We report a rationale for the formation of amyloid fibrils from globular proteins, and we infer about its possible generality by showing the formation of giant multistranded twisted and helical ribbons from both lysozyme and β-lactoglobulin. We follow the kinetics of the fibrillation under the same conditions of temperature (90 °C) and incubation time (0-30 h) for both proteins, and we assess the structural changes during fibrillation by single-molecule atomic force microscopy (AFM), circular dichroism (CD), and SDS-PAGE. With incubation time, the width of a multistranded fibril increases up to an unprecedented size, with a lateral assembly of as many as 17 protofilaments (173 nm width). In both cases, a progressive unfolding and hydrolysis of the proteins into very short peptide sequences occurs. The molecular weights of peptide fragments, the secondary structure evolution, and the morphology of the final fibrils present striking similarities between lysozyme and β-lactoglobulin. Because of additional analogies to synthetic peptide fibrils, these findings support a universal common fibrillation mechanism in which hydrolyzed fragments play the central role.

We report the investigation of the mechanical properties of different types of amyloid fibrils by the peak force quantitative nanomechanical (PF-QNM) technique. We demonstrate that this technique correctly measures the Young's modulus independent of the polymorphic state and the cross-sectional structural details of the fibrils, and we show that values for amyloid fibrils assembled from heptapeptides, a-synuclein, Ab(1-42), insulin, b-lactoglobulin, lysozyme, ovalbumin, Tau protein and bovine serum albumin all fall in the range of 2-4 GPa.

Amyloid fibrils are implicated in over 20 neurodegenerative diseases. The mechanisms of fibril structuring and formation are not only of medical and biological importance but are also relevant for material science and nanotechnologies due to the unique structural and physical properties of amyloids. We previously found that hen egg white lysozyme, homologous to the disease-related human lysozyme, can form left-handed giant ribbons, closing into nanotubes. By using matrix-assisted laser desorption ionization mass spectrometry analysis, we here identify a key component of such structures: the ILQINS hexapeptide. By combining atomic force microscopy and circular dichorism, we find that this fragment, synthesized by solid-phase peptide synthesis, also forms fibrillar structures in water at pH 2. However, all fibrillar structures formed possess an unexpected right-handed twist, a rare chirality within the corpus of amyloid experimental observations. We confirm by small- and wide-angle X-ray scattering and molecular dynamics simulations that these fibrils are composed of conventional left-handed β-sheets, but that packing stresses between adjacent sheets create this twist of unusual handedness. We also show that the right-handed fibrils represent a metastable state toward β-sheet-based microcrystals formation.

We study the fibrillation pathway of ovalbumin protein and report the simultaneous formation of several types of fibrils, with clear structural and physical differences. We compare the fibrillation mechanisms at low pH with and without salt, and follow the kinetics of fibrils growth by atomic force microscopy (AFM), static and dynamic light scattering (SLS, DLS), and small-angle X-ray scattering (SAXS). We show that, among the morphologies identified, long semiflexible amyloid fibrils (type I), with persistence length Lp∼3 μm, Young's modulus E∼2.8 GPa, and cross-β structure are formed. We also observe much more flexible fibrils (type III, Lp∼63 nm), that can assemble into multistranded ribbons with time. They show significantly lower intrinsic stiffness (1.1 GPa) and a secondary structure, which is not characteristic of the well-ordered amyloids, as determined by circular dichroism (CD), wide-angle X-ray scattering (WAXS), and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). In between these two main classes of fibrils, a third family, with intermediate flexibility (type II, Lp∼300 nm), is also resolved.

We show for the first time the possibility of using networks of amyloid fibrils, adsorbed to solid supports and with plasma polymer coatings, for the fabrication of chemically homogeneous surfaces with well-defined nanoscale surface features reminiscent of the topography of the extracellular matrix. The robust nature of the fibrils allows them to withstand the plasma polymer deposition conditions used with no obvious deleterious effect, thus enabling the underlying fibril topography to be replicated at the polymer surface. This effect was seen despite the polymer coating thickness being an order of magnitude greater than the fibril network. The in vitro culture of fibroblast cells on these surfaces resulted in increased attachment and spreading compared to flat plasma polymer films with the same chemical composition. The demonstrated technique allows for the rapid and reproducible fabrication of substrates with nanoscale fibrous topography that we believe will have applications in the development of new biomaterials allowing, for example, the investigation of the effect of extracellular matrix mimicking nanoscale morphology on cellular phenotype.