Interaction of the Second Binding Region of Troponin I with the Regulatory Domain of Skeletal Muscle Troponin C as Determined by NMR Spectroscopy

Two dimensional1H,15N-heteronuclear single quantum correlation NMR was used to monitor the resonance frequency changes of the backbone amide groups belonging to the 15N-labeled regulatory domain of calcium saturated troponin C (N-TnC) upon addition of synthetic skeletal N-acetyl-troponin I 115–131-amide peptide (TnI115–131). Utilizing the change in amide chemical shifts, the dissociation constant for 1:1 binding of TnI115–131 to N-TnC in low salt and 100 mm KCl samples was determined to be 28 ± 4 and 24 ± 4 μm, respectively. The off rate of TnI115–131 was determined to be 300 s−1 from observed N-TnC backbone amide1H,15N-heteronuclear single quantum correlation cross-peak line widths, which is on the order of the calcium off rates (Li, M. X., Gagné, S. M., Tsuda, S., Kay, C. M., Smillie, L. B., and Sykes, B. D. (1995) Biochemistry34, 8330–8340), and agrees with kinetic expectations for biological regulation of muscle contraction. The TnI115–131 binding site on N-TnC was determined by mapping of chemical shift changes onto the N-TnC NMR structure and was demonstrated to be in the “hydrophobic pocket” (Gagné, S. M., Tsuda, S., Li, M. X., Smillie, L. B., and Sykes, B. D. (1995) Nat. Struct. Biol. 2, 784–789). Two dimensional1H,15N-heteronuclear single quantum correlation NMR was used to monitor the resonance frequency changes of the backbone amide groups belonging to the 15N-labeled regulatory domain of calcium saturated troponin C (N-TnC) upon addition of synthetic skeletal N-acetyl-troponin I 115–131-amide peptide (TnI115–131). Utilizing the change in amide chemical shifts, the dissociation constant for 1:1 binding of TnI115–131 to N-TnC in low salt and 100 mm KCl samples was determined to be 28 ± 4 and 24 ± 4 μm, respectively. The off rate of TnI115–131 was determined to be 300 s−1 from observed N-TnC backbone amide1H,15N-heteronuclear single quantum correlation cross-peak line widths, which is on the order of the calcium off rates (Li, M. X., Gagné, S. M., Tsuda, S., Kay, C. M., Smillie, L. B., and Sykes, B. D. (1995) Biochemistry34, 8330–8340), and agrees with kinetic expectations for biological regulation of muscle contraction. The TnI115–131 binding site on N-TnC was determined by mapping of chemical shift changes onto the N-TnC NMR structure and was demonstrated to be in the “hydrophobic pocket” (Gagné, S. M., Tsuda, S., Li, M. X., Smillie, L. B., and Sykes, B. D. (1995) Nat. Struct. Biol. 2, 784–789). One of the first intracellular steps required for skeletal muscle contraction is release of Ca2+ ions in the muscle cell, leading to a protein-protein interaction cascade and sliding of the thin and thick filaments past one another in the contractile or power stroke (for reviews see Refs. 1Tobacman L.S. Annu. Rev. Physiol. 1996; 58: 447-481Crossref PubMed Scopus (460) Google Scholar, 2Leavis P.C. Gergely J. CRC Crit. Rev. Biochem. 1984; 16: 235-305Crossref PubMed Scopus (326) Google Scholar, 3Farah C.S. Reinach F.C. FASEB J. 1995; 9(9): 755-767Crossref Scopus (475) Google Scholar). Cardiac and skeletal muscle cells have similar cascades, although the individual proteins involved and the molecular mechanism of regulation differ. The target for calcium is the troponin complex consisting of troponin C (TnC), 1The abbreviations used are: TnC, troponin C; TnI, troponin I; N-TnC, N-terminal regulatory domain of chicken troponin C residues 1–90; TnIp, TnI region 104–115; TnI115–131, synthetic rabbit skeletalN α-acetyl-troponin I 115–131-amide peptide; high salt, NMR sample containing 100 KCl; low salt, NMR sample containing no added KCl; HSQC,1H,15N-heteronuclear single quantum correlation spectroscopy. troponin I (TnI), and troponin T. TnC is the calcium binding component and the best characterized member of the troponin complex. TnI inhibits the ATPase activity of myosin, whereas troponin T is thought to anchor the complex to actin/tropomyosin. Calcium binding alters the interaction