Figure 4.

The L27E/K28E Mutation Disrupts CaM Binding to the CNGC12 NT Motif.

(A) ND-PAGE of Ca²⁺/CaM with NT peptide or a peptide bearing the double mutation L27E/K28E (NT^mut) at molar ratios indicated. Closed triangles indicate the migration size of CaM alone, while open triangles indicate the migration size of CaM-peptide complex.

(B) Overlaid ¹H-¹⁵N HSQC-NMR spectra of 0.2 mM uniformly ¹⁵N-labeled CaM with increasing molar ratios (as indicated under the figure) of NT (top) or NT^mut (bottom) peptide. Spectra of 0.2 mM uniformly ¹⁵N-labeled CaM in the presence of increasing molar ratios of NT^mut peptide. NMR samples were prepared in 10 mM Tris-Cl, 150 mM NaCl, and 10 mM CaCl₂ pH 7.0. The peaks corresponding to three individual CaM residues (Gly-33, Ile-27, and Ala-57) are shown in enlarged panels.