NMR Structure of a Bifunctional Rhodamine Labeled N-Domain of Troponin C Complexed with the Regulatory "Switch" Peptide from Troponin I: Implications for in Situ Fluorescence Studies in Muscle Fibers

The structure of the calcium-saturated regulatory domain of skeletal troponin C (sNTnC) complexed with the switch peptide comprising residues 115−131 of troponin I (TnI), and with a bifunctional rhodamine fluorescent label attached to residues 56 (E56C) and 63 (E63C) on the C helix of sNTnC, has been determined using nuclear magnetic resonance (NMR) spectroscopy. The structure shows that the integrity of the C helix is not altered by the E(56,63)C mutations or by the presence of the bifunctional rhodamine and that the label does not interact with the hydrophobic cleft of sNTnC. Moreover, the overall fold of the protein and the position of the TnI peptide are similar to those observed previously with related cardiac NTnC complexes with residues 147−163 of cardiac TnI [Li et al. (1999) Biochemistry 38, 8289−8298] and including the drug bepridil [Wang et al. (2002) J. Biol. Chem. 277, 31124−31133]. The degree of opening of the structure is reduced as compared to that of calcium-saturated sNTnC in the absence...