Parallel Measurement of Ca2+ Binding and Fluorescence Emission upon Ca2+ Titration of Recombinant Skeletal Muscle Troponin C

Calcium binding to chicken recombinant skeletal muscle TnC (TnC) and its mutants containing tryptophan (F29W), 5-hydroxytryptophan (F29HW), or 7-azatryptophan (F29ZW) at position 29 was measured by flow dialysis and by fluorescence. Comparative analysis of the results allowed us to determine the influence of each amino acid on the calcium binding properties of the N-terminal regulatory domain of the protein. Compared with TnC, the Ca2+ affinity of N-terminal sites was: 1) increased 6-fold in F29W, 2) increased 3-fold in F29ZW, and 3) decreased slightly in F29HW. The Ca2+titration of F29ZW monitored by fluorescence displayed a bimodal curve related to sequential Ca2+ binding to the two N-terminal Ca2+ binding sites. Single and double mutants of TnC, F29W, F29HW, and F29ZW were constructed by replacing aspartate by alanine at position 30 (site I) or 66 (site II) or both. Ca2+ binding data showed that the Asp → Ala mutation at position 30 impairs calcium binding to site I only, whereas the Asp → Ala mutation at position 66 impairs calcium binding to both sites I and II. Furthermore, the Asp → Ala mutation at position 30 eliminates the differences in Ca2+ affinity observed for replacement of Phe at position 29 by Trp, 5-hydroxytryptophan, or 7-azatryptophan. We conclude that position 29 influences the affinity of site I and that Ca2+ binding to site I is dependent on the previous binding of metal to site II. Calcium binding to chicken recombinant skeletal muscle TnC (TnC) and its mutants containing tryptophan (F29W), 5-hydroxytryptophan (F29HW), or 7-azatryptophan (F29ZW) at position 29 was measured by flow dialysis and by fluorescence. Comparative analysis of the results allowed us to determine the influence of each amino acid on the calcium binding properties of the N-terminal regulatory domain of the protein. Compared with TnC, the Ca2+ affinity of N-terminal sites was: 1) increased 6-fold in F29W, 2) increased 3-fold in F29ZW, and 3) decreased slightly in F29HW. The Ca2+titration of F29ZW monitored by fluorescence displayed a bimodal curve related to sequential Ca2+ binding to the two N-terminal Ca2+ binding sites. Single and double mutants of TnC, F29W, F29HW, and F29ZW were constructed by replacing aspartate by alanine at position 30 (site I) or 66 (site II) or both. Ca2+ binding data showed that the Asp → Ala mutation at position 30 impairs calcium binding to site I only, whereas the Asp → Ala mutation at position 66 impairs calcium binding to both sites I and II. Furthermore, the Asp → Ala mutation at position 30 eliminates the differences in Ca2+ affinity observed for replacement of Phe at position 29 by Trp, 5-hydroxytryptophan, or 7-azatryptophan. We conclude that position 29 influences the affinity of site I and that Ca2+ binding to site I is dependent on the previous binding of metal to site II. recombinant chicken troponin C troponin I dithiothreitol 4-morpholinepropanesulfonic acid 5-hydroxytryptophan 7-azatryptophan Phe-29, used to indicate broad spectrum of Phe-29 mutations F29HW, and F29ZW, recombinant TnCs with phenylalanine at position 29 replaced by tryptophan, 5-hydroxytryptophan, or 7-azatryptophan, respectively HW, or ZW), any single mutant with phenylalanine at position 29 replaced by tryptophan, 5-hydroxytryptophan, or 7-azatryptophan any protein with phenylalanine at position 29 and at least one replacement of aspartate by alanine any double or triple mutant with tryptophan or an analog at position 29 and at least one replacement of aspartate by alanine N-terminal domain sites I and II of TnC or its mutants C-terminal domain sites III and IV of TnC or its mutants value of -log[Ca2+]free corresponding to 50% of the maximal associated transition One of the first events in