Nanostructured films from hierarchical self-assembly of amyloidogenic proteins

Abstract

In nature, sophisticated functional materials are created through hierarchical self-assembly of simple nanoscale motifs. In the laboratory, much progress has been made in the controlled assembly of molecules into one-, two- and three-dimensional artificial nanostructures, but bridging from the nanoscale to the macroscale to create useful macroscopic materials remains a challenge. Here we show a scalable self-assembly approach to making free-standing films from amyloid protein fibrils. The films were well ordered and highly rigid, with a Young's modulus of up to 5-7 GPa, which is comparable to the highest values for proteinaceous materials found in nature. We show that the self-organizing protein scaffolds can align otherwise unstructured components (such as fluorophores) within the macroscopic films. Multiscale self-assembly that relies on highly specific biomolecular interactions is an attractive path for realizing new multifunctional materials built from the bottom up.

Figure 1. Fabrication of nanostructured films through multiscale hierarchical self-assembly

a, Protein molecules are first assembled into amyloid fibrils, which are then stacked into films. b, Atomic force micrograph of the component lysozyme fibrils. c, Scanning electron micrograph of the resulting free-standing protein film. d, Optical images of plasticizer containing lysozyme amyloid films under crossed polarizers show low transmission through the protein film when the objective polarizer is parallel to the fibril alignment in the film (left) and maximal transmission at an angle of 45° (right).

Figure 2. Characterization of nanostructured protein films by X-ray diffraction studies

a–c, Top row shows the exposure geometry and bottom row shows the respective X-ray data. Film without (a) and with (b) plasticizer, illuminated from the top, showing an isotropic orientation of the characteristic inter-sheet and inter-strand repeats (a) and orientational order, with the inter-sheet and inter-strand orientations being perpendicular to each other as required by the fibril geometry (b). c, Same film as in a, illuminated from the side, demonstrating the alignment of the fibrils in the film plane as described in the main text.

Figure 3. Mechanical testing of nanostructured protein films in a three-point bending geometry

a, An oscillating load is applied (i) to the centre section (ii) of the film suspended between the supporting clamps (iii). b, Graph shows storage modulus as a function of frequency f for different film samples from lysozyme (green squares, three individual films) and β-lactoglobulin (blue triangles, two individual films) and the extrapolation f → 0 to give the Young’s modulus. c, Comparison of Young’s moduli of different materials shows that the artificial nanostructured protein films have moduli (dark red rectangle: three-point bending geometry, data from Fig. 3b; light red rectangle: cantilever geometry, data from Supplementary Information) in the upper range for biomaterials.

Figure 4. Nanoscale alignment of fluorophores through self-organizing protein scaffolds

a, Plot showing the intensity of the emission of light from nanostructured films containing aligned fluorophores. The emitted light was transmitted through a polarizing filter with a fixed orientation, and the orientation of the film was changed through 360° by rotating the sample in the plane (filled blue squares); as a guide to the eye, a fit to the function [const + A sin(α + φ)²]² is shown in blue, where const is an offset that includes contributions from the emission of the unoriented population of fluorophores, A represents an amplitude of the excitation wave and φ a shift between the measured film angle α and the true direction of fibril alignment. The green open squares represent data from an equivalent measurement without the presence of the polarizers. A constant offset was subtracted from the control data intensity to facilitate comparison. The relative error on the fluorescence intensity was assumed to be constant and was estimated from repeated measurements at a fixed angle. b, Fluorescence microscopy image of a non-functionalized (i) and functionalized (ii) fibril film.