The Role of the NH2- and COOH-terminal Domains of the Inhibitory Region of Troponin I in the Regulation of Skeletal Muscle Contraction*

The role of the inhibitory region of troponin (Tn) I in the regulation of skeletal muscle contraction was studied with three deletion mutants of its inhibitory region: 1) complete (TnI-(Δ96–116)), 2) the COOH-terminal domain (TnI-(Δ105–115)), and 3) the NH2-terminal domain (TnI-(Δ95–106)). Measurements of Ca2+-regulated force and relaxation were performed in skinned skeletal muscle fibers whose endogenous TnI (along with TnT and TnC) was displaced with high concentrations of added troponin T. Reconstitution of the Tn-displaced fibers with a TnI·TnC complex restored the Ca2+ sensitivity of force; however, the levels of relaxation and force development varied. Relaxation of the fibers (pCa 8) was drastically impaired with two of the inhibitory region deletion mutants, TnI-(Δ96–116)·TnC and TnI-(Δ105–115)·TnC. The TnI-(Δ95–106)·TnC mutant retained ∼55% relaxation when reconstituted in the Tn-displaced fibers. Activation in skinned skeletal muscle fibers was enhanced with all TnI mutants compared with wild-type TnI. Interestingly, all three mutants of TnI increased the Ca2+ sensitivity of contraction. None of the TnI deletion mutants, when reconstituted into Tn, could inhibit actin-tropomyosin-activated myosin ATPase in the absence of Ca2+, and two of them (TnI-(Δ96–116) and TnI-(Δ105–115)) gave significant activation in the absence of Ca2+. These results suggest that the COOH terminus of the inhibitory region of TnI (residues 105–115) is much more critical for the biological activity of TnI than the NH2-terminal region, consisting of residues 95–106. Presumably, the COOH-terminal domain of the inhibitory region of TnI is a part of the Ca2+-sensitive molecular switch during muscle contraction.

The inhibitory function of Tn has been studied in many different ways, yet the structure-function of the inhibitory region of TnI, responsible for this, is still under investigation. The role of the putative inhibitory region of TnI, which consists of 21 amino acids (residues 96 -116), has been studied previously utilizing synthetic peptides as well as proteolytic and recombinant fragments of TnI. The cyanogen bromide fragment (CN4) of TnI (residues 96 -116) was originally found to possess all of the inhibitory properties of intact TnI (6). Studies with synthetic peptides have demonstrated that residues 105-114 represent the minimal sequence necessary to produce inhibition of actomyosin ATPase activity and to retain TnC binding (7)(8)(9)(10); however, the CN4 fragment (TnI-(96 -116)) has been shown to have an 8-fold higher affinity for TnC compared with TnI-(104 -115) (11).
Several regions of TnI, including its inhibitory region (residues 96 -116), have been identified as interacting with actin-Tm and TnC (12)(13)(14)(15)(16). Sequence 104 -115 of TnI has been shown to share both the actin-Tm-and TnC-binding sites. Studies of Tripet et al. (17) suggest that the region of TnI that follows the inhibitory sequence (residues 96 -116) contains additional actin-Tm-and TnC-binding sites. A synthetic peptide consisting of residues 128 -148 was able to bind specifically to the actin-Tm filament and could induce a weak inhibitory activity on its own. Truncation of residues 140 -148 completely abolished the inhibitory effect of this region when compared with TnI-(96 -115), suggesting that region 140 -148 of TnI presumably contains the second actin-Tm-binding site (17). This is in agreement with Farah et al. (18), who demonstrated that residues 116 -156 are important for the Ca 2ϩ regulation of actomyosin ATPase activity. The recombinant fragment TnI-  failed to inhibit ATPase activity in the absence of Ca 2ϩ compared with other fragments (TnI-(1-156) and TnI-(103-182)) that were able to regulate actomyosin ATPase activity in a Ca 2ϩ -dependent manner (18). Furthermore, Tripet et al. (17) demonstrated that residues 116 -131 are not important for inhibition, but are critical for the interaction with TnC and designated this region to be the second TnC-binding site (17). Several studies have shown that residues 96 -116 of TnI are primarily responsible for the binding to the COOH-terminal domain of TnC and residues 117-148 for the binding of TnI to the NH 2 -terminal domain of TnC (19,20). Reconstituted TnI fragments containing the inhibitory region of TnI (residues 96 -116) plus either the NH 2 -terminal (TnI-(1-116)) or COOHterminal (TnI-(96 -148)) region of TnI were shown to be responsible for either maintaining the maximal level of actomyosin ATPase activity or the Ca 2ϩ dependence of ATPase, respectively (20). In summary, the regulatory complex containing TnT, TnC, and TnI-(96 -148) retained all of the full regulatory properties of troponin, suggesting that TnI-(96 -148) contains the major sequence of TnI responsible for inhibitory activity (17,20). Based upon these experiments, an extended inhibitory region of TnI has been proposed, containing residues 96 -148 (17, 19 -22).
