Figure 5.

¹H-¹⁵N HSQC NMR spectra of WT CIB2 in its apo form and in the presence of Mg²⁺ and Ca²⁺ highlight an allosteric communication between E64 and N121. (A) Superimposition of the two-dimensional ¹H-¹⁵N HSQC NMR spectra of the apo- (black), and Ca²⁺-bound (green) ¹⁵N-WT CIB2. (B) Superimposition of the HSQC spectra of apo (black), and Mg²⁺-bound (blue) ¹⁵N-WT CIB2. In the insets, zoom of the HSQC spectra of the downfield peaks of the metal-bound forms of ¹⁵N-WT CIB2. Metal ions were present at a protein:metal ratio of 1:15. (C) Superimposition of downfield region of the ¹H-¹⁵N HSQC NMR spectra recorded on ¹⁵N-WT CIB2 containing 15 equivalents of Mg²⁺ (blue), 15 eq Mg²⁺ + 15 eq Ca²⁺ (red), and 15 eq Ca²⁺ (green). (D–F) Variation of ¹H-¹⁵N HSQC peak intensities of WT CIB2 as a function of Ca²⁺ concentration. The peak intensities were normalized with respect to the maximum value. The continuous lines represent the data fitted against equations as indicated in section Materials and Methods. The plots refer to the amide peaks of residues E64 (D), G162 (E), and N121 (F). All the spectra were recorded at 600 MHz and 25°C. All samples were at protein concentration of 320 μM in 20 mM Hepes, 100 mM KCl, 1 mM DTT, pH 7.5.