It is worth noting that the association coefficient (𝑘 𝑜𝑛 ) measured by our QD-FRET assay is almost 1000 fold faster than that measured from SPR using surface immobilized DC-SIGN (e.g. ~10 vs. ~10 4 M -1 s -1 ). We attribute this difference to the different binding environments. As our QD-FRET assays are performed in solution, both binding partners are able to diffuse freely, greatly increasing the likelihood of collision and thus association. Whereas, in SPR, as one binding partner (e.g. DC-SIGN) is immobilized on surface and unable to diffuse, it must rely on the diffusion of the other partner to the surface target sites for any binding to occur. This would result in a significantly slower on-rate In summary, we have developed a new sensitive QD-FRET based technique for successful dissection of the thermodynamic and kinetic contributions behind affinity enhancing mechanisms in MLGIs with distinct binding modes, and for identification of lectin structure-function relationships. We have revealed that the lower QD-DiMan binding affinity for the inter-cross-linking DC-SIGNR, over that of the simultaneous binding DC-SIGN, is a consequence of a larger binding entropy penalty. We have further demonstrated that the removal of a 16 amino acid Cterminal segment in DC-SIGN, absent in DC-SIGNR, greatly affects its QD-DiMan binding thermodynamic profiles, and completely changes the binding from an enthalpy driven into an entropy driven MLGI. These results have allowed us to hypothesize that the entropic gain in removing the Cterminal segment is the result of an increased freedom of the CRDs which is not present in DC-SIGN naturally. This result supports the idea that the C-terminal segment may play a key role in maintaining CRD orientation and therefore facilitating the multivalent specificity of DC-SIGN in binding to multivalent glycans.