FIGURE 3.

Chemical shift changes in the I-domain accompanying the D302A and H305A substitutions at a temperature of 20 °C. A, superposition of the ¹H,¹⁵N HSQC spectra for the WT I-domain (green) and the D302A mutant (red). (B) Superposed ¹H,¹⁵N HSQC spectra for the WT I-domain (green) and the H305A mutant (red). In A and B large chemical shift changes are indicated by arrows, drawn from the position of the cross-peak in the WT to the position of the cross-peak in the mutant spectrum. Composite chemical shift differences were calculated as abs [ΔdHN + 0.1(ΔdN)] for D302A (C) and H305A (D). Errors in the composite chemical shift differences are estimated to be 0.08 ppm based on the acquired digital resolutions of the ¹H and ¹⁵N dimensions. Segments with the largest chemical shift perturbations are labeled. E, mapping of the chemical shift differences between WT and D302A onto the I-domain structure: red, Val-300–His-305 reverse turn between strands β4-β5; orange, Thr-258–Gly-266 reverse turn between strands β2-β3; pink, long range effects for the Ser-333–Ala-335 segment. The corresponding structural mapping for H305A is nearly identical to that for D302A and is not shown.