FIGURE 1.

Titration of cCTnC·2Ca²⁺ with EGCg. Two-dimensional ¹H,¹⁵N HSQC (a) and ¹H,¹³C-HSQC (b) spectra arising from backbone and side chain amide groups (a) and side chain methyl groups (b) are overlaid for a series of EGCg additions. Each titration point represents the titration points described under “Experimental Procedures.” The titration was made into ¹³C,¹⁵N-labeled cCTnC·2Ca²⁺, and both the ¹H,¹⁵N HSQC and ¹H,¹³C-HSQC spectra were acquired at each titration point. Assignments of some of the cross-peaks are labeled. The multiple contours (●) represent the initial point in the titration, with no EGCg added, and each open contour (○) represents a specific point in the titration for a given residue. b, red contours represent cross-peaks with negative intensity, a feature of the constant time ¹H,¹³C-HSQC experiment. The direction the peaks shift is indicated with arrows, for example see Gly¹²⁵. c, curves represent the amide resonances belonging to residues affected by ligand binding, as shown in a. The curves were fit as a function of normalized total chemical shift perturbation versus [EGCg]_total/[cCTnC·2Ca²⁺]_total. The total chemical shift changes were calculated in hertz as follows: Δδ = ((Δδ¹H)² + (Δδ¹⁵N)²)^1/2. Because hertz is used instead of parts/million, a correction factor of 1/5 for the ¹⁵N dimension is not used. d, chemical shift mapping on the structure of cCTnC·2Ca²⁺. The ribbon representation of cCTnC·2Ca²⁺ is shown in yellow, and residues that were perturbed greater than the mean chemical shift change for all backbone amide resonances of cCTnC are colored in red.