skeletal muscle contraction is calcium binding to the N-terminal sites of troponin C (TnC).1 Structural modifications resulting from this association are thought to be transmitted to other components of the thin filament, which ultimately allows the effective association of myosin to actin and force generation (for review, see Refs. 1Perry S.V. Cole H.A. Head J.F. Wilson F.J. Cold Spring Harbor Symp. Quant. Biol. 1972; 37: 251-262Google Scholar, 2Zot A.S. Potter J.D. Annu. Rev. Biophys. Biophys. Chem. 1987; 16: 535-559Google Scholar, 3Farah C.S. Reinach F.C. FASEB J. 1995; 9: 755-767Google Scholar, 4Tobacman L.S. Annu. Rev. Physiol. 1996; 58: 447-481Google Scholar). TnC has a dumbbell-shaped structure with the globular N-terminal and C-terminal domains separated by a nine-turn α-helix (5Herzberg O. James M.N. Nature. 1985; 313: 653-659Google Scholar, 6Herzberg O. James M.N. J. Mol. Biol. 1988; 203: 761-779Google Scholar). Calcium binds with high affinity (Kd ≈ 10−7m) to the two EF-hand Ca2+/Mg2+ binding sites located at the TnC C-terminal domain (sites III and IV, the C-terminal sites) and with low affinity (Kd ≈10−5m) to the two EF-hand Ca2+-specific binding sites located in the TnC N-terminal domain (sites I and II, the N-terminal sites) (7Potter J.D. Gergely J. J. Biol. Chem. 1975; 250: 4628-4633Google Scholar). Because of the affinity and kinetic properties of the C-terminal sites they are believed to be always occupied by Ca2+ or Mg2+ in vivo and are involved in the stable TnC binding to TnI (8Farah C.S. Miyamoto C.A. Ramos C.H. da Silva A.C. Quaggio R.B. Fujimori K. Smillie L.B. Reinach F.C. J. Biol. Chem. 1994; 269: 5230-5240Google Scholar). Ca2+ binding to the two TnC N-terminal sites results in a greatly increased affinity for the TnI C-terminal and inhibitory regions, resulting in their dissociation from actin and disinhibition of the actomyosin interaction. The affinity and cooperativity of calcium binding to TnC have been studied extensively. These parameters are determined through direct measurement of free calcium concentration in association systems (direct techniques) (7Potter J.D. Gergely J. J. Biol. Chem. 1975; 250: 4628-4633Google Scholar, 9Iida S. J. Biochem. (Tokyo). 1988; 103: 482-486Google Scholar, 10Fujimori K. Sorenson M. Herzberg O. Moult J. Reinach F.C. Nature. 1990; 345: 182-184Google Scholar, 11da Silva A.C. de Araujo A.H. Herzberg O. Moult J. Sorenson M. Reinach F.C. Eur. J. Biochem. 1993; 213: 599-604Google Scholar) or through spectroscopically monitored titrations (indirect techniques) (12–31). These studies largely agree on the value of the binding constants but disagree with regard to the cooperativity parameter. Cooperativity between N-terminal sites has been detected only by indirect techniques, raising the possibility that potentially damaging conditions, the extensive time needed to prepare samples, or the intrinsically high errors involved in direct techniques disturb or conceal the N-terminal site cooperativity. In one of the studies using indirect techniques (25Pearlstone J.R. Borgford T. Chandra M. Oikawa K. Kay C.M. Herzberg O. Moult J. Herklotz A. Reinach F.C. Smillie L.B. Biochemistry. 1992; Scholar) constructed a mutant of recombinant skeletal chicken TnC, in which Phe-29 was replaced by position is to Ca2+ binding site I and the was to only in both and of the N-terminal domain Ca2+ binding to N-terminal sites is a 3-fold in the of fluorescence at mutant binds a of a high of cooperativity between sites I and II. of cooperativity for N-terminal sites were detected by of chicken recombinant TnC K. J.R. Borgford T. Oikawa K. Kay C.M. Smillie L.B. J. Biol. Chem. Scholar, J.R. Kay C.M. Smillie L.B. Biochemistry. 1992; Scholar). that the properties of the N-terminal domain are from of TnC M. da Silva Sorenson J.R. Borgford T. Kay C.M. Smillie L.B. J. Biol. Chem. 1994; 269: Scholar, S. Kay C.M. Smillie L.B. Biochemistry. 