Carbohydrate-based biopolymers are found abundantly across all forms of life and play a major role in biomolecular recognition processes. For example, cellulolytic microbial enzymes adsorption to carbohydrate polymers like cellulose plays a critical role in the deconstruction of lignocellulosic biomass to fermentable sugars. Hence, characterization of protein adsorption to carbohydrates is often the focus of various fields of research ranging from targeted drug delivery to cellulosic biofuels production. Several bulk-ensemble based analytical methods have been developed to characterize the adsorption/desorption kinetics of proteins to carbohydrate polymers. However, many such methods rely on over-simplified models to characterize binding interactions that often do not reflect the underlying molecular mechanism of protein binding to highly multivalent carbohydrate-based ligands. But we have so far lacked access to suitable analytical methods to carefully probe protein-carbohydrate binding interactions from a first-principles point of view.<br/>In this study, we use a highly multiplexed Acoustic Force Spectroscopy technique to measure the single-molecule binding lifetimes and unbinding rupture forces of carbohydrate-binding module (CBM) proteins to model cellulosic films. We characterize the dependence of the piconewton force loading rates on the non-equilibrium unbinding forces of various CBM proteins and use established theory to extract relevant equilibrium binding constants. Furthermore, we elucidate the role of conserved aromatic residues in the CBM carbohydrate recognition sites on the unbinding process to develop binding models that provide insight into the molecular mechanisms of protein adsorption and orientation on multivalent cellulosic polymers. In summary, our single-molecule force spectroscopy method allows us to systematically probe the structure-function relationships driving multivalent protein adsorption to carbohydrate-based polymers.<br/>

Single-molecule protein-carbohydrate dissociation characterization using acoustic force spectroscopy

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.