Abstract
We used resonance energy transfer and site-directed photo-cross-linking to probe the Ca(2+)-dependent proximity relationships between residue 117 next to the C-terminus of the inhibitory region in rabbit skeletal troponin-I (TnI) and residues in troponin-C (TnC) and in actin. A mutant TnI that contains a single cysteine at position 117 (I117) was constructed, and the distance between TnI residue 117 and TnC residue 98 was measured with the following results: for both the binary TnC-TnI complex and the ternary troponin complex, this distance was 30 and 41 A in the presence and absence of Ca(2+), respectively. The distance between TnI residue 117 and Cys374 of actin was 48 and 41 A in the presence and absence of Ca(2+), respectively. Six additional distances from this TnI residue to cysteines in TnC mutants were measured and used to localize this residue with respect to the crystal structure of TnC. The results show that in the presence of Ca(2+) it is localized near the B and C helices of TnC's N-terminal domain. In the absence of Ca(2+) this residue moves away from this location by approximately 8 A. Photo-cross-linking experiments show that I117 labeled with 4-maleimidobenzophenone photo-cross-linked to TnC but not to actin in both the presence and absence of Ca(2+). Taken together these results provide independent experimental support for the proposal (Y. Luo, J. L. Wu, B. Li, K. Langsetmo, J. Gergely, and T. Tao, 2000, J. Mol. Biol. 296:899-910) that upon Ca(2+) removal the region comprising TnI residues 114-125 triggers the movements of residues 89-113 and 130-150 toward actin, but does not itself interact with actin.