Defining the region of troponin-I that binds to troponin-C

The kinetics and energetics of the binding of three troponin-I peptides, co rresponding to regions 96-131 (TnI(96-131)), 96-139 (TnI(96-139)), and 96-1 48 (TnI(96-148)), to skeletal chicken troponin-C were investigated using mu ltinuclear, multidimensional NMR spectroscopy. The kinetic off-rate and dis sociation constants for TnI(96-131) (400 s(-1), 32 mu M), TnI(96-139) (65 s (-1), <1 mu M), and TnI(96-148) (45 s(-1), <1 mu M) binding to TnC were det ermined from simulation and analysis of the behavior of H-1,N-15-heteronucl ear single quantum correlation NMR spectra taken during titrations of TnC w ith these peptides, Two-dimensional N-15-edited TOCSY and NOESY spectroscop y were used to identify 11 C-terminal residues from the N-15-labeled TnI(96 -148) that were unperturbed by TnC binding. TnI(96-139) labeled with C-13 a t four positions (Leu(102), Leu(111), Met(121), and Met(134)) was complexed with TnC and revealed single bound species for Leu(102) and Leu(111) but m ultiple bound species for Met(121) and Met(134). These results indicate tha t residues 97-136 (and 96 or 137) of TnI are involved in binding to the two domains of troponin-C under calcium saturating conditions, and that the in teraction with the regulatory domain is complex. Implications of these resu lts in the context of various models of muscle regulation are discussed.