1995; Scholar, A. Smillie J. Silva J. A. J. Biophys. and studies using the N-terminal domain of TnC that sites I and II are occupied a affinity with cooperativity between S. Kay C.M. Smillie L.B. Biochemistry. 1995; Scholar). this and to the influence of position 29 of chicken TnC on the properties of N-terminal the amino acid 5-hydroxytryptophan and 7-azatryptophan position 29 of TnC S. O. J. Scholar, C.A. S. A. 1992; Scholar, 1992; Scholar, Biophys. Chem. 1993; Scholar, Scholar, Biochemistry. 37: Scholar). Ca2+ binding by mutants and F29ZW, and TnC was using a direct flow dialysis calcium binding and by fluorescence. We have constructed and mutants with Asp → Ala at position of sites I or II or both sites to the of calcium binding properties on each These mutants were to flow dialysis fluorescence data indicate 1) tryptophan at position 29 the N-terminal site calcium affinity 6-fold with 2) at position 29 the N-terminal site calcium affinity to slightly of 3) at position 29 the N-terminal site to an between the and TnC N-terminal site the flow dialysis data with Asp → Ala mutants indicate that Asp at the first position of site II is replaced by site I calcium binding to site II is by Asp → Ala mutation at position of site The fluorescence data indicate of cooperativity between N-terminal sites and a N-terminal site cooperativity value for F29HW. at position 29 the binding of calcium to the affinity N-terminal site (site II) by fluorescence and to the the of affinity N-terminal site (site I) by the fluorescence in a bimodal fluorescence calcium binding to the regulatory N-terminal sites. In the data with TnC and its mutants a for Ca2+ binding to N-terminal that site I Ca2+ site II is results indicate that mutations in position 29 the properties of the regulatory sites. The and (8Farah C.S. Miyamoto C.A. Ramos C.H. da Silva A.C. Quaggio R.B. Fujimori K. Smillie L.B. Reinach F.C. J. Biol. Chem. 1994; 269: 5230-5240Google Scholar) and mutants (25Pearlstone J.R. Borgford T. Chandra M. Oikawa K. Kay C.M. Herzberg O. Moult J. Herklotz A. Reinach F.C. Smillie L.B. Biochemistry. 1992; Scholar) were used to the mutants and using the Scholar). The to the Asp → Ala mutation at position 30 on the were and The Asp → Ala mutation at position 66 was with the and The were from low and used in the These were and and to J. Mol. Biol. Scholar). were by The and of TnC and the mutant have been K. Sorenson M. Herzberg O. Moult J. Reinach F.C. Nature. 1990; 345: 182-184Google Scholar, J.R. Borgford T. Chandra M. Oikawa K. Kay C.M. Herzberg O. Moult J. Herklotz A. Reinach F.C. Smillie L.B. Biochemistry. 1992; F.C. J. Biol. Chem. 1988; Scholar). The was used to and and or TnC mutants a of J. Mol. Biol. Scholar) was constructed by this with J. Mol. Biol. Scholar) and with A.H. 1990; Scholar). The the for tryptophan from the the from of the and the from this with or the of of the the in containing and the were at for at and in and or by of at with 1992; Scholar). The protein was the of that of TnC K. Sorenson M. Herzberg O. Moult J. Reinach F.C. Nature. 1990; 345: 182-184Google of of analog was on a analysis of the and of the The of and free amino were in The F29W, F29HW, and F29ZW were by the TnC spectrum to the of the The of mutant was using the and are the between and of the of F29W, and F29HW, The F29ZW is the is with a We the two F29ZW by at or and the a was to was with that of a with a concentration and to the of the F29ZW dialysis were Silva A.C. de Araujo A.H. Herzberg O. Moult J. Sorenson M. Reinach F.C. Eur. J. Biochem. 1993; 213: 599-604Google Scholar) with The used in dialysis to Ca2+ from which 2) with 3) but with The were with each for with two We have that using dialysis in the concentration was determined Biochem. 