Mapping Subdomains in the C-terminal Region of Troponin I Involved in Its Binding to Troponin C and to Thin Filament*

Troponin I (TnI) is the inhibitory component of troponin, the ternary complex that regulates skeletal and cardiac muscle contraction. Previous work showed that the C-terminal region of TnI, when linked to the “inhibitory region” (residues 98–116), possesses the major regulatory functions of the molecule (Farah, C. S., Miyamoto, C. A., Ramos, C. H. I., Silva, A. C. R., Quaggio, R. B., Fujimori, K., Smillie, L. B., and Reinach, F. C. (1994)J. Biol. Chem. 269, 5230–5240). To investigate these functions in more detail, serial deletion mutants of the C-terminal region of TnI were constructed. These experiments showed that longer C-terminal deletions result in lower inhibition of the actomyosin ATPase activity and weaken the interaction with the N-terminal domain of troponin C (TnC), consistent with the antiparallel model for the interaction between these two proteins. The conclusion is that the whole C-terminal region of TnI is necessary for its full regulatory activity. The region between residues 137 and 144, which was shown to have homology with residues 108–115 in the inhibitory region (Farah, C. S., and Reinach, F. C. (1995) FASEB J. 9, 755–767), is involved in the binding to TnC. The region between residues 98 and 129 is involved in modulating the affinity of TnC for calcium. The C-terminal residues 166–182 are involved in the binding of TnI to thin filament. A model for the function of TnI is discussed.

The binding of Ca 2ϩ to the troponin complex initiates muscle contraction (1)(2)(3)(4)(5). The troponin complex is composed of three subunits: troponin I (TnI), 1 troponin C (TnC), and troponin T (TnT) (6). The three subunits are necessary for the complete regulation by calcium of muscle contraction. TnC possesses two low affinity Ca 2ϩ binding sites (I and II) located in the Ndomain and two high affinity Ca 2ϩ binding sites (III and IV) located in the C-domain (7). In physiological conditions, the C-domain of TnC always has Ca 2ϩ bound, whereas Ca 2ϩ binding to the N-domain triggers muscle contraction (8). TnT, the tropomyosin (TM) binding subunit, is necessary for the Ca 2ϩ -dependent regulation of the Tn complex (9) and is required for complete inhibition of the ATPase activity of actomyosin (10).
TnI is the subunit that inhibits the actomyosin Mg 2ϩ -ATPase in the presence of TM, and its inhibition is removed by TnC. The inhibitory action of TnI has been associated with a central part of its amino acid sequence (residues 98 -116) named the "inhibitory region" (11). Models suggest that TnI domains movement from TnC to thin filament are involved in the regulation of muscle contraction (reviewed in 4 and 5). By studying N-and C-terminal deletion mutants of TnI, Farah et al. (10) showed that the C-terminal region of TnI (residues 117-182), when linked to the inhibitory region, has the major regulatory functions of the molecule. The C-terminal region is involved in both the TnC-TnI and actin-TnI interactions. These interactions are responsible for both the calcium regulation of the inhibitory action of TnI and the maintenance of TnI inhibition in the presence of TnC and in the absence of Ca 2ϩ (10).
At this time, little is known about the TnI structure, even though information from low resolution structures is available (12,13) and part of its N-terminal region of TnI (residues 1-47) has been crystallized in a complex with TnC (14). However, the use of deletion mutants (10,15,16) and peptides (11,(17)(18)(19) containing regions of TnI has been shown to be a powerful tool for studying the functions of different regions of this protein and for connecting the functions of TnI with its primary structure.
The problem studied here is to map the functions of the C terminus of TnI by constructing serial deletion mutants of this region. The understanding of the mechanism of actomyosin ATPase inhibition by TnI can be improved by studying how these deletion mutants interact with proteins in the thin filament (actin-TM). The measurement of how these deletions affect the inhibition by TnI of the actomyosin ATPase activity and how they affect the binding of TnI to TnC shows that the entire C-terminal region of TnI is important for maintaining the properties of TnI.

EXPERIMENTAL PROCEDURES
Muscle Proteins-Chicken actin was purified as described by Pardee and Spudich (20). Cardiac ␣-TM was purified as described by Smillie (21). Chicken myosin was purified as described by Reinach et al. (22). The purifications of recombinant TnT and TnC were made as described by Farah et al. (10). Tn complex reconstitution was made as described by Farah et al. (10).