This study was undertaken to determine the effect of the NH 2 -or COOH-terminal deletions of inhibitory region 96 -116 of TnI on the contractile properties of skinned skeletal muscle fibers. We have expressed and purified wild-type TnI (WTnI) and three deletion mutants of TnI: TnI-(⌬95-106), TnI-(⌬105-115), and TnI-(⌬96 -116). We have applied the improved method of Shiraishi and Yamamoto (23) and Hatakenaka and Ohtsuki (24) to displace the endogenous Tn complex with high concentrations of added TnT, followed by functional reconstitution of the fibers with preformed complexes of TnC and the TnI mutants. This system allowed us to study the effect of these deletions on the Ca 2ϩ regulation of force development. These proteins were also tested for their ability to inhibit actin-Tm-activated myosin ATPase activity as well as their ability to regulate ATPase activity when complexed with TnT and TnC. Our results suggest that the COOH-terminal end of the inhibitory region of TnI (residues 105-115) is much more critical for the biological activity of TnI than its NH 2 -terminal region (residues 95-106). Presumably, the COOH-terminal domain of the inhibitory region of TnI is a component of the Ca 2ϩsensitive molecular switch that regulates muscle contraction.
Wild-type TnI and the TnI inhibitory region deletion mutants were expressed in Escherichia coli BL21(DE3) (Novagen) using the protocol provided by the manufacturer. The expression was checked by SDS-15% PAGE (28) and Western blotting. The culture for bacterial expression was collected and centrifuged at 7000 rpm (J-10, Beckman). Bacterial pellets were dissolved in a solution containing 6 M urea, 10 mM sodium citrate, pH 7.0, 1 mM dithiothreitol, 2 mM EDTA, and 0.01% NaN 3 and sonicated (Sonicator Heat Systems, Inc.) twice at setting 8 for 2 min at 4°C. After the sonication, the pH of the solution was adjusted to 5.0, and the mixtures were then centrifuged again at 18,000 rpm (J-20, Beckman) at 4°C for 30 min. Supernatants, containing the TnI proteins, were loaded onto a CM52 ion-exchange column equilibrated with the same buffer used to dissolve bacterial pellets, except that the pH was 5.0. The proteins were eluted with a linear salt gradient of 0 -0.6 M KCl in the equilibration buffer. The fractions containing the TnI proteins (identified by SDS-PAGE and Western blotting) were pooled together and dialyzed against 0.5 M NaCl, 50 mM Tris, 2 mM CaCl 2 , and 1 mM dithiothreitol, pH 7.5, and loaded onto a TnC affinity column equilibrated with the same buffer. Pure TnI proteins were eluted with a double gradient of urea (0 -6 M) and EDTA (0 -3 mM). The average yield was 3-5 mg of pure TnI/liter of culture.