among the components of the troponin complex and with other proteins in the thin filament (4Heeley D.H. Golosinska K. Smillie L.B. J. Biol. Chem. 1987; 262: 9971-9978Abstract Full Text PDF PubMed Google Scholar). Calcium-saturated TnC binds to TnI relieving the inhibition of muscle contraction (Refs. 5Syska H. Wilkinson J.M. Grand R.J.A. Perry S.V. Biochem. J. 1976; 153: 375-387Crossref PubMed Scopus (192) Google Scholar and 6Head J.F. Perry S.V. Biochem. J. 1974; 137: 145-154Crossref PubMed Scopus (164) Google Scholar and references therein). The crystal structures of TnC revealed a dumbbell shaped molecule with two distinct domains joined by a helical linker (7Herzberg O. James M.N.G. J. Mol. Biol. 1988; 203: 761-779Crossref PubMed Scopus (291) Google Scholar, 8Satyshur K.A. Rao S.T. Pyzalska D. Drendal W. Greaser M. Sundaralingam M. J. Biol. Chem. 1988; 263: 1628-1647Abstract Full Text PDF PubMed Google Scholar). TnC contains four EF-hand calcium binding motifs, two in each of the N- and C-terminal domains (9Leavis P.C. Rosenfeld S.S. Gergely J. Grabarek Z. Drabikowski W. J. Biol. Chem. 1978; 253: 5452-5459Abstract Full Text PDF PubMed Google Scholar). The N-terminal or regulatory domain of TnC (N-TnC) was devoid of calcium under the crystallization conditions and showed a “closed” structure. This domain was postulated to open upon calcium binding in the Herzberg-Moult-James model for calcium-saturated conformation of TnC exposing nonpolar residues and creating a hydrophobic pocket (10Herzberg O. Moult J. James M.N.G. J. Biol. Chem. 1986; 261: 2638-2644Abstract Full Text PDF PubMed Google Scholar). The opening of the regulatory domain in response to calcium binding was demonstrated by the NMR solution structures of the calcium-saturated N-terminal domain and whole TnC molecules (11Gagné S.M. Tsuda S. Li M.X. Smillie L.B. Sykes B.D. Nat. Struct. Biol. 1995; 2: 784-789Crossref PubMed Scopus (251) Google Scholar, 12Slupsky C.M. Sykes B.D. Biochemistry. 1995; 34: 15953-15964Crossref PubMed Scopus (185) Google Scholar). Comparison of these structures demonstrated that isolation of the N-terminal domain does not significantly alter the effects of Ca2+ binding. Interestingly, calcium-saturated cardiac N-TnC remains in the closed conformation (13Sia S.K. Li M.X. Spyracopoulos L. Gagné S.M. Liu W. Putkey J.A. Sykes B.D. J. Biol. Chem. 1997; 272 (12221): 18216Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Although there have been several studies investigating the interaction of TnC and TnI, no high resolution three-dimensional structures of TnI or the TnC·TnI complex are available (3Farah C.S. Reinach F.C. FASEB J. 1995; 9(9): 755-767Crossref Scopus (475) Google Scholar, 14Olah G.A. Rokop S.E. Wang C.-L.A. Blechner S.L. Trewhella J. Biochemistry. 1994; 33: 8233-8239Crossref PubMed Scopus (82) Google Scholar, 15Olah G.A. Trewhella J. Biochemistry. 1994; 33: 12800-12806Crossref PubMed Scopus (108) Google Scholar, 16Campbell A.P. Sykes B.D. J. Mol. Biol. 1991; 222: 405-421Crossref PubMed Scopus (73) Google Scholar, 17Ngai S.-M. Sönnichsen F.D. Hodges R.S. J. Biol. Chem. 1994; 269: 2165-2172Abstract Full Text PDF PubMed Google Scholar, 18Tao T. Scheiner C.J. Lamkin M. Biochemistry. 1986; 25: 7633-7639Crossref PubMed Scopus (32) Google Scholar, 19Tripet B.P. Van Eyk J.E. Hodges R.S. J. Mol. Biol. 1997; 271: 728-750Crossref PubMed Scopus (184) Google Scholar, 20Pearlstone J.R. Smillie L.B. Biophys. J. 1995; 68 (abstr.): 166Google Scholar, 21Pearlstone J.R. Smillie L.B. Biophys. J. 1997; 72 (abstr.): 331Google Scholar, 22Kobayashi T. Grabarek Z. Gergely J. Collins J.H. Biochemistry. 1995; 34: 10946-10952Crossref PubMed Scopus (21) Google Scholar, 23Pearlstone J.R. Smillie L.B. Biochemistry. 1995; 34: 6932-6940Crossref PubMed Scopus (42) Google Scholar, 24Leszyk J. Grabarek Z. Gergely J. Collins J.H. Biochemistry. 