Proximity relationships between residue 6 of troponin I and residues in troponin C: Further evidence for extended conformation of troponin c in the troponin complex

Skeletal muscle troponin C (TnC) adopts an extended conformation when cryst allized alone and a compact one when crystallized with an N-terminal tropon in I (TnI) peptide, TnI(1-47) [Vassylyev et al. (1998) Proc. Natl. Acad. Sc i. U.S.A. 95, 4847-4852]. The N-terminal region of TnI (residues 1-40) was suggested to play a functional role of facilitating the movement of TnI's i nhibitory region between TnC and actin [Tripet et al. (1997) J. Mel. Biol. 271, 728-750]. To test this hypothesis and to investigate the conformation of TnC in the intact troponin complex and in solution, we attached fluoresc ence and photo-cross-linking probes to a mutant TnI with a single cysteine at residue 6. Distances from this residue to residues of TnC were measured by the fluorescence resonance energy transfer technique, and the sites of p hoto-cross-linking in TnC were determined by microsequencing and mass spect rometry following enzymatic digestions. Our results show that in the tropon in complex neither the distance between TnI residue 6 and TnC residue 89 no r the photo-cross-linking site in TnC, Ser133, changes with Ca2+, in suppor t of the notion that this region plays mainly a structural rather than a re gulatory role. The distances to residues 12 and 41 in TnC's N-domain are bo th considerably longer than those predicted by the crystal structure of TnC .TnI(1-47), supporting an extended rather than a compact conformation of Tn C. In the binary TnC TnI complex and the presence of Ca2+, Met43 in TnC's N -domain was identified as the photo-cross-linking site, and multiple distan ces between TnI residue 6 and TnC residue 41 were detected. This was taken to indicate increased flexibility in TnC's central helix and that TnC dynam ically changes between a compact and an extended conformation when troponin T (TnT) is absent. Our results further emphasize the difference between th e binary TnC TnI and the ternary troponin complexes and the importance of u sing intact proteins in the study of structure-function relationships of tr oponin.