Actin-Tm-activated Myosin ATPase Assays-Wild-type TnI and the TnI mutants were first tested for their ability to inhibit actin-Tmactivated myosin ATPase activity, and then the whole Tn complexes (TnI ϩ TnC ϩ TnT formed at a molar ratio 1:1:1) were examined for their ability to regulate (ϮCa 2ϩ ) the ATPase activity. The ATPase assays were performed with actin (3.5 M), Tm (1 M), and myosin (0.6 M) in the presence (0.5 mM CaCl 2 ) or absence (1 mM EGTA) of Ca 2ϩ . The ATPase reaction (in 10 mM MOPS, 50 mM KCl, and 4 mM MgCl 2 , pH 7.0) was initiated by the addition of 2.5 mM ATP and terminated after 6 min with 5% trichloroacetic acid. Inorganic phosphate was measured according to Fiske and SubbaRow (29).
Airfuge Binding Assays-The binding of the TnI proteins or troponins containing TnI, TnT, and TnC (1:1:1) to actin-Tm was measured at a molar ratio of 7 actin (14 M)/1 Tm (2 M)/1 Tn (2 M) or 2 TnI (4 M) in a buffer containing 10 mM MOPS, 50 mM KCl, and 4 mM MgCl 2 , pH 7.0. The complexes of Tn and actin-Tm were additionally mixed with either 0.5 mM CaCl 2 or 1 mM EGTA. Protein complexes were incubated for 10 min at room temperature and Airfuged (maximum speed) for 30 min. In parallel, the control samples of the TnI proteins alone or complexed with TnT and TnC without actin-Tm were sedimented. The samples of TnI proteins, troponins, and their complexes with actin-Tm (prior to sedimentation) as well as the supernatants and pellets were run on SDS-15% polyacrylamide gels (28) and analyzed.

Displacement of the Endogenous Troponin Complex in Skinned Skeletal Muscle Fibers with
TnT: Steady-state Force and the Ca 2ϩ Sensitivity of Force Development-Displacement of the endogenous Tn complex in skinned skeletal muscle fibers was performed according to method of Shiraishi and Yamamoto (23) and Hatakenaka and Ohtsuki (24). We have slightly modified this method (described below) to achieve complete Tn displacement, as judged by SDS-PAGE, and the measurements of Ca 2ϩ -unregulated force. Briefly, rabbit psoas skinned muscle fiber bundles (three to five fibers) were mounted on a force transducer and treated with a pCa 8 relaxing solution containing 1% Triton X-100 for 20 min. The composition of the pCa 8 solution was 10 Ϫ8 M Ca 2ϩ , 1 mM Mg 2ϩ , 7 mM EGTA, 5 mM MgATP 2ϩ , 20 mM imidazole, pH 7.0, 20 mM creatinine phosphate, and 15 units/ml creatinine phosphokinase, I ϭ 150 mM. The Ca 2ϩ dependence of force development was tested twice to make certain that it was stable. Following the initial testing, the fibers were then incubated in a solution containing 250 mM KCl, 20 mM MOPS, pH 6.2, 5 mM MgCl 2 , 5 mM EGTA, 0.5 mM dithiothreitol, and Ϸ1.6 -2 mg/ml rabbit skeletal TnT for 1 h at room temperature. The fibers were then washed with the same solution without the protein (10 min at room temperature) and tested for the Ca 2ϩ -unregulated force that results from the displacement of the endogenous Tn complex from the fibers. The Ca 2ϩ regulation of steady-state force development was then restored with a preformed TnI⅐TnC complex. The reconstitution with the TnI⅐TnC complex (20 M) was performed in the pCa 8 solution, generally for 1 h at room temperature or long enough for the force to reach a stable level. Wild-type TnI and the various TnI mutants were complexed with TnC and used to reconstitute Ca 2ϩ -regulated force. Control fibers were run in parallel and treated with the same solutions minus the proteins. The purpose of this was to get an estimate of fiber rundown and to be able to determine the extent of reconstitution of the TnT-treated fibers. The Ca 2ϩ dependence of force development was determined before the TnT treatment and after TnI⅐TnC reconstitution, and the data were analyzed with the Hill equation (30): % relative force ϭ 100 ϫ [Ca 2ϩ ] n /([Ca 2ϩ ] n ϩ pCa 50 n ), where pCa 50 is the pCa of a solution in which 50% of the change is produced and n is the Hill coefficient.
