(164k) Acoustic Force Spectroscopy Enables Multiplexed Single-Molecule Characterization of Protein-Carbohydrate Binding

To rationally engineer more efficient enzymes for cellulosic biomass deconstruction for industrial biotechnology applications or develop protein binders targeting extracellular matrix enriched in carbohydrates for therapeutic applications, it is necessary to better understand the complex protein-carbohydrate interfacial interactions. Carbohydrate binding module (CBM) proteins or lectins are often associated with driving such surface-tethered protein-ligand interfacial interactions. However, it is not well known how proteins recognize, bind, and dissociate from polysaccharide surfaces due to the lack of suitable toolkit to study CBM-substrate binding interactions. Here, we outline a general approach to methodically study the unbinding behavior of CBMs from model polysaccharide surfaces using a highly multiplexed single-molecule force spectroscopy assay technique called acoustic force spectroscopy. We use this technique to study how CBM dissociate from cellulose surface to infer the molecular mechanism governing substrate recognition and dissociation. Our approach can be broadly applied to study multivalent protein-polysaccharide binding interactions relevant to other carbohydrates such as starch, chitin, or hyaluronan to engineer more efficient ligand binders or biocatalysts.