Nanocellulose aerogels as 3D amyloid templates

Abstract

Proteins in solution tend to coat solid surfaces upon exposure. Depending on the nature of the surface, the environmental conditions, and the nature of the protein these adsorbed proteins may self-assemble into ordered, fibre-like structures called amyloids. Nanoparticulate surfaces, with their high surface to volume ratio, are particularly favourable to amyloid formation. Most prior research has focussed on either inorganic or organic nanoparticles in solution. In this research, we instead focus on aerogels created from TEMPO-oxidized cellulose nanofibers (TO-CNF) to serve as bio-based, three-dimensional amyloid templates with a tuneable surface chemistry. Previous research on the use of cellulose as a protein adsorption template has shown no evidence of a change in the secondary protein structure. Herein, however, with the aid of the reducing agent TCEP, we were able to induce the formation of amyloid-like 'worms' on the surface of TO-CNF aerogels. Furthermore, we demonstrate that the addition of the TO-CNF aerogel can also induce bulk aggregation under conditions where it previously did not exist. Finally, we show that the addition of the aerogel increases the rate of 'worm' formation in conditions where previous research has found a long lag-phase. Therefore, TO-CNF aerogels are shown to be excellent templates for inducing ordered protein aggregation.

Fig. 1. Schematic illustrating the unfolding, aggregation, and self-assembly of the HEWL monomer in the presence of TCEP and the TO-CNF aerogel and as a function of varying pH.

Fig. 2. (a) HEWL-TCEP solutions at various pH values in the presence and absence of the TO-CNF aerogel. Solutions with the aerogel are marked with an asterisk (*). (b) Z-Average diameter of the particles dispersed in the HEWL-TCEP solution at pH 3.5 incubated in the presence (black and red) and absence (blue) of the aerogel. Black and red data correspond to two independent replicates. (c) AFM height image of the aggregates found in the HEWL-TCEP solution at pH 3.5 incubated for 24 hours in the presence of the aerogel.

Fig. 3. IR spectra of various TO-CNF aerogel incubated in HEWL solutions with or without TCEP at various pH values. The spectra were normalized to the C–O peak of cellulose. Arrows indicate the positions of different peaks and the corresponding vibration.

Fig. 4. TIRF (left) and Brightfield (right) images of TO-CNF aerogels incubated in HEWL-TCEP solutions at pH 2.5 stained with FSB. Red arrows indicate various worm-like protein fibres formed on the aerogel surface.

Fig. 5. SEM image of a TO-CNF aerogel incubated in a HEWL-TCEP (left) or TCEP solution (right) at pH 2.5 for 24 hours. Red arrows indicate a fibre-like structure on the surface of each aerogel that was investigated via EDX spectroscopy.