Expression and Purification of Recombinant Proteins-The deletions and expression of the proteins were made as described by Farah et al. (10). The mutant proteins have deletions in the C-terminal region of TnI: TnI 103-156 consists of residues 103-156; TnI 1-107 consists of residues 1-107, and so on (Fig. 1). Purification of the mutants was performed as described for WT-TnI, the exception being the mutant TnI 103-156, which was performed as described for mutant TnI 103-182 (10,23). Protein concentration was determined as described by Hartree (24).
Actin Binding Experiments-Ultracentrifugation was performed for 10 min at 272,000 ϫ g and 25°C in a Beckman TL100 ultracentrifuge.  (25) after and before centrifugation. The protein was judged as co-sedimented or not by its presence in the pellet and the results are described in Tables I (for TnI only) and  II (for Tn complex).
Mg 2ϩ -ATPase Measurements-Mg 2ϩ ATPase measurements were made by combining actin (4 M), TM (1.14 M), myosin (0.2 M), and mutant TnIs or TnCs (see Figs. 2, 3, 4 and 5 for concentration) or Tn complexes (1.14 M). This experiment was performed in 20 mM imidazole-HCl, pH 7.0, 60 mM KCl, 3.5 mM MgCl 2 , 1 mM DTT, and 0.5 mM EDTA on ice. CaCl 2 was added, when necessary, to give the free Ca 2ϩ concentration desired. Sodium ATP was added to 2 mM, and the ATPase reaction was run at 25°C. After 25 min, the phosphate released was detected as described by Heinohen and Lahti (26).
Urea Gel Electrophoresis-TnI, TnC, or TnC-TnI complex (2:1 molar ratio) was loaded in 8% polyacrylamide gels containing 6 M urea and 0.5 mM CaCl 2 (see Refs. 27 and 10 for details). The TnC-TnI complexes run in between TnI, which remains in the origin, and TnC, which has a high mobility. The complexes were prepared using the TnI mutants described above and TnC wild-type and mutants. TnC mutants were described elsewhere (10) and consist of two categories: DA-TnC mutants (of which the calcium binding site I, II, III, or IV was destroyed by mutation), and TnC domains (either N-domain (residues 1-90) or Cdomain (residues 89 -162)). The strength of binding of the TnI mutants was compared with WT-TnI by comparing the bands of the complexes and free TnC in the gel, and the results are described in Table III.
Fluorescence Measurements-The TnC mutant F29W was produced in a Trp-defective Escherichia coli strain, resulting in a protein containing 5-hydroxy-Trp (TnCF29W5OH). 2 The preparation of TnCF29W5OH and details of its fluorescence behavior are described elsewhere. 2 5-Hydroxy-Trp has red-shifted excitation wavelength when compared with the Trp residue, which allows the fluorescence spectrum of TnC 5OHTrp to be distinguished from TnI Trp. Fluorescence measurements using TnCF29W5OH were collected on an F-4500 Hitachi spectrofluorometer at 25°C. The excitation wavelength was 315 nm (slit band with 5 nm), and the emission wavelength was 335 nm (slit band with 5 nm). TnC mutant F29W5OH alone or combined with TnI was diluted with 50 mM MOPS, pH 7.0, 6.5 mM KCl, 1 mM EDTA, and 1 mM DTT to a final concentration of 3 M. CaCl 2 was added to give the free Ca 2ϩ concentration desired. The fluorescence data were analyzed using the equation, where F is the relative fluorescence observed, f is the change in fluorescence caused by binding Ca 2ϩ to the low-affinity sites, [Ca] is the free calcium concentration, n is the Hill coefficient, and K d is the apparent dissociation constant (28).

C-terminal Sequences and Deletion
Mutants-Investigation of the amino acid sequences of the inhibitory and C-terminal regions of TnI from different organisms ( Fig. 1) shows that there are two domains where the homology is high: the inhibitory region (residues 101-115) and the region between residues 137 and 144 (see also Ref. 5). To study the C-terminal functions, mutants were constructed to produce serial deletions in this region (Fig. 1). The proteins were purified, and their solubilities are as follows. WT-TnI and TnI 103-156 remain soluble in 0.5 M NaCl, and TnI 1-165 , TnI 1-156 , TnI 1-147 , and TnI 1-107 remain soluble in 1.0 M NaCl. TnI 1-136 and TnI 1-129 are soluble in 1.0 M NaCl only at low concentrations. Tn complexes remain soluble in 6.5 mM NaCl.
