FIGURE 7.

Effects of the bound Ca²⁺ to the C2 quad(410–526) upon interaction with DPC micelles. A, changes in chemical shifts, Δδ¹H, of indicated amide proton resonances of the C2 quad(410–526) are plotted against [DPC]. Solid lines show the fits of the data to Equation 3 (see “Experimental Procedures”). Filled triangles, diamonds, circles, and squares showed Δδ¹H values of residues Asp-429, Asp-489, Asp-490 and Asp-485, respectively. The average dissociation constant (K_d) was determined as 1.1 ± 0.3 mm. B–F, overlay of a portion of ¹H-¹⁵N SOFAST-HMQC spectra of 150 μm ¹⁵N-labeled C2 D491N (B), D429N (C), D435N (D), D483N (E), and D490N (F) mutants in the presence of 30 mm [Ca²⁺] recorded with increasing [DPC]. Spectra were processed identically and plotted with the same contour levels. Resonance colors correspond to different [DPC] shown in the same color on the bottom right side of spectra. In the D491N mutant, no significant CSPs upon adding DPC were observed in the resonances of backbone amides, but a side chain amide resonance of Asn-454Δ was clearly perturbed. In the D429N, D435N, and D483N mutants, resonances indicative of degradation or aggregation gradually emerged with increasing [DPC]. In the D490N mutant, the same resonances of the C2 quad(410–526) were perturbed with increasing [DPC].