To probe how pH may affect the MLGI properties between Gx-DiMan and DC-SIGN/R, we have further monitored the hydrodynamic diameters (Dhs) of the resulting lectin-GNP complexes by dynamic light scattering (DLS). Similarly, all measurements were performed under a fixed lectin (wild-type, no dye labelling): Gx-DiMan molar ratio of 10: 1 and 60:1 for G13-and G27-DiMan, respectively, in the MES As shown in Fig. , the pH dependences of Dh of binding induced lectin-GNP complexes were very different between DC-SIGN and DC-SIGNR. For DC-SIGN, it formed compact complexes at normal physiological pH (7.4) with Dhs of ∼50 and ∼80 nm with G13-DiMan and G27-DiMan, respectively. These Dhs match well to what expected for single Gx-DiMan particles bounded with a single layer of DC-SIGN molecules. This result is fully consistent to our previous observation that DC-SIGN binds tetravalently with all four CRDs to a single GNP-glycan particle. This binding mode for DC-SIGN was maintained, from pH 7.4 to 5.8 with G13-DiMan, or pH 7.4 to 7.2 with G27-DiMan. Further reducing pH to 5.4 (and below) produced two distinct particle distributions with Dh of ∼25 nm and ∼560 nm at pH 5.4 (Fig. ). Interestingly, the Dh of the smaller species matched well to that of individual G13-DiMan particles, indicating the possible release of some G13-DiMan particles from the G13-DiMan-DC-SIGN complexes at this pH. The larger Dh species were attributed to extensively crosslinked DC-SIGN-G13-DiMan assemblies, likely due to a change of DC-SIGN binding mode from simultaneous tetravalent binding with G13-DiMan to cross-linking via pH induced conformational changes (Dh of Gx-DiMan alone did not change over pH 4.6 to 7.4). This was further supported by that the overall Dh distribution of DC-SIGN-G13-DiMan complexes at pH 5.4 (Fig. ) closely resembled that of the DC-SIGNR-G13-DiMan complexes at pH 7.4 (Fig. ), where DC-SIGNR was known to cross-link with GNP-DiMan particles at pH 7.4. Moreover, based on a much weaker GNP-DiMan affinity of DC-SIGNR over DC-SIGN at pH 7.4 despite identical monovalent binding motifs, changing DC-SIGN binding mode from simultaneous tetravalent binding to crosslinking would thus expect to weaken its G13-DiMan affinity at ≤ pH 5.4. This is exactly what has been observed in the fluorescence quenching experiments described in the previous section (Fig. ).