Defining the region of troponin-I that binds to troponin-C.
The kinetics and energetics of the binding of three troponin-I peptides, corresponding to regions 96-131 (TnI96-131), 96-139 (TnI96-139), and 96-148 (TnI96-148), to skeletal chicken troponin-C were investigated using multinuclear, multidimensional NMR spectroscopy. The kinetic off-rate and dissociation constants for TnI96-131 (400 s-1, 32 microM), TnI96-139 (65 s-1, <1 microM), and TnI96-148 (45 s-1, <1 microM) binding to TnC were determined from simulation and analysis of the behavior of 1H,15N-heteronuclear single quantum correlation NMR spectra taken during titrations of TnC with these peptides. Two-dimensional 15N-edited TOCSY and NOESY spectroscopy were used to identify 11 C-terminal residues from the 15N-labeled TnI96-148 that were unperturbed by TnC binding. TnI96-139 labeled with 13C at four positions (Leu102, Leu111, Met 121, and Met134) was complexed with TnC and revealed single bound species for Leu102 and Leu111 but multiple bound species for Met121 and Met134. These results indicate that 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 interaction with the regulatory domain is complex. Implications of these results in the context of various models of muscle regulation are discussed.
McKay
R T
RT
MRC Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Canada.
Tripet
B P
BP
Pearlstone
J R
JR
Smillie
L B
LB
Sykes
B D
BD
D000595:Amino Acid Sequence; D000818:Animals; D002247:Carbon Isotopes; D002645:Chickens; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D019209:Troponin C / Q000737:chemistry* / Q000378:metabolism; D019210:Troponin I / Q000737:chemistry* / Q000378:metabolism
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D010446:Peptide Fragments; D019209:Troponin C; D019210:Troponin I