1972; Scholar) using a TnC concentration was determined through amino acid analysis and concentration in the dialysis was and was with the used for the flow dialysis The of was used for curve The of calcium sites of each protein was from the with between and the of in the curve associated with calcium binding at The data from TnC or or were using The data from and were using The data from and were using and are the dissociation and for the C-terminal sites and the N-terminal sites. The fluorescence were in a with and for and for and for other The used was for and and for and The of the was at We used the protein used in the flow dialysis The of the protein in the fluorescence was and calcium was to its concentration the in the flow dialysis In this the in the flow dialysis to to the data in the fluorescence for F29W, F29HW, and for was the data to for and was the data to is the fluorescence the dissociation for calcium binding to the N-terminal sites the dissociation to the site for the and the the cooperativity between the two sites. for F29ZW was the data to is the fluorescence is the fluorescence associated with the of the site II, the dissociation for site is the fluorescence associated with the of the site is the dissociation for site the of the amino Trp, and and have with is in the of the corresponding recombinant TnC F29W, F29HW, and F29ZW the between the free amino acid and the corresponding protein The and of the amino and the mutants are in The fluorescence of of Ca2+ (25Pearlstone J.R. Borgford T. Chandra M. Oikawa K. Kay C.M. Herzberg O. Moult J. Herklotz A. Reinach F.C. Smillie L.B. Biochemistry. 1992; Scholar). at the fluorescence of of Ca2+ to the is The fluorescence of F29ZW a bimodal to from and from to analysis of the and of the recombinant TnC analog for and for F29ZW and Because of this the results with both analog mutants are by with We used the in of the other and F29ZW the calcium binding to TnC and to the F29W, F29HW, and F29ZW determined by flow Calcium binding indicate that and that is between and is an in the calcium binding which is related to calcium binding to was to be the Ca2+ concentration to which any dependent parameter. The TnC and are the and the the binding is the of the and F29ZW with TnC and F29HW. an in the affinity of the low affinity N-terminal sites. The C-terminal sites have the N-terminal sites and are by the of and the fluorescence of F29W, F29HW, and The data were the the flow dialysis that in of The fluorescence showed that the of the the calcium affinity of N-terminal sites with to the N-terminal sites. The F29ZW fluorescence is with the value at and a of which at In flow dialysis and fluorescence data for each mutant to the fluorescence with calcium and to determine the in the free calcium which to the fluorescence In the of and C and the of the fluorescence the are two calcium is for for The in affinity between C-terminal and N-terminal sites is between C-terminal and N-terminal sites of These fluorescence direct that calcium binding to the C-terminal sites the fluorescence of the tryptophan or at position 29 of TnC and that calcium binding to the regulatory N-terminal sites of fluorescence of F29ZW with The maximal fluorescence value at and is associated in the calcium binding curve with in the of this the be used to between the binding of the and the Ca2+ calcium binding measured by flow dialysis to TnC and its mutants and mutants and mutants and F29ZW and F29ZW mutants The data for TnC and its mutants that the Asp → Ala mutations at position 30 or 66 or the mutations the calcium binding at in a associated with binding to N-terminal sites. is in with the of the and of the TnC and binding from the and binding The and binding are and indicate that mutants only two Ca2+ The TnC and binding only at the binding curve a a of Ca2+ The TnC binding curve to at is in the calcium binding of the mutants The is that the of the the at which the for