Co-sedimentation in Presence of Actin and Actin-TM-The binding of TnI deletion mutants in actin and actin-TM was tested by co-sedimentation. The presence of TnI mutants in the pellet after ultracentrifugation was analyzed by SDS-polyacrylamide gel electrophoresis (data not shown), and a description of the results is given in Table I. Neither TnI or TM is present in the pellet in the absence of actin. WT-TnI is present in the pellet in the presence of actin, and its concentration in the pellet increases when TM is present, as seen by a stronger band in the presence of TM than in its absence (data not shown). The deletion mutants TnI 1-165 , TnI 1-147 , TnI 1-136 , TnI 1-129 , and TnI 103-156 show behavior similar to WT-TnI. TnI 1-107 deletion mutant, which lacks part of the inhibitory region, is not present in the pellet. This experiment shows that the whole inhibitory region is necessary to bind TnI to actin.
Inhibition of Actomyosin ATPase Activity-The inhibitory properties of TnI deletion mutants were tested by measuring their effects in the actomyosin ATPase activity (Fig. 2). Maxi-  (10). TnI 1-107 contains the N terminus and part of the inhibitory region. TnI 1-129 contains the N terminus, the inhibitory region, and 13 residues of the C terminus. TnI 1-136 contains the N terminus, the inhibitory region, and 20 residues of the C terminus. TnI 1-147 contains the N terminus, the inhibitory region, and the region between residues 137 and 145. TnI 1-156 contains the N terminus, the inhibitory region, the region between residues 137 and 145, and 12 more residues of the C terminus. TnI 1-165 contains the N terminus, the inhibitory region, the region between residues 137 and 145, and 21 more residues of the C terminus. mum inhibition of WT-TnI (about 80%) was reached when its concentration was 4 M (1 actin: 1 TnI). The deletion mutants TnI 1-165 , TnI 1-156 , and TnI 103-156 showed behavior similar to WT-TnI. Maximum inhibition of TnI 1-147 (about 80%) was reached around 6 M (1 actin: 1.5 TnI). The TnI 1-147 curve of inhibition showed a much more cooperative shape than the WT-TnI curve. The deletion mutants TnI 1-136 and TnI 1-129 did not show the same behavior as WT-TnI, because the inhibition is weaker at the concentrations tested; testing at higher concentrations was not possible due to poor solubility of these mutants. The deletion mutant TnI 1-107 did not show any inhibition at all concentrations tested. This experiment shows that there is a region between residues 147 and 156 that is important to achieving inhibition of the ATPase activity at the same levels show by WT-TnI.
Effect of TnC and Ca 2ϩ on the Inhibitory Action of TnI- Fig.  3 shows release of the TnI inhibitory effect by increased con-centrations of TnC in the absence (Fig. 3A) or in the presence (Fig. 3B) of Ca 2ϩ . The TnI deletion mutants used in this test were the ones that reached the maximum inhibition of ATPase activity shown by WT-TnI; the concentration used was 6 M (1 actin:1.5 TnI), where this inhibition is maximum even for TnI 1-147 (see above). TnC did not release the inhibition of WT-TnI in the absence of calcium: inhibition of the ATPase activity was about 60% in all TnC concentrations tested. Total liberation of the inhibitory activity of WT-TnI, in the presence of calcium, occurred when TnC/inhibitor ratio is 1. The mutants TnI 1-165 , TnI 1-147 , and TnI 1-156 failed to inhibit the ATPase activity in the absence of calcium: TnC can release most of the inhibition when the TnC/inhibitor ratio is 0.5. In the presence of calcium, the TnI 1-165 , TnI 1-147 , and TnI 1-156 inhibition was released at a lower TnC concentration than shown by WT-TnI. In the absence of calcium, the deletion mutant TnI 103-156 inhibited the ATPase activity in a manner similar to WT-TnI. In the presence of calcium, however, the inhibition caused by TnI 103-156 was released at a lower TnC concentration than observed for WT-TnI. This experiment shows that in the presence of the N-terminal region, the region between residues 166 and 182 of TnI is necessary to maintain the ATPase inhibition in the presence of TnC and in the absence of calcium.