Inhibition of Actin-Tm-activated Myosin ATPase Activity by TnI and Its Inhibitory Region Deletion Mutants-The inhibitory properties of rabbit skeletal TnI, WTnI, and its deletion mutants are presented in Fig. 2. Actin-Tm-activated myosin ATPase activity was measured as a function of increasing concentrations of the TnI proteins. As illustrated, recombinant TnI (WTnI) had essentially the same activity as rabbit skeletal TnI, and both inhibited Ϸ90 -95% of the ATPase activity. The NH 2terminal inhibitory deletion mutant, TnI-(⌬95-106), only partially inhibited actin-Tm-activated myosin ATPase activity, and Ϸ40 -50% inhibition occurred at a 3-4-fold molar excess of TnI-(⌬95-106) over actin. The COOH-terminal deletion mu- tant, TnI-(⌬105-115), and the entire inhibitory region deletion mutant, TnI-(⌬96 -116), completely lost the ability to inhibit ATPase activity. The latter also gave a slight activation. In summary, the TnI mutants alone could not inhibit actin-Tmactivated myosin ATPase activity when the COOH-terminal region or the entire inhibitory region was deleted.
Regulation of Actin-Tm-activated Myosin ATPase Activity by Tn Containing TnT, TnC, and the TnI Inhibitory Mutants- Fig. 3 (A and B) illustrates the effect of the TnI inhibitory region deletion mutations on actin-Tm-activated myosin ATPase activity in reconstituted thin filaments. TnI and its deletion mutants were complexed with TnT and TnC, and the actomyosin ATPase activity was measured in the presence (  Fig. 3, the troponin complex containing wild-type TnI or rabbit skeletal TnI regulated the ATPase activity in a similar way (ϮCa 2ϩ ), suggesting that recombinant TnI is functional. Tn containing the NH 2 -terminal inhibitory region deletion mutant, TnI-(⌬95-106), activated the ATPase activity in the presence of Ca 2ϩ , but its inhibitory function (in the absence of Ca 2ϩ ) was lost. The COOH-terminal inhibitory region deletion mutant, TnI-(⌬105-115), activated the ATPase activity in the presence or absence of Ca 2ϩ , with ϳ1.3-fold higher activation in Ca 2ϩ . A similar, Ca 2ϩindependent activation of ATPase activity was observed for the complete inhibitory region deletion mutant, TnI-(⌬96 -116); however, the extent of activation was not as high as for TnI-(⌬105-115). In summary, in the presence of Ca 2ϩ (Fig. 3A), all the activation curves were not significantly different, whereas the inhibition curves (in the absence of Ca 2ϩ ) were dramatically different among the various TnI mutants (Fig.  3B). TnI-(⌬105-115) as well as TnI-(⌬96 -116) not only did not inhibit the ATPase activity in the absence of Ca 2ϩ , but gave a 1.4-fold activation of the ATPase. TnI-(⌬95-106) did not inhibit the ATPase activity in the absence of Ca 2ϩ and also lacked the activation seen with the other mutants (Fig. 3B).
Sedimentation Studies (Airfuge)-To examine the binding of TnI proteins alone or complexed with TnC and TnT to actin-Tm, the complexes were Airfuged, and the pellets and supernatants were analyzed by SDS-PAGE. In parallel, the controls of the TnI proteins alone or complexed with TnT and TnC without actin-Tm were sedimented. No pellets of the TnI proteins alone or troponins containing the TnI mutants were observed (data not shown). Fig. 4A shows the binding of WTnI and TnI mutants to actin-Tm, whereas Fig. 4B demonstrates the binding of the Tn complexes in the presence or absence of Ca 2ϩ . The analysis of the pellets and supernatants indicated that all of the troponin complexes containing either WTnI or the TnI deletion mutants bound well to actin-Tm in the presence or absence of Ca 2ϩ (Fig. 4B). Their binding to actin-Tm was weaker for the TnI mutants alone than when they were complexed with TnT and TnC (Fig. 4, A and B).
