Probing Multivalent Protein-Carbohydrate Interactions by Quantum Dot-Förster Resonance Energy Transfer

Cell surface carbohydrate-binding proteins (also known as lectins) recognize surface carbohydrates of viral, bacterial, and fungal pathogens to regulate host immune responses; however, such interactions are also often exploited by pathogens to enhance infection. Multivalent binding is typically involved to compensate for the intrinsically weak nature of protein-carbohydrate interactions. The spatial orientation of carbohydrate recognition domains (CRDs) in multimeric lectins plays a central role in governing their binding affinity and specificity. Such information is also key for designing potent, multivalent inhibitors, but is difficult to obtain in the absence of valid lectin crystal structures. In this chapter, we describe a method to allow controlled polyvalent display of carbohydrates on the surface of fluorescent quantum dots (QDs), which enables the use of Förster resonance energy transfer (FRET) efficiency between the QD and dye-labeled lectins to probe how well the displayed sugars match the presentation of the CRDs. A highly efficient ligand-exchange method has been developed to construct compact, polyvalent QD-mannose conjugates (QD-Man), which are essential for sensitive FRET readout and effective valency tuning. A rapid, sensitive, and ratiomeric FRET readout has been developed to quantify their binding affinity and specificity, which, in combination with the geometry of QD-Man, allow us to derive the spatial orientation of the CRDs. A good correlation between the specific binding of QD-Man to recombinant lectins and its effective inhibition of virus cell entry mediated by native cell surface lectins validates QD-FRET as a reliable technique to study multivalent receptor-ligand recognition mechanisms.