1990; 29: 299-304Crossref PubMed Scopus (65) Google Scholar, 25Slupsky C.M. Shaw G.S. Campbell A.P. Sykes B.D. Protein Sci. 1992; 1: 1595-1603Crossref PubMed Scopus (16) Google Scholar, 26Talbot J.A. Hodges R.S. J. Biol. Chem. 1979; 254: 3720-3723Abstract Full Text PDF PubMed Google Scholar). We know that TnI binds TnC and actin and inhibits the actomyosin ATPase in the absence of calcium. However, it was not known exactly which residues of TnI bind to TnC nor where this complex occurs on TnC. Mutation studies (23Pearlstone J.R. Smillie L.B. Biochemistry. 1995; 34: 6932-6940Crossref PubMed Scopus (42) Google Scholar), cross-linking experiments (17Ngai S.-M. Sönnichsen F.D. Hodges R.S. J. Biol. Chem. 1994; 269: 2165-2172Abstract Full Text PDF PubMed Google Scholar, 18Tao T. Scheiner C.J. Lamkin M. Biochemistry. 1986; 25: 7633-7639Crossref PubMed Scopus (32) Google Scholar, 22Kobayashi T. Grabarek Z. Gergely J. Collins J.H. Biochemistry. 1995; 34: 10946-10952Crossref PubMed Scopus (21) Google Scholar, 24Leszyk J. Grabarek Z. Gergely J. Collins J.H. Biochemistry. 1990; 29: 299-304Crossref PubMed Scopus (65) Google Scholar, 27Jha P.K. Mao C. Sarkar S. Biochemistry. 1996; 35: 11026-11035Crossref PubMed Scopus (26) Google Scholar), and low angle x-ray diffraction structures (15Olah G.A. Trewhella J. Biochemistry. 1994; 33: 12800-12806Crossref PubMed Scopus (108) Google Scholar) have suggested that the exposed TnC hydrophobic pockets are the site of TnC·TnI interaction. Interestingly the TnC·TnI complex formation was insensitive to calcium unless tropomyosin and other members of the troponin complex were present, although calcium was found to stabilize the isolated TnC·TnI complex (6Head J.F. Perry S.V. Biochem. J. 1974; 137: 145-154Crossref PubMed Scopus (164) Google Scholar, 28Van Eyk J.E. Hodges R.S. J. Biol. Chem. 1988; 263: 1726-1732Abstract Full Text PDF PubMed Google Scholar). Farah et al. used deletion mutants to determine that TnC and TnI bind in an anti-parallel fashion (29Farah C.S. Miyamoto C.A. Ramos C.H.I. da Silva A.C.R. Quaggio R.B. Fujimori K. Smillie L.B. Reinach F.C. J. Biol. Chem. 1994; 269: 5230-5240Abstract Full Text PDF PubMed Google Scholar). In regards to TnI, Syska et al. showed that TnI bound actin, and they were the first to show that only a portion of TnI (specifically region 96–117) was needed for full inhibition of ATPase activity (5Syska H. Wilkinson J.M. Grand R.J.A. Perry S.V. Biochem. J. 1976; 153: 375-387Crossref PubMed Scopus (192) Google Scholar). Subsequently, synthetic TnI peptides provided an attractive alternative to the use of highly insoluble whole TnI in studies of muscle protein interactions. Talbot and Hodges showed that the synthetic TnI peptide corresponding to region 96–116 behaved identically to the cyanogen bromide-cleaved fragment (26Talbot J.A. Hodges R.S. J. Biol. Chem. 1979; 254: 3720-3723Abstract Full Text PDF PubMed Google Scholar). Talbot and Hodges also identified the TnIp peptide (TnI residues 104–115), which was the minimum length inhibitory peptide of TnI still regulated by TnC, and able to inhibit ATPase activity (30Talbot J.A. Hodges R.S. J. Biol. Chem. 1981; 256: 2798-2802Abstract Full Text PDF PubMed Google Scholar). Campbell et al. solved the structures of the synthetic TnIp peptide while bound to intact skeletal and cardiac TnC using the transferred nuclear Overhauser effect technique (16Campbell A.P. Sykes B.D. J. Mol. Biol. 1991; 222: 405-421Crossref PubMed Scopus (73) Google Scholar, 31Campbell A.P. Van Eyk J.E. Hodges R.S. Sykes B.D. Biochim. Biophys. Acta. 1992; 1160: 35-54Crossref PubMed Scopus (30) Google Scholar). TnIp had an amphiphilic α-helical structure distorted around the central proline residues in both cases. Ngai et al. determined that the TnIp fragment cross-linked to the C-terminal domain of TnC and modeled the NMR-derived TnIp structure into the TnC crystal structure (17Ngai S.