Calcium Regulation of the ATPase Activity of Actomyosin-The effect of TnI deletion mutants on the Tn complex was studied by testing its regulation of the ATPase activity of actomyosin as a function of calcium concentration (pCa) (Fig.  4A). WT-Tn complex inhibited the ATPase activity about 70% at pCa 9.0 and activated to about 20% at pCa 4.5. At pCa 9.0, Tn complex reconstituted with TnI 103-156 (Tn-TnI 103-156 ) showed the same inhibition as WT-Tn. Tn-TnI 1-165 , Tn-TnI 1-156, or the Tn-TnI 1-147 complex inhibited the ATPase activity about 55%, Tn-TnI 1-136 inhibited the ATPase activity about 35%, and the Tn-TnI 1-129 inhibited the ATPase activity about 25%. Tn-TnI 1-107 did not inhibit the ATPase activity at any pCa tested. At pCa 4.5, Tn-TnI 1-147 , Tn-TnI 103-156 , and the Tn-TnI 1-136 complex showed a little lower activation than WT-Tn, whereas Tn-TnI 1-156 and Tn-TnI 1-165 showed a little higher activation of the ATPase activity than WT-Tn. The   midpoints of the transition between inhibition and activation for Tn complexes reconstituted with TnI deletion mutants occurred around the same pCa as for WT-Tn, the exception being Tn-TnI 1-107 , which did not activate. Fig. 4B shows actomyosin ATPase inhibition (pCa 9.0) and activation (pCa 4.5) as a function of TnI chain extension and includes results for Tn-TnI 1-98 and Tn-TnI 1-116 described by Farah et al. (10). The greater the TnI chain extension, the greater the inhibition at pCa 9.0. This experiment shows that in the presence of the N terminus, deletions in the C terminus of TnI decrease the inhibitory action of the Tn complex at pCa 9.0.
Co-sedimentation with Actin-TM of Tn Complexes Reconstituted with TnI Deletion Mutants-Tn complexes were ultracentrifugated with actin-TM in the presence and in the absence of calcium, to test the effect of the TnI deletion mutants on the binding of Tn in the thin filament. The presence of the Tn components in the pellet was analyzed by SDS-polyacrylamide gel electrophoresis (data not shown), and a description of the results is given in Table II. About half of TM and Tn components (TnI, TnC, and TnT) co-sediment with actin in the presence or in the absence of calcium. The results obtained with the Tn complexes made with the TnI deletion mutants are the same as for the WT-Tn, the exception being the Tn complex reconstituted with TnI 103-156 (Tn-TnI 103-156 ). Tn-TnI 103-156 did not show the same behavior as WT-Tn because TnC did not co-sediment in the absence of calcium, although about half of the TnT and TnI 103-156 did, and neither TnC or TnI 103-156 co-sedimented in the presence of calcium, although about half of the TnT did. This experiment shows that there is a balance of affinity in TnI C-terminal region for binding either TnC or actin-TM and that this affinity is calcium-dependent.
Complex Formation with DA-TnC, N-domain, and C-domain of TnC Mutants-Analysis in urea gels of complex formation between TnI deletion mutants and TnC was made to test the binding between these proteins. TnI loaded alone remains at the origin of the gel, and TnCs loaded alone show higher electrophoretic mobility than the complexes (27, 10); the results are described in Table III. WT-TnI and TnI 1-147 formed strong complexes with TnC mutants III or IV and with the TnC N-domain. TnI 1-136 or TnI 1-129 formed weak complexes with TnC mutants III or IV and with the TnC N-domain: the bands representing the complex are weaker than the band formed with WT-TnI, and free TnC was present. There is no complex formation between TnI 1-116 and TnI 1-107 with the TnC III or IV mutants or with the TnC N-domain. TnI 103-156 formed weaker complexes with the TnC mutants I, II, III, or IV and with the TnC N-or C-domains than the complexes formed with WT-TnI. This experiment shows that in presence of the N terminus, deletion of the region between residues 147 and 182 of TnI does not affect the complex formation between TnI and TnC in urea gels. Deletion of the region between residues 129 and 147 of TnI decreases its interaction with the TnC N-domain, and deletion of the entire C-terminal region removes this interaction.
