FIGURE 6.
Titration of cCTnC·2Ca2+·cTnI-(34–71) 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+·cTnI-(34–71), 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 the open contours (○) represent the end 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 that the peaks shift is indicated by arrows, for example see Met120. c, curves represent a number of 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+·cTnI-(34–71)]total. d, cCTnC·2Ca2+·cTnI-(34–71) complex is shown in lime green with cCTnC·2Ca2+ and cTnI-(34–71) shown in schematic representation. Chemical shift perturbations of the backbone amide resonances induced by EGCg binding to cCTnC·2Ca2+·cTnI-(34–71) are colored in red for residues that shifted greater than the mean shift of all residues of cCTnC. Total chemical shift changes are 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.