Unravelling Protein–Fungal Hyphae Interactions at the Nanoscale

Fungal hyphae have demonstrated their importance in developing environmentally friendly, multiscale, composite assemblies where animal-derived proteins have been predominantly used as binders. Now, an ongoing challenge is to replace those high-performance animal protein binders with ecofriendly, plant-based alternatives. While the majority of studies have focused on the binding implied by rheological observations, relatively little is known about how such animal proteins bind to hyphal surfaces at nanometric length scales, and this knowledge is required to replace animal-derived binders with plant protein alternatives. Here, we decode intermolecular interactions of plant protein-based binders such as potato protein (<i>PoP)</i> to fungal (<i>Fusarium venenatum</i><i>)</i> hyphae in comparison to a classic animal protein-based binder (egg white protein, <i>EWP</i>) using a suite of theoretical and experimental approaches. Self-consistent field calculations modeling fungal hyphae as weakly hydrophobic, parallel cylinders predicted differences in the interaction potentials between the model protein layers, showing that <i>EWP</i> had an attractive potential across a broad range of conditions, in contrast to <i>PoP</i> that was mainly repulsive. Stimulated emission depletion (STED) microscopy of protein-coated fungal hyphae confirmed that <i>EWP</i> delivers a uniform and complete coverage, while <i>PoP</i> naturally aggregates, resulting in more patchy coverage. Experimental interaction forces were measured using colloidal probe atomic force microscopy, confirming the influence of non-Coulombic forces particularly dominating in <i>PoP</i>, and attractive forces in <i>EWP</i>, further differentiating their respective binding mechanisms. Collectively, this multimethodological study provides a first-hand molecular explanation of the weaker hyphal-binding properties of aggregated plant proteins at the nanoscale, consistent with the previously reported macroscale observations.