Cell surface carbohydrate-binding-proteins (also known as lectins) recognize pathogen (e.g. including viruses, bacteria and fungi) surface carbohydrates to regulate host immune responses, however but, the such interactions are also often exploited by pathogens to enhance for infection. Because of the intrinsically weak nature of protein-carbohydrate interactions, multivalent bindings are typically invloved. The spatial orientation of carbohydrate-recognition-domains (CRDs) in multimeric lectins plays a centralvery important role in governing their binding high affinity and specificity recognition. 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 a of controlled display of ingpolyvalent mulitple carbohydrates onto fluorescent quantum dots (QDs) surface to probe how they match the CRDs presentation by measuring Förster Resonance Energy Transfer (FRET) efficiency between the QD and fluorescent dye-labelled lectins. A highly efficient ligand-exchange method has been developed here allows to construct compact, polyvalent QD-mannose conjugates (QD-Man) with high copies of mannose which are essential for sensitive FRET application readout and effective valency tuning. A rRapid, sensitive and ratiomeric FRET readout has been used to quantify their binding affinity and specicity, which in combination with QD-Man’s geometry, quantifies the interactions allows us to derive the CRD`s spatial orientation. A good correlation of between QD-Man’s nose specifically binding to recombinatly expressed lectins in solution and with its effective inhibition of cell surface native lectin mediated virus cell entry proves validates QD-FRET being a reliable technique to study multivalent receptor-ligands recognition mechanisms.
CORE (COnnecting REpositories)
CORE (COnnecting REpositories)