-M. Sönnichsen F.D. Hodges R.S. J. Biol. Chem. 1994; 269: 2165-2172Abstract Full Text PDF PubMed Google Scholar). Recently Tripet et al. have mapped a second TnC binding site on TnI and postulated that the region corresponding to residues 115–131 (TnI115–131) interacts with the N-terminal domain of TnC in the Ca2+-regulated hydrophobic pocket (19Tripet B.P. Van Eyk J.E. Hodges R.S. J. Mol. Biol. 1997; 271: 728-750Crossref PubMed Scopus (184) Google Scholar). In this paper the interaction of calcium-saturated N-TnC with TnI115–131 was explored with multinuclear, multi-dimensional NMR spectroscopy. We monitored15N-labeled N-TnC upon the addition of TnI115–131 to determine the stoichiometry of binding, dissociation constant of the complex, and location of chemical shifts induced in the N-TnC molecule. We have mapped those chemical shift changes onto the structure of Ca2+-bound N-TnC. Further we monitored the change in cross-peak line width to determine the reaction rate constants. These results provide direct evidence for TnI binding in the hydrophobic pocket of the regulatory domain and have implications for the kinetic competence of the complex with respect to muscle contraction. The cloning, expression, and purification of 50% deuterated, uniformly 15N-labeled N-TnC 2R. T. McKay, J. R. Pearlstone, S. M. Gagné, D. C. Corson, L. B. Smillie, and B. D. Sykes, manuscript in preparation. was done following the protocols described in Gagné et al. for nondeuterated N-TnC (32Gagné S.M. Tsuda S. Li M.X. Chandra M. Smillie L.B. Sykes B.D. Protein Sci. 1994; 3: 1961-1974Crossref PubMed Scopus (176) Google Scholar). The synthetic N α-acetyl-TnI (115–131)-amide rabbit skeletal peptide was prepared as described previously (19Tripet B.P. Van Eyk J.E. Hodges R.S. J. Mol. Biol. 1997; 271: 728-750Crossref PubMed Scopus (184) Google Scholar) and lyophilized repeatedly to remove residual organic solvents. The sequence was confirmed by amino acid analysis, and the mass was verified by electrospray mass spectrometry. Two NMR samples (500 μl) of U-15N; 50% 2H-N-TnC were prepared for titration with the TnI115–131 peptide differing only in the concentration of added KCl. The first sample (designated high salt) 100 mm whereas the second had no added KCl The N-TnC was in a containing and mm with as an The only other were the of or to the of the samples to The of N-TnC and TnI115–131 were determined by amino acid in The concentration of the high salt sample was ± mm N-TnC, whereas the low salt sample was ± mm N-TnC. The of for the high and low salt samples were and respectively. experiments were on a The of the high salt sample were with a width of for complex and complex with whereas the low salt were using a width of for and for complex with 28 was experiments were using the S. J. J. 1995; PubMed Scopus Google Scholar) and using the R. J. 1991; Scopus Google Scholar). were to complex were by to complex and to complex were using a added in to N-TnC samples to TnI115–131 to N-TnC of and for the high and low salt respectively. R. J. 1991; Scopus Google Scholar, T. J. Chem. 1992; Scopus Google Scholar) were TnI115–131 to N-TnC of and for the high salt sample and of and for the low salt TnI115–131 was in the as N-TnC, and the of the NMR sample was each N-TnC backbone amide were each of the and the chemical shift change was determined as in where N and are the and chemical shift changes for a backbone amide The chemical shift change for the N-TnC molecule is in The is residues that were The dissociation constant for the was determined from the by an C.M. Shaw G.S. Campbell A.P. Sykes B.D. Protein Sci. 1992; 1: 1595-1603Crossref PubMed Scopus (16) Google Scholar, G.S. Hodges R.S. Sykes B.D. J. Chem. 1991; Scopus Google Scholar) using the In is the N-TnC is the TnI115–131 and is the complex. The of the results also the stoichiometry of the Two backbone amide were from the low salt titration were shifts were to the NMR and the demonstrated line changes and were not by in the 50% N-TnC was it showed one of the of whereas was it showed a change of The S. The Scholar) was used to the line for the protein and and bound chemical shifts, and and bound line for each of using the for the effect of site on NMR on NMR Scholar). these the off rate for the TnI115–131 peptide in the complex was The interaction of TnI115–131 with calcium-saturated N-TnC was using NMR spectroscopy. The TnI115–131 was whereas the N-TnC was for of the N-TnC component