Force Activation, Relaxation, and the Ca 2ϩ Sensitivity of Force in Skinned Skeletal Muscle Fibers Reconstituted with the TnI Inhibitory Region Deletion Mutants-The physiological significance of the NH 2 -and COOH-terminal domains of the inhibitory region of TnI was examined using rabbit psoas skinned fibers, in which steady-state force, relaxation, and the Ca 2ϩ sensitivity of force development were measured. Following Tn displacement, the fibers were reconstituted with preformed complexes of TnC and WTnI and its deletion mutants. Fig. 5 illustrates a typical experiment on the TnT-treated fiber (panel A) compared with the control buffer-treated fiber (panel B), which had been tested in parallel to estimate the time-dependent rundown of the fibers. Fig. 6 illustrates the Tn displacement procedure. As shown, incubation of the fibers with TnT resulted in a complete loss of Ca 2ϩ dependence of force, and the fibers became unregulated (Fig. 5A). This is illustrated in Fig.  6 by a transition from step 1 to step 2. When Tn-displaced fibers were incubated with a preformed TnI⅐TnC complex, dissolved in the relaxing solution (pCa 8), the fibers underwent a gradual relaxation as the Tn activity was reconstituted (Fig. 5A). This step (step 3) restored the Tn complex (Fig. 6), and the fibers became entirely regulated by Ca 2ϩ . The level of force relaxation depended on the TnI mutant used for the TnI⅐TnC complex. In Fig. 5A, WTnI or rabbit skeletal TnI was used, and full relaxation in the pCa 8 solution was achieved. Fig. 7 summarizes the effect of the TnI mutations on the level of relaxation and force development following reconstitution of the fibers with preformed TnI⅐TnC complexes. The dashed line in Fig. 7 represents the level of Ca 2ϩ -unregulated force after RSTnT treatment (this step and the level of Ca 2ϩ -unregulated force are also shown in Fig. 5A). Arrows indicate the percentage of force inhibition relative to Ca 2ϩ -unregulated force. As shown, the relaxation in the fibers could be restored upon incubation with TnI⅐TnC; however, the level of relaxation varied depending on the TnI mutant utilized in the reconstitution. The NH 2 -terminal inhibitory region deletion mutant, TnI-(⌬95-106), inhibited Ϸ54.9 Ϯ 6% of the force following reconstitution. On the other hand, the COOH-terminal inhibitory region deletion mutant, TnI-(⌬105-115), as well as the complete inhibitory region deletion mutant, TnI-(⌬96 -116), inhibited only 28.3 Ϯ 10 and 28.0 Ϯ 7% of the force, respectively. Interestingly, all three TnI deletion mutants gave an elevated maximal level of force recovery in the high Ca 2ϩ solution (pCa 4) (Fig. 7). The extent of activation over that recovered with WTnI⅐TnC was Ϸ114 Ϯ 16, 116 Ϯ 15, and 123 Ϯ 5% for TnI-(⌬96 -116), TnI-(⌬105-115), and TnI-(⌬95-106), respectively (Fig. 7). The Ca 2ϩ sensitivity of force development measured after fiber reconstitution is demonstrated in Fig. 8. All three deletion mutants of TnI increased slightly the Ca 2ϩ dependence of force (⌬pCa 50 Х 0.1-0.2) compared with WTnI⅐TnC-reconstituted fibers. A Western blot of the experimental fibers performed with the TnI antibodies is presented in Fig. 9. As shown, all of the experimental fibers were reconstituted with the respective TnI deletion mutants. Amino acid deletions within the inhibitory region of TnI did not prevent the binding of TnI-(⌬95-106)⅐TnC, TnI-(⌬105-115)⅐TnC, or TnI-(⌬96 -116)⅐TnC complexes to the Tndisplaced fibers.