the and the corresponding mutant ≈ for and ≈ for and and ≈ for F29ZW and The corresponding and and calcium binding largely the the fluorescence calcium binding to the which only one or Ca2+ in their N-terminal the fluorescence of each mutant calcium binding to the the fluorescence is that observed in the mutants that two N-terminal Ca2+ binding sites. In the of the fluorescence slightly Ca2+ and the fluorescence was or mutants were used in the fluorescence calcium the fluorescence and flow dialysis Ca2+ binding for the and of the fluorescence of and the calcium two and is the calcium that to the single N-terminal site in (site II) is the associated with the fluorescence the between and of and the fluorescence calcium of the mutants with and the These results are in II. the calcium binding by flow dialysis for and The binding are that the of the at position 29 on to site II is in the the fluorescence calcium of the The are for and and for the calcium titrations by fluorescence of the mutants with and the and have to and the only slightly at low free calcium of tryptophan is at position the mutation has a of is whereas the of is The of both are of for and to for the F29ZW and The bimodal of the F29ZW curve is replaced by a in with ≈ constants and for F29W, F29HW, F29ZW, and its mutants measured by has an the fluorescence curve showed an The the but with cooperativity the F29ZW mutant are dissociation for sites II and the is the site II dissociation has an the fluorescence curve showed an The the but with cooperativity in a of dissociation and for TnC and its mutants measured by flow of for mutant was determined of the of the for one of the data The for mutant was determined of the of the for one of the data in a the F29ZW mutant are dissociation for sites II and the is the site II dissociation is to that the of and and are only the and are the are by the We the from the and on the to on We that the of sequential binding is the bimodal of the fluorescence curve and that the of site II and site I site II is to be to be in with sequential skeletal chicken TnC is of and (25Pearlstone J.R. Borgford T. Chandra M. Oikawa K. Kay C.M. Herzberg O. Moult J. Herklotz A. Reinach F.C. Smillie L.B. Biochemistry. 1992; Scholar) replaced Phe at position 29 with Trp, Ca2+ binding to regulatory N-terminal sites) to be using fluorescence that the properties of TnC were in F29W, the Ca2+ affinity of the N-terminal sites. In the fluorescence data a high of cooperativity of with an of Ca2+ binding to the two N-terminal sites. Chandra M. da Silva Sorenson J.R. Borgford T. Kay C.M. Smillie L.B. J. Biol. Chem. 1994; 269: Scholar) that the mutant is in its to TnI of actomyosin in a the a in calcium affinity was S. Kay C.M. Smillie L.B. Biochemistry. 1995; Scholar) this affinity determined by an increased of the at A. Smillie J. Silva J. A. J. Biophys. Scholar) showed that the of is stable at with the TnC used direct binding to that the N-terminal site calcium affinity is at least 6-fold that of TnC N-terminal sites The of a at position of this affinity is at position the N-terminal site affinity 3-fold with the calcium binding properties of TnC are to the of the amino acid at this position in the Trp, or in the influence of position 29 on TnC Ca2+ affinity is in the structure of chicken an extensive of Phe-29, and be In both the and C.M. Biochemistry. 1995; Scholar, S. Smillie L.B. Biol. 1995; Scholar, M. Smillie L.B. James M.N. J. Mol. Biol. Scholar) the Phe-29 is to and Phe-29 and the a that is calcium binding to the N-terminal TnC from the to the is that the structure of TnC be to the at position of a with or and in the is that in the thin filament, this with TnI in the of In the of calcium binding to TnC force the to the calcium affinity of the N-terminal sites in free TnC with that measured in the (7Potter J.D. Gergely J. J. Biol. Chem. 1975; 250: 4628-4633Google Scholar). (25Pearlstone J.R. Borgford T. Chandra M. Oikawa K. Kay C.M. Herzberg O. Moult J. Herklotz A. Reinach F.C. Smillie L.B. Biochemistry. 