Figure 7.

Chemical shift perturbation upon binding of GlcNAc₃ to the CRD. (A) Total chemical shift perturbation per residue (Δδ_total) upon addition of GlcNAc₃ was calculated from Eqn (1) in Experimental procedures. The dashed horizontal line represents the 1 × standard deviation cut‐off for the dataset, above which a change was considered to be significant. (B) Chemical shift perturbations upon binding of GlcNAc₃ mapped onto the CRD crystal structure. Residues showing chemical shift perturbations above the 1 × standard deviation cut‐off (see dashed line) are shown in red on the CRD crystal structure (PDB ID http://www.rcsb.org/pdb/search/structidSearch.do?structureId=1K9J). These agree well with the regions of the CRD that responded most strongly to binding of the small glycans (Man₃, Man₅, and GlcNAc₂Man₃) previously reported, namely α‐helix 2, β‐strands 6 and 7, and both Ca²⁺‐binding loops. (C) The total chemical shift perturbation per residue (Δδ_total) is plotted against glycan concentration, and fitted to a single‐site binding model. All significantly perturbed residues fit the same single‐site model, but here only three residues in α‐helix 2 (a proposed binding site for small glycan fragments) are shown as an illustration. The fit was used to calculate the dissociation constant of the CRD for GlcNAc₃.