of the complex from TnI N-TnC was to the line width of the observed show backbone amide and that be used to the titration in cross-peak to changes in the of the and this of each amino of an region of the the titration are in and for the high salt and low salt respectively. N-TnC amide are not by the addition of TnI115–131 whereas other are significantly in the or both and amide shift in both a first line these are in the NMR a single resonance is observed for each amide is the of the and bound chemical shifts 4 where is the observed chemical shift of the backbone amide are the and bound of N-TnC, and and are the chemical shifts for the and bound respectively. of N-TnC backbone amide and were the of the high salt The NMR chemical shift change was for each amide for each in the and individual were the of N-TnC backbone amide and to be the titration were from Gagné et al. (32Gagné S.M. Tsuda S. Li M.X. Chandra M. Smillie L.B. Sykes B.D. Protein Sci. 1994; 3: 1961-1974Crossref PubMed Scopus (176) Google Scholar). and were not the titration to amide or resonance a backbone amide and does not show a cross-peak in the in a region in the high salt sample and was not in the and were not in the N-TnC NMR was to these two residues in the complex and to where the cross-peak from in the N-TnC the a of 24 ± 4 was determined for a 1:1 binding complex The low salt N-TnC titration was as described a of were In the low salt sample was isolated to the titration and not shift upon TnI115–131 of ± for a 1:1 complex was the titration not a TnI115–131 to N-TnC of the was with the shift constant of the high salt of 28 ± 4 for a 1:1 complex was This was both samples had to binding of the peptide and the backbone amide groups were The only is which was for the low salt not alter the binding it does not shift the The of the titration is a of the chemical shift changes the of the of individual NMR the titration of (for see and the of N-TnC residues and are in and to the effect of the addition of where the was the line only the the other which a of the titration and the is the effect of chemical on line width in the a on the chemical shift the and bound where is the observed line and are the of and bound N-TnC, and is the as The line of the and bound N-TnC and the and resonance were determined from the NMR TnI115–131 and the TnI115–131 to N-TnC The line width of the N-TnC of 24 to for the bound complex. This results N-TnC from a to a complex C.M. C.M. Reinach F.C. Smillie L.B. Sykes B.D. Biochemistry. 1995; 34: PubMed Scopus Google Scholar). were using the for and The results of using off rates of and s−1 were in for and respectively. off of s−1 the to the and for both The binding site of TnI115–131 on N-TnC was mapped by following backbone amide chemical shift changes the The was for each TnI115–131 under both salt conditions and is in 4 The was and for the high and low salt respectively. of and was for the of the high salt and low salt respectively. of or the to one the were to be and In the high salt sample residues and showed whereas residues and significantly in the low salt N-TnC that show a change in chemical shift upon addition of TnI115–131 are in The structure used is that of N-TnC (11Gagné S.M. Tsuda S. Li M.X. Smillie L.B. Sykes B.D. Nat. Struct. Biol. 1995; 2: 784-789Crossref PubMed Scopus (251) Google Scholar). N-TnC residues that have a are in and line the hydrophobic We have used NMR to show that the peptide TnI115–131 binds N-TnC with 1:1 stoichiometry under both high salt and low salt to the dissociation and off rate for the complex, and to that binding occurs in the hydrophobic pocket of N-TnC (10Herzberg O. Moult J. James M.N.G. J. Biol. Chem. 1986; 261: 2638-2644Abstract Full Text PDF PubMed Google Scholar, S.M. Tsuda S. Li M.X. Smillie L.B. Sykes B.D. Nat. Struct. Biol. 1995; 2: 784-789Crossref PubMed Scopus (251) Google Scholar). The high resolution a for each individual in the This one to protein complex formation in solution the of a chemical or sequence in the which are used to a single Interestingly, the high and low salt samples showed similar had that salt a in (19Tripet B.P. Van Eyk J.E. Hodges R.S. J. Mol. Biol. 1997; 271: 728-750Crossref PubMed Scopus (184) Google Scholar). Van Eyk et al. showed the of residues in TnC studies Eyk J.E. C.M. Hodges R.S. Biochemistry. 