Fluorescence Measurements-The effect of TnI deletion mutants on calcium binding to TnC is tested by fluorescence using the TnC mutant F29W5OH (TnCF29W5OH). 2 Residue Phe-29 is at the regulatory calcium binding site I, and it is a good probe with which to study the effects of calcium binding to TnC (28). The relative fluorescence of TnCF29W5OH alone, or when combined with TnI deletion mutants, as a function of pCa is shown in Fig. 5A, and calculated values of pCa 1/2 , K d , and n are given in Table IV (see under "Experimental Procedures" for details of the calculation of these parameters). When combined with deletion mutant TnI 1-98 or TnI 1-107 , TnCF29W5OH fluorescence showed almost no change from that of TnCF29W5OH alone. When combined with deletion mutant TnI 1-116 or TnI 103-156 , increasing in relative fluorescence occurred at a lower calcium concentration than for TnCF29W5OH alone. When combined with WT-TnI, TnI 1-129 , TnI 1-136 , or TnI 1-147 deletion mutants, increasing in relative fluorescence occurred at an even lower calcium concentration than for TnCF29W5OH combined with deletion mutant TnI 1-116 . The variation of the pCa 1/2 and K d as a function of increased chain extension of the TnI C terminus is given in Fig. 5B, showing that the main changes in the calcium binding properties of TnC occur between residues 98 and 129 (including the inhibitory region and part of the C-terminal region). This experiment shows that the region between these residues is involved in modulating the affinity of TnC for calcium. DISCUSSION Use of deletion mutants to study TnI has been shown to be useful in understanding which regions interact with TnC and with the thin filament (10, 15, 16). With the aim of studying these interactions in more detail, I looked for conserved regions inside the C-terminal region of TnI. The region between residues 137 and 145 is well conserved, and as pointed out in Farah and Reinach (5), this region shares homology with part of the inhibitory region, residues 108 -115. Some studies (29 -31) have shown that this region also shares homology with one of the two calmodulin binding regions of the regulatory unit of the phosphorylase kinase. This homology deserves attention because calmodulin can substitute for TnC in neutralizing the inhibition by TnI of actomyosin ATPase in presence of calcium (32). The importance of the C terminus in the regulation by TnI of muscle contraction is investigated here by mutations that create serial deletions in the C terminus of TnI. The results show that the entire C-terminal region of TnI is necessary for the regulatory properties of this protein.
The Region between the Inhibitory Region and Residue 147 of TnI Interacts with the N-domain of TnC-Deletion of residues 148 -182 of the C terminus of TnI does not affect complex formation between TnI and TnC, and there is no complex formation if the whole C terminus of TnI is deleted. This implies that the region between the inhibitory region and residue 147 of TnI interacts with the N-domain of TnC. TnC can be cross-linked to residues 96 -145 of TnI, but not to residues 146 -182 (33,34). Tripet et al. (35) showed that fragments consisting of regions 96 -131 and 96 -148 interact with TnC. Analysis of the fluorescence results shown here gives the same conclusion. The presence of the inhibitory region increases the affinity of TnC by calcium, and this affinity is similar to the affinity of the dimer formed between TnC and WT-TnI when the region between residues 117 and 129 is present. There is a region between residues 98 and 129 that is important in modulating the affinity of TnC for calcium. Those results complement the results of Farah et al. (10) showing that the inhibitory region is involved in the Ca 2ϩ -dependent binding to TnC and that TnI binds TnC in an antiparallel manner: the C-terminal region (regulatory) of TnI interacts with the N terminus (reg-ulatory) of TnC, the C-terminal region (structural) of TnC interacts with the N terminus (structural) of TnI, and the inhibitory region of TnI interacts with both the N-and Cterminal regions of TnC. Support to this model was also given by Sheng et al. (15) and by Jha et al. (16). McKay et al. (19), using a peptide corresponding to residues 115-131 of TnI, showed that this region binds to the hydrophobic pocket of N-domain of TnC (36). Pearlstone et al. (37), using a recombinant fragment of TnI containing residues 96 -148, showed that residues 96 -116 are responsible for binding to the C-domain of TnC, that residues 117-148 are responsible for binding the N-domain of TnC, and that fragment 96 -148 binds with higher affinity than fragment 96 -116.