1992; Scholar) and da Silva A.C. de Araujo A.H. Herzberg O. Moult J. Sorenson M. Reinach F.C. Eur. J. Biochem. 1993; 213: 599-604Google Scholar) TnC mutants with increased N-terminal site Ca2+ affinity by one by the and from the by and These were to the between the and the calcium affinity of the N-terminal sites. The increased observed for the N-terminal sites be the of a of the of a of the of the of the at least of the of the in the The that the Ca2+ affinity of the N-terminal sites was to that of TnC constants and I) that the or of the N-terminal domain structure by is for both the and The mutant F29ZW displayed an N-terminal site Ca2+ between that of and TnC that of and that of TnC, These data indicate that the in of the at position from Phe to Trp, be a to the influence of that position on N-terminal sites Ca2+ by Biochemistry. Scholar) for a of mutants with replaced by In the of possibility is an the of 5-hydroxytryptophan and the of the of the which is for calcium to to site I M. Smillie L.B. James M.N. J. Mol. Biol. Scholar). to a the replacement of at position by a mutation to the site I calcium dissociation from to and impairs of with site I Chandra M. Smillie L.B. Biochemistry. Scholar). The fluorescence data for F29W, F29HW, and F29ZW II) were with from flow dialysis The of fluorescence for F29ZW was the of are the of the with high and Scholar). and J. Biol. Chem. 1985; Scholar) a bimodal fluorescence using skeletal TnC at which at low and was at The F29ZW titrations were at which that the bimodal of the Ca2+ titrations by fluorescence is the of in the which the of Furthermore, the of Mg2+ the of the transition a bimodal curve to the the first and of the curve be related to the calcium binding to the N-terminal sites II and The F29ZW fluorescence is by calcium binding to C-terminal sites at and Ca2+ and the Ca2+ binding the site (site II of each The influence of the at position 29 on the Ca2+ binding is Ca2+ to site that site II Ca2+ affinity is by any of the at position 29 in mutants with a site that the transition from the to in the is associated with Ca2+ binding to site by Chandra M. Smillie L.B. Biochemistry. the in affinity the TnC and Phe-29 N-terminal sites be related to the of the mutants with alanine at position 66 and or with at position 66 and 30 and only two and have calcium binding determined in the flow dialysis In mutants with a site I the of is at the TnC and We conclude that the sites I and II to the properties of the One is that the replacement of aspartate at position 66 by alanine both whereas the replacement at position 30 is to site is that previous of site II is for calcium binding to site I at calcium by Biochemistry. Scholar) and M. Smillie L.B. James M.N. J. Mol. Biol. Scholar). to this Ca2+ binding to site II the for calcium binding to site site I in two one previous to Ca2+ binding to site II and other Ca2+ binding to site II. The data indicate that site I is previous site II site I on site II is with the determined and of cooperativity of the N-terminal domain S. Kay C.M. Smillie L.B. Biochemistry. 1995; Scholar). The Ca2+ binding of TnC and TnCs be a of differences the sites I or the of the which be to at position The and fluorescence Ca2+titration in in the but transition a is with the that binding to site II site I sequential binding and that Ca2+ binding to site I the fluorescence the other the fluorescence transition the fluorescence site II in has an affinity in with only site II Furthermore, the site I affinity site II is is site II affinity in the the F29ZW and fluorescence Ca2+ The fluorescence has a of corresponding to a dissociation is of the of that observed for the and The bimodal of the F29ZW