1991; PubMed Scopus Google Scholar), and high salt was to and complex (19Tripet B.P. Van Eyk J.E. Hodges R.S. J. Mol. Biol. 1997; 271: 728-750Crossref PubMed Scopus (184) Google Scholar, J.R. Sykes B.D. Smillie L.B. Biochemistry. 1997; PubMed Scopus Google Scholar). However, this was not for the in results be that high salt in the of N-TnC was a in et al. had previously that the in the hydrophobic pocket of N-TnC C.M. C.M. Reinach F.C. Smillie L.B. Sykes B.D. Biochemistry. 1995; 34: PubMed Scopus Google Scholar). this we binding with an not The first two with dissociation of 100 and mm that this be to complex dissociation constants. of there was a in the observed an of to a of et al. showed of mm for intact TnC, and line an N-TnC dissociation for N-TnC of was determined using the observed line width for both the complex and N-TnC molecule and that the N-TnC have a line width that of the the for the complex are to the We used the chemical shift changes of the backbone amide to the of the TnI peptide binding. This technique been used to and binding for 1996; PubMed Scopus Google Scholar). that one from the chemical shift change were in 4 and and on the N-TnC structure in These residues are hydrophobic and line the N-TnC hydrophobic These results with studies that demonstrated that residues as and were for regulation of muscle contraction J.R. C.M. Sykes B.D. Smillie L.B. Biochemistry. 1992; PubMed Scopus Google Scholar, J.R. T. Chandra M. K. C.M. O. Moult J. Reinach F.C. Smillie L.B. Biochemistry. 1992; PubMed Scopus Google Scholar). In addition results with cross-linking studies that the of the TnI residues to whole TnC T. Grabarek Z. Gergely J. Collins J.H. Biochemistry. 1995; 34: 10946-10952Crossref PubMed Scopus (21) Google Scholar, 24Leszyk J. Grabarek Z. Gergely J. Collins J.H. Biochemistry. 1990; 29: 299-304Crossref PubMed Scopus (65) Google Scholar, T. P.C. Collins J.H. Biochim. Biophys. Acta. 1996; PubMed Scopus Google Scholar). TnIp was to to TnC residues and or The interaction of TnIp with the C-terminal domain of TnC (17Ngai S.-M. Sönnichsen F.D. Hodges R.S. J. Biol. Chem. 1994; 269: 2165-2172Abstract Full Text PDF PubMed Google Scholar) and the interaction of TnI115–131 with N-TnC (19Tripet B.P. Van Eyk J.E. Hodges R.S. J. Mol. Biol. 1997; 271: 728-750Crossref PubMed Scopus (184) Google Scholar) the anti-parallel model of Farah et al. (3Farah C.S. Reinach F.C. FASEB J. 1995; 9(9): 755-767Crossref Scopus (475) Google Scholar). The of an the two TnI binding a bound TnC structure by low resolution experiments G.A. Rokop S.E. Wang C.-L.A. Blechner S.L. Trewhella J. Biochemistry. 1994; 33: 8233-8239Crossref PubMed Scopus (82) Google Scholar, 15Olah G.A. Trewhella J. Biochemistry. 1994; 33: 12800-12806Crossref PubMed Scopus (108) Google Scholar). bound TnC structure also with the M. 1992; 256: PubMed Scopus Google Scholar, C. Sci. S. 1995; PubMed Scopus Google Scholar) and regulatory and crystal structures K. R. 261: PubMed Scopus Google Scholar, M. M. 261: PubMed Scopus Google Scholar). We determined from line of The of cross-peak and show that one off rates differing by a of However, the is to ± 100 was to off and of the line was the of the as are as resolution and also the the of the which to in the The off is to be for muscle contraction. the contraction frequency of muscle be as as J.R. The Scholar). Interestingly, the off for complex is of the order as the off for calcium Calcium and Scholar and references that the steps of calcium binding, opening of the hydrophobic and TnI binding on similar This a for kinetic of other troponin interactions. These be with kinetic on TnC TnI protein and is only the of the molecular of muscle contraction. We the Protein of the of for use of the and and for of the L. B. of is for use of the amino acid to Li, and Gagné for of the