FIGURE 2. Titration of rCBD with P713.

Panel A. HSQC spectra of 0.5 mM ¹⁵N-labeled CBD in 25 mM sodium phosphate buffer, 5% D₂O/H₂O, pH 7.0 were recorded before (black) and after titration with collagen peptide P713 at concentrations of 0.15 mM (red), 0.3 mM (green), and 1.5 mM (blue). Residues that exhibited shift perturbations of backbone amides upon P713 peptide addition are shown and labeled with their one letter amino acid code and residue number. Residues which were selected for alanine substitution are underlined. Residues selected as negative controls are labeled in italics. The insert shows the change in the chemical shift of F297 as P713 is added. Panel B. The concentration dependent chemical shifts of three representative CBD residues upon binding P713 were plotted against the concentration of P713. Panel C. Magnitudes of chemical shift change (Δδ) for CBD residues in the presence of a P713 concentration of 1.5 mM. Chemical shift perturbations, Δδ, were calculated from proton and nitrogen shift by using the formula {[(δΔH)²+(δΔN/5)²]/2}½. Panel D. Three-dimensional surface representation of individual CBD modules showing the positions of residues that underwent shift changes in the presence of P713. The intensity of blue color in each CBD modules is proportional to the magnitude of the chemical shifts changes induced by peptide P713. The residues are labeled with the one letter amino acid code and residue number. The structure shown is from the reported MMP-2 crystal structure, PDB 1CK7.