fluorescence be related to an in fluorescence by Ca2+ binding to site II and a fluorescence associated with Ca2+ binding to site I the fluorescence of the is the fluorescence of the with only site II The affinity of the site I site II is be to that of site II results related to cooperativity between TnC sites I and II or III and IV are (7Potter J.D. Gergely J. J. Biol. Chem. 1975; 250: 4628-4633Google Scholar, Gergely J. J. Biol. Chem. Scholar, J.D. Potter J.D. J. Biol. Chem. Scholar, J.D. J.D. Scholar) the cooperativity from the studies J. Gergely J. J. Biol. Chem. Scholar) cooperativity between the sites in each data were allowed to to the detected cooperativity between C-terminal sites but cooperativity between N-terminal sites S. J. Biochem. (Tokyo). 1988; 103: 482-486Google Scholar, J. T. Gergely J. Nature. 1990; 345: Scholar, Potter J.D. Scholar, K. Potter J.D. J. Biol. Chem. Scholar, A.S. Potter J.D. J. Biol. Chem. 1987; Scholar, A.S. Potter J.D. Biochemistry. Scholar, K. Potter J.D. J. Biol. Chem. 1987; Scholar, Potter J.D. J. Biol. Chem. 1988; Scholar, K. J.R. Borgford T. Oikawa K. Kay C.M. Smillie L.B. J. Biol. Chem. Scholar, Gergely J. J. Biol. Chem. Scholar, J.R. Borgford T. Chandra M. Oikawa K. Kay C.M. Herzberg O. Moult J. Herklotz A. Reinach F.C. Smillie L.B. Biochemistry. 1992; Scholar, J.R. Kay C.M. Smillie L.B. Biochemistry. 1992; Scholar, K. Borgford T. Biochemistry. 1992; Scholar, Chandra M. J.R. Borgford T. Kay C.M. Smillie L.B. Biochemistry. 1994; Scholar, M. da Silva Sorenson J.R. Borgford T. Kay C.M. Smillie L.B. J. Biol. Chem. 1994; 269: Scholar, M. Oikawa K. Kay C.M. Smillie L.B. Biochemistry. 1994; Scholar, C.A. Biochemistry. 1996; Scholar, O. T. S. Eur. J. Biochem. Scholar, Gergely J. J. Biol. Chem. Scholar). but one of studies S. J. Biochem. (Tokyo). 1988; 103: 482-486Google Scholar) to the free calcium (25Pearlstone J.R. Borgford T. Chandra M. Oikawa K. Kay C.M. Herzberg O. Moult J. Herklotz A. Reinach F.C. Smillie L.B. Biochemistry. 1992; Scholar) and Biochemistry. Scholar) have the value for cooperativity between N-terminal sites. J.R. Chandra M. Sorenson Smillie L.B. J. Biol. Chem. Scholar) the in to cooperativity from Ca2+ titrations using indirect is of which were for to be related to the of only one site J.R. Chandra M. Sorenson Smillie L.B. J. Biol. Chem. Scholar). fluorescence data in to from that is to of or cooperativity. direct Ca2+ binding data from flow dialysis in from to I) that to the or of cooperativity between sites I and II or III and Furthermore, the sequential binding the between and cooperativity. The associated with calcium binding indicate that the and mutants in at calcium J.R. Chandra M. Sorenson Smillie L.B. J. Biol. Chem. Scholar) that this is associated with of the on the of TnC and the mutant of These the the of calcium binding Biochemistry. Scholar, J. Biol. Chem. Scholar). In the data that the replacement of Asp by Ala at position 30 impairs of the of mutants and The fluorescence of mutants be related to the of metal binding to site II on the of the at position 29 and the of the of the fluorescence of and mutants calcium Because is in fluorescence of and mutants calcium binding to the sites I which at high calcium to in mutant and in results indicate that position 29 influences the Ca2+ affinity of TnC N-terminal sites. has N-terminal sites with affinity TnC, and the of this of results in N-terminal site between of and of The direct binding data of mutants that site I is previous Ca2+ binding to site II and that the differences TnC and site are the of differences in the site I only site II is The fluorescence data of and mutants be sequential Ca2+ binding to site II) with the Ca2+ binding to site I the of the fluorescence and The bimodal F29ZW fluorescence is the of a fluorescence by Ca2+ binding to site II and a fluorescence associated with Ca2+ binding to site