The Affinity of the C-terminal Region of TnI for TnC and Thin Filament Is Calcium-dependent-TnI 103-156 lacks the last residues (157-182) of the C terminus, and its inhibition of actomyosin ATPase activity is not released by TnC in the absence of calcium as happens with TnI 1-156 . This happens because TnI 103-156 also lacks the N-terminal region that binds TnC in a Ca 2ϩ -independent manner (10,15), making the affinity of TnI 103-156 for the thin filament be larger than for TnC. The absence of the N-terminal region of TnI is the reason why Tn-TnI 103-156 has a different behavior than Tn-TnI 1-156 (or WT-Tn) in the co-sedimentation experiments: in the absence of calcium, TnC does not bind TnI 103-156 and does not co-sediment, whereas TnI 103-156 does co-sediment; however, in the presence of calcium, TnC binds TnI 103-156 and prevents this mutant from binding to actin-TM. This result shows that there is a balance of affinity in the C-terminal region of TnI between binding either to TnC or to actin-TM, and this balance of affinity is Ca 2ϩ -dependent. It is interesting to notice that TnI 103-182 still binds actin-TM, in the Tn co-sedimentation experiments, even in the presence of calcium, but with lower affinity than WT-TnI (38). This happens because the region between residues 165 and 182 is important in binding the thin TABLE II Co-sedimentation of Tn complexes with actin-TM in the presence or in the absence of calcium Tn binding to actin-TM is assayed by ultracentrifugation. A "yes" means co-sedimentation, and a "no" means no co-sedimentation.

TABLE III Complex formation between TnI deletion mutants and TnC mutants (DA-, N-, or C-domain)
Complex formation between TnI deletion mutants with TnC mutants (sites I (D30A), II (D66A), III (D106A), or IV (D142A) destroyed; or N-domain, amino acids 1-90, or C-domain, amino acids 88 -162) are described as follows: ϩϩϩ, strong complex formation resembling WT-TnI; ϩϩ, weaker complex formation than WT-TnI and part of TnC is free; ϩ, weaker complex formation than WT-TnI and most of TnC is free; Ϫ, no complex formation visible and all TnC is free; and a blank means experiment not done. This table shows results both of this paper and of Farah et al. (10). filament (see below).
In the fluorescence experiments, the dimer formed between TnC and TnI 103-156 has a lower affinity for calcium than the dimer formed with TnI 1-156 . The absence of the N-terminal region weakens the binding to TnC, as shown by Farah et al. (10) and in this study. In the Tn complex, a higher affinity for binding TnI  to TnC than to thin filament causes a weakening of the inhibition of the ATPase activity at lower Ca 2ϩ concentrations; Tn-TnI 103-156 has a higher inhibitory activity at pCa 9.0 than Tn-TnI  .
The Whole C-terminal Region of TnI Is Important for Interacting with Thin Filament-There is a region between residues 148 and 156 that is important for obtaining maximum inhibition at the same levels as WT-TnI: higher molar ratios of deletion mutant TnI 1-147 to actin-TM are necessary than for WT-TnI to obtain maximum inhibition of the ATPase activity; deletion of the last 26 residues of TnI, TnI 1-156 (10), causes no change in its ability to inhibit ATPase activity. However, Van Eyk et al. (39) and Tripet et al. (35) showed that a fragment of TnI encompassing residues 96 -148 inhibits the ATPase activity at the same molar ratio as the WT-TnI. This difference between the results with TnI 1-147 and TnI 96 -148 is probably caused by the absence of the N-terminal region in the latter. The inhibitory region is a poorer inhibitor than whole TnI (18) but a better inhibitor than when it is linked to the N-terminal region, as shown for TnI 1-116 by Farah et al. (10). Van Eyk et al. (39) tested WT-TnI, TnI 96 -148 , TnI 1-116 , and TnI 104 -115 in the same conditions (Fig. 1A of Ref. 39), and one can see that the inhibitory region alone has most of the TnI inhibitory activity, whereas when linked to the N-terminal region, this activity is reduced. This behavior could be caused by some misbinding of the N terminus of TnI because, in the Tn complex, this region binds TnT (40 -42) or TnC (10,11,15,16), but further investigation is needed to determine whether this is the case.
