under the action of sound or from natively fibrillated lysozyme, share identical structural characteristics. Thus, these results provide insights into the effects of ultrasound on fibrillar protein self-assembly and lay the foundation for the potential use of sound energy in protein chemistry.

Keywords: amyloid; fibrillar protein self-assembly; molecular dynamics simulations; small-angle X-ray scattering; ultrasound.

Fig. 1.

(A) Schematic representation describing amyloid fibril formation and growth under the action of low-amplitude ultrasound (see Materials and Methods, Formation of Lysozyme Fibrils under the Action of Ultrasound). (B and C) Atomic force microscope (AFM) images of lysozyme aggregates (see Materials and Methods, Morphological Analysis by Atomic Force Microscopy) and (D) scanning electron microscopy (SEM) images of aggregates formed by US. (E–G) Mechanical properties of the obtained fibrils measured by AFM nanoindentation. Separate images were prepared with biorender.com, Blender 2.93, and Autodesk Fusion 360.

Fig. 2.

(A) A schematic representation of the model dynamics features: the pale red circles represent the free monomers and the gray structures represent fibrils (or their fragments). The arrows highlight transition processes within the system, and their respective rates are given. (B) The probability density function (PDF) of the numerical results (the red and pink lines) compared to the experimentally measured histogram (the gray bars). The basic model contains processes (1) to (3) in panel (A), which can only lead to long fibrils transiently. In contrast, a modified model that contains process (4) in panel (A) can also lead to long fibrils in steady state. (C) Fibril mass concentration at the moment of observation (i.e., after the sonication) plotted against the initial concentration of monomers. The model curves are obtained by a numerical solution of SI Appendix, Eqs. S1 and S12, with parameters reproducing the data in panel (B). An Inset includes a table detailing the initial protein monomer concentrations and the fraction of fibrils obtained under specific ultrasonication conditions (20 kHz, 2 W, 10 min). A photograph illustrating the samples treated by ultrasound, which vary depending on the protein concentration is presented in SI Appendix, Fig. S2. For a detailed explanation of the equation symbols, refer to SI Appendix.

Fig. 3.

Kinetics of self-assembly and FTIR analysis of lysozyme monomers and nanofibrils formed due to low-amplitude US. (A) Analysis of the chemical kinetics of lysozyme nanofibrils formed by US at different protein concentrations (1, 5, 10, 20, 40 mg mL⁻¹) compared to seeds formed according to the standard protocol (see Experimental section), and native lysozyme monomers at the same concentrations (ratio 1:1). Nonnormalized data are shown in SI Appendix, Fig. S3. (B) Amide I and Amide II bands of the FTIR spectra of lysozyme monomers (cyan), and a nanofibril solution formed after 10 min of US (red). (C) Deconvoluted Amide I band of lysozyme monomers, (D) nanofibril solution, (E) A comparative analysis of the secondary structure in %.

Fig. 4.

Small X-ray scattering (SAXS) analysis. (A) Azimuthally integrated background-subtracted scattering intensity I, as a function of q, the magnitude of the momentum transfer vector $\overrightarrow{\mathrm{q}}$ (the black symbols in Fig. 3B) from soluble lysozyme monomers (adapted from ref. 17) Copyright 2023 the Author(s). Published by PNAS), (B) I vs. q from lysozyme assemblies formed after applying US irradiation to a solution of lysozyme monomers. (C) The mass fraction of the coexisting structures that formed after US irradiation (where 2DS denotes a 2D sheet). Models of the narrow (D) and flat (E) lysozyme fibrils formed after US exposure, based on the SAXS analysis. Fibril and protein visualization: ChimeraX 1. 7 (23, 24). Data analysis was performed as explained in our earlier publication (17).

Fig. 5.

(A) Chemical structure of hen egg white lysozyme (HEWL, PDB ID: 193L, 129 amino acid residues) represented as a cartoon model. The α-helix and β-sheet structures are shown in rainbow colors and are denoted by the letters “H” and “S”, corresponding to their positions in the protein structure and the amino acid sequence. Numeration of elements started from the N-terminal; (B) the superposition of three protein structures under different conditions and different simulation time frame snapshots: blue—at 300 K, cyan—at 338 K (t = 449,693 ps), and red—at 338 K 25 bar (t = 1,843,799 ps, state 2; see also SI Appendix, Figs. S19–S22), pointing to key structural elements; visualization: ChimeraX 1.7 (23, 24); diagrams of cumulative (0 to 3,000,000 ps) RMSD of C_α (SI Appendix, Fig. S4) over two runs for (C), the whole protein sequence, and (D) for H7 (Fig. 5A) under different simulation conditions (referred to in SI Appendix, Figs. S5 and S14, correspondingly).