In summary, our results show that the putative inhibitory region of skeletal muscle TnI, consisting of residues 96 -116, is not functionally uniform along its amino acid sequence. The inhibitory activity of TnI in the reconstituted thin filaments and in skinned skeletal muscle fibers was less affected by the deletion of the NH 2 -terminal inhibitory region residues (amino acids 95-106) than by the deletion of its COOH-terminal inhibitory sequence (amino acids 105-115). Deletion of residues 105-115 was functionally analogous to the removal of the entire inhibitory region of TnI (residues 96 -116).

DISCUSSION
This study characterizes the effect of NH 2 -and COOH-terminal deletions within the inhibitory region of TnI on the Ca 2ϩ regulation of the actin-Tm-activated myosin ATPase activity and force development in skinned skeletal muscle fibers. The lack of ATPase inhibition with some additional activation for the troponins containing TnI-(⌬105-115) or TnI-(⌬96 -116) indicates that the inhibitory region of TnI, especially its COOH terminus, is directly involved in the regulation of ATPase activity. Applying the three-state model of muscle regulation (31), one can speculate that this depleted region of TnI is the minimum sequence necessary to maintain the "blocked" state of the thin filament, where the interaction between actin and the myosin heads is highly inhibited. Removing this part of the inhibitory region of TnI (residues 105-115) resulted in a dramatic alteration of the blocked state of the thin filament, facilitating a transition from the blocked to the "closed" and/or "open" state (31). Our ATPase and force development results support the above hypothesis and suggest that the COOH terminus of the inhibitory region of TnI is much more critical for its regulatory function than the NH 2 terminus.
The binding experiments of the TnI inhibitory region mutants to actin-Tm filaments and to TnI⅐TnC-depleted fibers also suggest that the inhibitory region of TnI (residues 96 -116) is not the only interaction site for actin-Tm and that the other TnI site may be involved in the interaction of TnI with TnC. Even removing the entire inhibitory region of TnI did not eliminate the binding of the TnI-(⌬96 -116) mutant to actin-Tm or the TnI⅐TnC-depleted fibers.
These results are in accord with studies of Tripet et al. (17) that suggest that the second site on TnI for actin is located in the region COOH-terminal to inhibitory sequence 96 -116, somewhere between residues 140 and 148. These authors also suggested that this region is involved in conferring an inhibitory activity to the thin filaments. Interestingly, the region containing residues 116 -131, adjacent to sequence 96 -116 and preceding region 140 -148, was found not to be important for actin-Tm binding, but significant for the Ca 2ϩ -dependent TnI-TnC interaction (17). Moreover, the studies of Pearlstone et al. (19) and Van Eyk et al. (20) demonstrated that the inhibitory residues (positions 96 -116) of TnI are primarily responsible for the binding to the COOH-terminal domain of TnC (and to actin-Tm), whereas residues 117-148 of TnI are responsible for the binding to the NH 2 -terminal regulatory domain of TnC.
Our results with the TnI-(⌬105-115) and TnI-(⌬96 -116) mutants suggest, however, that the inhibitory region of TnI or at least its COOH terminus (residues 105-116) is important for the Ca 2ϩ -dependent interaction of TnI with TnC. Removal of this region eliminated the Ca 2ϩ sensitivity of the TnI-TnC interaction and caused a Ca 2ϩ -unregulated activation of the actin-Tm-activated myosin ATPase activity. This agrees with the postulated earlier (1-3) antiparallel interaction between TnI and/or its inhibitory region and TnC and indicates that the COOH terminus of the inhibitory region of TnI is necessary for maintaining the Ca 2ϩ -specific interactions with the regulatory domain of TnC. Lack of additional activation of actin-Tm-acti- vated myosin ATPase activity in the absence of Ca 2ϩ for TnI-(⌬95-106) suggests that this region is not that critical to maintain the Ca 2ϩ -dependent interactions with TnC. These results support the above conclusion that the inhibitory region of TnI is not functionally uniform and give rise to the question of the structural basis for this functional heterogeneity seen in the NH 2 and COOH termini of the inhibitory region of TnI.