The entire C-terminal region of TnI is important to maintain the inhibition by TnI in the presence of TnC and in the absence of calcium. If the region between residues 166 and 182 is deleted, the affinity of TnI for thin filament decreases: TnC, even in the absence of calcium, can remove the inhibitory activity of the mutant TnI 1-165 . The loss of this region corresponds to decreasing the affinity of TnI for thin filament. With less affinity of TnI for thin filament, TnC can sequester TnI, and its inhibitory activity is lost. TnI has a balance of affinity for thin filament and for TnC, and the last residues of TnI are important for binding TnI to thin filament.
The entire C-terminal region of TnI is shown be important for the regulation by Tn of the actomyosin ATPase activity: serial deletions in the C-terminal region of TnI affect the inhibitory activity of Tn gradually and the loss of certain regions has larger effects. The region between residues 148 and 182 (results for Tn complexes reconstituted with TnI 1-165 , TnI 1-156 , or TnI 1-147 can be grouped together), the region between residues 130 and 147, and the region between residues 117 and 129. Deletion of the entire C-terminal region of TnI, TnI 1-116 and TnI 1-98 (10), and TnI 1-107 (this report) results in loss of the ability of Tn complexes to inhibit the ATPase activity.
Deletion of the part of the inhibitory region, residues 107-115, that shares homology with the region between residues 137 and 145 destroys the TnI inhibitory activity: the deletion mutant TnI 1-107 does not show inhibitory activity even when present in a 2:1 molar ratio with actin. This result is in agreement with those of Syska et al. (11) and Talbot and Hodges (17,43), who showed that the whole inhibitory region is necessary for the inhibition activity. This can be seen also in the cosedimentation experiments: all mutants that do not have the whole inhibitory region do not bind actin. This experiment also shows that TM increases the affinity of TnI for thin filament, as expected because inhibition of the actomyosin ATPase activity  by TnI is higher in the presence of TM (11).
Concluding Remarks and a Model for the Action of the TnI C Terminus-The current model for regulation of skeletal muscle contraction suggests that TnI switches between TnC and the thin filament in a calcium-dependent manner (11, 44 -46). In the presence of calcium, the inhibitory region and the C terminus of TnI disconnect from the actin and bind to the N-and C-terminal regions of TnC, releasing the inhibition of the actomyosin ATPase activity (10,47,48). This work shows that the C-terminal region participates in various TnI functions: (i) the region between the inhibitory region and residue 148 is involved in TnC binding; (ii) the region between residues 98 and 129 is involved in modulating the affinity of TnC for calcium; (iii) the region between residues 166 and 182 is involved in binding to thin filament and is important for the inhibition of the actomyosin ATPase activity by Tn complex; and (iv) the region between the inhibitory region and residue 156 is important for the inhibition of the actomyosin ATPase activity by TnI alone.
With these points in mind, a model for the regulatory activity of the C-terminal region of TnI can be described in more detail. The region between the inhibitory region and residue 147 is involved in the binding to TnC, and the last residues of TnI are involved in its binding to thin filament. In the absence of calcium, the last residues of TnI help TnI to bind to thin filament, contributing to the inhibition of the actomyosin ATPase activity. In the presence of calcium, the region between the inhibitory region and residue 147 binds to TnC, increasing the TnI-TnC interaction and releasing the inhibitory activity.
The complexity of the interactions of TnI is evident. The different regions of TnI interact, which complicates the interpretation of the results: the presence of the N-terminal region influences the inhibitory region function and the binding of the C terminus to actin. More structural studies, such as the recent work by Sykes and co-workers (49), will be necessary to analyze the overall functions of these regions. The use of deletion mutants and peptides is a powerful approach to studying the functions of regions inside the C terminus, but the conclusions must be reached carefully. Deletions can decrease the stability of a protein (50,51), and the functions of TnI depend on its stability and hence on the amount deleted. Possibly, permutation of the regions in TnI will prove to be a powerful tool in elucidating the complex functions of this protein and their structural origins.