Covalent linkage of cello-oligosaccharides to borosilicate glass for single molecule binding measurements using acoustic force spectroscopy

Carbohydrate binding modules (CBMs) are proteins that bind to various carbohydrate constructs. CBMs are often components of modular cellulase enzymes that bind to polysaccharides and specifically break them down into smaller subunits. These cellulases are important protein complexes in the field of biomass processing, where they are used to decompose materials like cellulose into useful compounds that are raw materials for industrial processes. The engineering of CBMs for improved binding to polysaccharides is one of the main methods for enhancing efficiency of cellulases. For this reason, the binding behavior between CBMs and the polysaccharides they target has been an area of interest for some time. CBMs are categorized into three types based on the polysaccharides they bind to. Type-B CBMs typically bind to oligosaccharides with a degree of polymerization of three or more. Since many of these shorter oligosaccharides are soluble in water, previous studies of Type-B CBMs have been performed with both components of the binding complex free in solution. Force spectroscopy is one of the most powerful tools for measuring the binding interactions at the single molecule level. Acoustic force spectroscopy (AFS) is a high throughput method that can measure the rupture forces of hundreds of bound complexes simultaneously. The AFS requires both components of the protein-ligand complex to be bound to either a borosilicate glass surface, or a polystyrene microsphere. Given the lack of force spectroscopy measurements of type-B CBMs and cello-oligosaccharides, the purpose of this project was to develop and validate a novel method for covalently linking cello-oligosaccharides to a borosilicate glass surface. This method has enabled the first force spectroscopy study of bound cello-oligosaccharides and type-B CBMs. By chemically modifying cello- oligosaccharides, and using multiple bioconjugation techniques, cellotetraose, cellopentaose, and cellohexaose were successfully linked to a borosilicate glass surface and used for AFS measurements of binding rupture forces. In the future, this novel technique can be used to further the understanding of binding interactions between various proteins and soluble polysaccharides.M.S.Includes bibliographical reference