Localization Of Cys(133) Of Rabbit Skeletal Troponin-L With Respect To Troponin-C By Resonance Energy Transfer

We have used the technique of resonance energy transfer in conjunction with distance geometry analysis to localize Cys(133) of troponin-l (Tnl) with respect to troponin-C (TnC) in the ternary troponin complex and the binary TnC.Tnl complex in the presence and absence of Ca2+. Cys(133) of Tnl was chosen because our previous work has shown that the region of Tnl containing this residue undergoes Ca2+-dependent movements between actin and TnC, and may play an important role in the regulatory function of troponin. For this purpose, a Tnl mutant with a single Cys at position 133, and TnC mutants, each with a single Cys at positions 5, 12, 21, 41, 49, 89, 98, 133, and 158, were constructed by site-directed mutagenesis. The distances between Tnl Cys(133) and each of the nine residues in TnC were then measured. Using a least-squares minimization procedure, we determined the position of Tnl Cys(133) in the coordinate system of the crystal structure of TnC. Our results show that in the presence of Ca2+, Tnl Cys(133) is located near residue 12 beneath the N-terminal lobe of TnC, and moves away by 12.6 Angstrom upon the removal of Ca2+. Tnl Cys(133) and the region of TnC that undergoes major change in conformation in response to Ca2+ are located roughly on opposite sides of TnC's central helix. This suggests that the region in Tnl that undergoes Ca2+-dependent interaction with TnC is distinct from that interacting with actin.