FIGURE 4. Equilibrium binding of PEP-19 polypeptides to CaM.

Panel A shows the fractional change in amide chemical shifts in the C-domain of Ca²⁺-CaM as a function of increasing concentrations of either PEP-19 (closed circles) or PEP(28–62) (open circles). The data represent the average fractional change for ¹H and or ¹⁵N nuclei for residues 99, 105, 109, 116, 121, 146, and 147. The line represents the fit to a single-site binding model described under “Experimental Procedures.” Panel B shows the average fractional change in chemical shifts for residues 5, 17, 19, 21 29, 33, 44, 53, 55, 57, 64, 70, and 73 in the N-domain (open squares) and residues 94, 105, 106, 110, 116, 117, 130, 137, 146, 147, and 148 in the C-domain (open triangles) of Ca²⁺-CaM as a function of increasing concentrations of PEP(39–62). Chemical shift changes in response to PEP(39–62) did not fit well to a single-site binding model. Panel C shows the change in fluorescence from CaM_ACR upon titration with PEP-19 or PEP(39–62). Panel D shows titration of IAEDANS-labeled CaM(T110C) with DDPM-labeled PEP-19 or PEP(39–62). Labeled CaM for experiments in both panels C and D was present at 0.05 or 0.5 μM for titration with PEP(39–62) or PEP-19, respectively.