Oat Plant Amyloids for Sustainable Functional Materials

Abstract

Amyloid functional materials from amyloid fibril building blocks, produced in vitro from amyloidogenic natural proteins or synthetic peptides, show diverse functionalities ranging from environmental science and biomedicine, to nanotechnology and biomaterials. However, sustainable and affordable sources of amyloidogenic proteins remain the bottleneck for large-scale applications, and to date, interest remains essentially limited to fundamental studies. Plant-derived proteins would be an ideal source due to their natural abundance and low environmental impact. Hereby oat globulin, the primary protein of oat plant (Avena sativa), is utilized to yield high-quality amyloid fibrils and functional materials based thereof. These fibrils show a rich multistranded ribbon-like polymorphism and a fibrillization process with both irreversible and reversible pathways. The authors furthermore fabricate oat-amyloid aerogels, films, and membranes for possible use in water purification, sensors, and patterned electrodes. The sustainability footprint of oat-amyloids against other protein sources is demonstrated, anticipating an environmentally-efficient platform for advanced materials and technologies.

Keywords: amyloid fibrils; functional amyloid materials; plant protein; reversible amyloid; sustainability.

Conflict of interest statement

The authors declare no conflict of interest.

© 2021 The Authors. Advanced Science published by Wiley-VCH GmbH.

Figures

Figure 1

Oat globulin extraction process and comparison with proteins from other plant sources. a) Schematic of extraction and purification of oat globulin from oat grain. b) SDS‐PAGE of oat globulin and reduced oat globulin. Oat globulin in the presence of 1% SDS, 31.3 mm Tris‐HCl, 12.5% glycerol (oat globulin), or of 1% SDS, 2.5% β‐mercaptoethanol, 31.3 mm Tris‐HCl, 12.5% glycerol (reduced oat globulin) were tested, respectively. c) Protein content from different sources including oat, rice, cereal (average), fabaceae, soy, and milk. Oat has a protein content of 14%, the highest in all cereal grains[ 18 ] and comparable to bean and soy. d) Protein diversity of oat, pea, cowpea, chickpea, soy, rice, wheat, quinoa, and barley. Oat globulin is the dominating fraction in oat protein, while other plants have a broader diversity in their protein composition.

Figure 2

Characterization and analysis on the oat globulin mature amyloid fibril. a) AFM height image of oat globulin amyloid fibrils. Hierarchical fibrils show a clear periodic height fluctuation with different periodicities. b) ThT fluorescence assay of oat globulin formation. ThT signal immediately increased after incubation at 90 °C and gradually reached a plateau after 18 h. c) SDS‐PAGE pattern of protein solution during incubation, indicating a gradual hydrolysis of monomeric oat globulin. d) Evolution of FTIR spectra showing the transition from helical and random coil structures (1645 cm−1) to β‐sheets (1623 cm−1). e) Distribution of oat globulin fibril maximum heights, showing three main peaks, 2.2, 4.1, and 6.0 nm, that are related to different families of fibrils. Inset shows the linear correlation between persistence length (Lp) and fibril height. f) Proposed schemes and corresponding AFM images of multistranded left‐handed twisted ribbon fibrils with one, two, three, and four protofilaments.

Figure 3

Formation and coexistence of mature fibrils and flexible worm‐like fibrils. a) TEM and AFM images of the mature fibril, coexistence of mature and worm‐like fibril, and elongated worm‐like fibrils. b) ThT assay of oat globulin solution during a heating and following cooling treatment corresponding to mature (green) and worm‐like (blue) fibrillization, respectively. To automatically sampling ThT signal, the protein solution was first incubated at 65 °C in the reader; ThT signal reached the equilibrium phase after 60 h. Then, the following incubation at room temperature enabled a further aggregation of worm‐like fibrils, with detailed curves in the inset. c) Histogram of worm‐like fibril maximum height, that shows two families of worm‐like fibrils with different heights as illustrated in the inset. d) Persistence length of each analyzed family of worm‐like fibrils (blue) and mature rigid fibrils (green). The worm‐like fibrils, irrespectively of the family they belong to, have a persistence length of ≈100 nm. Diversely, the persistence length of rigid fibrils is a function of the family they belong to, and can be as high as several µm. Families of worm‐like and rigid fibrils that have a similar average height, show instead a difference of one order of magnitude between their persistence lengths (as highlighted by the red circle). Comparison of molecular and morphological features between mature fibril and worm‐like fibril, including e) contour length, f) curvature histograms, g) periodicity analysis and h) pitch estimation.

Figure 4

The reversibility of worm‐like fibrils and oat globulin fibrillization mechanism. Forming and dissembling of worm‐like fibrils over the cooling and heating processes respectively in the a) presence and b) absence of mature fibrils. ThT signal during the cooling (blue) and heating (red) incubation processes in the c) presence and d) absence of mature fibrils. The insets show the details in a higher time resolution. e) CD spectra of worm‐like fibril solutions overheating from 20 to 90 °C. Inset shows secondary structural variation obtained from the analysis of the collected CD spectra. f) MALDI‐MS spectra of mature rigid and worm‐like fibrils. g) WAXS 2D‐scattering patterns, azimuthal and radial WAXS profile of the film of mature oat fibrils (MF), and the hybrid of mature and worm‐like fibrils (MF and WF). h) Energy landscape of oat globulin mature rigid fibrils and worm‐like fibrils.

Figure 5

Functional material applications and sustainability of oat amyloid fibrils. a) Oat amyloid based aerogel on the top of the awns of an oat spikelet. b) Photo (left), SEM images (middle), and schematics (right) of freeze‐dried aerogel from oat globulin mature fibril (top) and hybrid of mature and worm‐like fibrils. Scale bar: 100 nm. c) Water purification performance of β‐lactoglobulin/oat globulin fibrils‐active carbon hybrid membranes, with purification efficiency indicated (blue). d) Heavy metal adsorption capacity of oat mature fibril and hybrid of mature fibrils and different amounts of worm‐like fibrils incubated from 2 to 50 days. e) Au‐amyloids interdigital electrode and its capacitance variation with a finger approaching to and retracting from the electrode. Scale bar = 500 nm. f) Sustainability footprint of meat protein, milk protein, and oat protein with multiple factors considered, including affordability, GFG emissions, land use, eutrophication, freshwater withdrawals, terrestrial acidification, protein content, and public acceptability. Scores in each discriminant are shown by red (low), yellow (medium), and green (high performance). The overall sustainability footprint indicates that oat is the most sustainable source of protein in this comparison.