Characterizing the Cellulose Binding Interactions of Type-A Carbohydrate-Binding Modules using Acoustic Force Spectroscopy

A critical understanding of carbohydrate binding modules (CBMs) is vital for the manipulation of a variety of biological functions they support, including biomass deconstruction, polysaccharide biosynthesis, pathogen defence, and plant development. The unbinding characteristics of CBMs from a polysaccharide substrate surface can be studied using rupture force measurements since it enables a quantitative inference of binding properties through the application of dynamic force spectroscopy (DFS) theory. With the increase in usage of CBMs for diverse applications, it is important to engineer and characterize CBMs that have desired sets of interactions with various carbohydrate substrates. However, though the effect of mutations in the binding motif residues is known to influence CBM binding affinity, its effect on the rupture forces is still not well quantified. This is primarily due to the low experimental throughput of most single-molecule DFS techniques available to characterize the force-induced dissociation of protein-ligand interactions. Here, we have determined the rupture forces of microscopic beads functionalized with various wild-type and mutant CBMs using a highly multiplexed DFS technique called Acoustic Force Spectroscopy (AFS). We have characterized the acoustic force-induced dissociation of specifically two type A CBMs (i.e., CBM3a and CBM64) and relevant seven binding motif targeting CBM mutants unbinding from a nanocellulose surface, over a broad range of DFS loading rates (i.e., 0.1 pN/s to 100 pN/s). Our analysis of the rupture force DFS data yields apparent CBM-cellulose bond interaction parameters, which enables a quantitative comparison of the effect of corresponding mutations on cellulose-CBM binding interactions that compares favorably with results from classical bulk ensemble based binding assays. In summary, detailed insights into the rupturing mechanism of multi-CBM fused domains provides motivation for usage of specific constructs for industrial biotechnological applications.