The structure of the inhibitory peptide (residues 96 -115) was presented in a recent study of Hernandez et al. (32). Utilizing CD and NMR spectroscopy, the authors demonstrated a predominantly extended conformation of the free TnI peptide with no significant change on binding to TnC. This is opposite to what was found by Campbell and Sykes (10), who demonstrated that a smaller inhibitory peptide of TnI (residues 104 -115) forms two helical regions (residues 104 -108 and 112-115) upon binding TnC. Important structural information on the interaction between TnI and TnC comes from the crystal structure of TnC complexed with a TnI peptide comprising the first 47 amino acids of TnI (33). This structure of TnI-(1-47)⅐TnC confirmed the antiparallel interaction between the COOH-terminal domain of TnC and the NH 2 -terminal region of TnI and also revealed a more compact organization of TnC in this complex compared with the free state of TnC (34). Based on this newly resolved TnI-(1-47)⅐TnC structure, atomic interactions were modeled between the inhibitory region of TnI-(96 -127) (TnI reg ) and the NH 2 -terminal lobe of TnC, implying that this region of TnI acts as a Ca 2ϩ -sensitive switch in muscle contrac-tion that moves between actin-Tm and the hydrophobic pocket of the NH 2 -terminal domain of TnC (33).
A similar mechanism was proposed by McKay et al. (22) based upon the NMR structure of the regulatory NH 2 -terminal domain of TnC and the elongated inhibitory peptide of TnI containing residues 96 -148. These authors demonstrated that this region of TnI interacts with the NH 2 -terminal domain of TnC in a Ca 2ϩ -sensitive manner and that this regulatory domain of TnC does not undergo a major structural change upon binding to TnI-(96 -148). A large-scale in situ movement of mass within TnI, in response to Ca 2ϩ binding to the regulatory sites of TnC, was observed in the neutron scattering experiments of Stone et al. (35). TnI, when bound to TnC and TnT, underwent a significant conformational change upon binding of Ca 2ϩ to the regulatory Ca 2ϩ -specific sites of TnC. Therefore, the TnI molecule was identified as part of the Ca 2ϩ -sensitive molecular switch during muscle contraction (35,4,5). Modeling of TnI indicated that the rod-like portion of the molecule, containing ϳ35% of the mass, moved closer to the larger oblate ellipsoid portion (65% of the mass) upon Ca 2ϩ binding (35).
Our actomyosin ATPase results and activation instead of inhibition in the absence of Ca 2ϩ for TnT⅐TnC⅐TnI-(⌬105-115) support these interesting findings and suggest that the Ca 2ϩsensitive molecular switch identified by Stone et al. (35) could be located somewhere in the COOH-terminal domain of the inhibitory sequence of TnI. This region would move between its inhibitory position on actin-Tm in the absence of Ca 2ϩ and activate the ATPase activity in the presence of Ca 2ϩ , possibly through an interaction with TnC. Deletion of this region from TnI would result in an activation of the ATPase activity in the absence of Ca 2ϩ or a very impaired relaxation when reconstituted in skinned skeletal muscle fibers. Future studies are planned to investigate the physiological significance of the second interaction site on TnI for actin-Tm and its importance for force development and the Ca 2ϩ regulation of skeletal muscle fibers.  9. Western blotting of TnI⅐TnC-reconstituted fibers performed with the TnI-specific antibodies. After force measurements, the fibers reconstituted with TnI inhibitory region deletion mutants were run on SDS-12.5% polyacrylamide gel and tested for the presence of the TnI mutants with Western blotting and TnI-specific antibodies (mouse anti-TnI IE7 and goat anti-mouse IgG-peroxidase). Under these conditions (12.5% gel), there was no difference in the migration of the TnI deletion mutants.