Conformational changes of troponin C within the thin filaments detected by neutron scattering

Regulation of skeletal and cardiac muscle contraction is associated with structural changes of the thin filament-based proteins, troponin consisting of three subunits (TnC, TnI, and TnT), tropomyosin, and actin, triggered by Ca2+-binding to TnC. Knowledge of in situ structures of these proteins is indispensable for elucidating the molecular mechanism of this Ca2+-sensitive regulation. Here, the in situ structure of TnC within the thin filaments was investigated with neutron scattering, combined with selective deuteration and the contrast matching technique. Deuterated TnC (dTnC) was first prepared, this dTnC was then reconstituted into the native thin filaments, and finally neutron scattering patterns of these reconstituted thin filaments containing dTnC were measured under the condition where non-deuterated components were rendered "invisible" to neutrons. The obtained scattering curves arising only from dTnC showed distinct difference in the absence and presence of Ca2+. These curves were analyzed by model calculations using the Monte Carlo method, in which inter-dTnC interference was explicitly taken into consideration. The model calculation showed that in situ radius of gyration of TnC was 23 A (99% confidence limits between 22 A and 23 A) and 24 A (99% confidence limits between 23 A and 25 A) in the absence and presence of Ca2+, respectively, indicating that TnC within the thin filaments assumes a conformation consistent with the extended dumbbell structure, which is different from the structures found in the crystals of various Tn complexes. Elongation of TnC by binding of Ca2+ was also suggested. Furthermore, the radial position of TnC within the thin filament was estimated to be 53 A (99% confidence limits between 49 A and 57 A) and 49 A (99% confidence limits between 44 A and 53 A) in the absence and presence of Ca2+, respectively, suggesting that this radial movement of TnC by 4A is associated with large conformational changes of the entire Tn molecule by binding of Ca2+.     

Photocrosslinking of benzophenone-labeled single cysteine troponin I mutants to other thin filament proteins

The interaction sites of rabbit skeletal troponin I (TnI) with troponin C (TnC), troponin T (TnT), tropomyosin (Tm) and actin were mapped systematically using nine single cysteine residue TnI mutants with mutation sites at positions 6, 48, 64, 89, 104, 121, 133, 155 or 179 (TnI6, TnI48 etc.). Each mutant was labeled with the heterobifunctional photocrosslinker 4-maleimidobenzophenone (BP-Mal), and incorporated into the TnI.TnC binary complex, the TnI.TnC.TnT ternary troponin (Tn) complex, and the Tn.Tm.F-actin synthetic thin filament. Photocrosslinking reactions carried out in the presence and absence of Ca(2+) yielded the following results: (1) BP-TnI6 photocrosslinked primarily to TnC with a small degree of Ca(2+)-dependence in all the complex forms. (2) BP-TnI48, TnI64 and TnI89 photocrosslinked to TnT with no Ca(2+)-dependence. Photocrosslinking to TnC was reduced in the ternary versus the binary complex. BP-TnI89 also photocrosslinked to actin with higher yields in the absence of Ca(2+) than in its presence. (3) BP-TnI104 and TnI133 photocrosslinked to actin with much higher yields in the absence than in the presence of Ca(2+). (4) BP-TnI121 photocrosslinked to TnC with a small degree of Ca(2+)-dependence, and did not photocrosslink to actin. (5) BP-TnI155 and TnI179 photocrosslinked to TnC, TnT and actin, but all with low yields. All the labeled mutants photocrosslinked to TnC with varying degrees of Ca(2+)-dependence, and none to Tm. These results, along with those published allowed us to construct a structural and functional model of TnI in the Tn complex: in the presence of Ca(2+), residues 1-33 of TnI interact with the C-terminal domain hydrophobic cleft of TnC, approximately 48-89 with TnT, approximately 90-113 with TnC's central helix, approximately 114-125 with TnC's N-terminal domain hydrophobic cleft, and approximately 130-150 with TnC's A-helix. In the absence of Ca(2+), residues approximately 114-125 move out of TnC's N-terminal domain hydrophobic cleft and trigger the movements of residues approximately 89-113 and approximately 130-150 away from TnC and towards actin.     

Differential regulation of the actomyosin interaction by skeletal and cardiac troponin isoforms

There are significant isoform differences between the skeletal and cardiac troponin complexes. Studies of the regulatory properties of these proteins have previously shown only significant differences in the calcium dependence of their regulation. Using a sensitive myosin subfragment 1 (S1) binding assay we show that in the presence of calcium, thin filaments reconstituted with either skeletal or cardiac troponin produce virtually identical S1 binding curves. However in the absence of calcium the S1 binding curves differ considerably. Combined with kinetic measurements, curve fitting to the three-state thin filament regulatory model shows the main difference is that calcium produces a 4-fold change in K(T) (the closed-open equilibrium) for the skeletal system but little change in the cardiac system. The results show a significant difference in the range of regulatory effect between the cardiac and skeletal systems that we interpret as effects upon actin-troponin (Tn)I-TnC binding equilibria. As structural data show that the Ca(2+)-bound TnC structures differ, the additional counter-intuitive result here is that with respect to myosin binding the +Ca(2+) state of the two systems is similar whereas the -Ca(2+) state differs. This shows the regulatory tuning of the troponin complex produced by isoform variation is the net result of a complex series of interactions among all the troponin components.     

Inhibitory region of troponin I: Ca(2+)-dependent structural and environmental changes in the troponin-tropomyosin complex and in reconstituted thin filaments

In muscle thin filaments, the inhibitory region (residues 96-117) of troponin I (TnI) is thought to interact with troponin C (TnC) in the presence of Ca(2+) and with actin in the absence of Ca(2+). To better understand these interactions, we prepared mutant TnIs which contained a single Cys-96 or Cys-117 and labeled them with the thiol-specific fluorescent probe N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine (IAEDANS). We characterized the microenvironments of the AEDANS labels on TnI in the presence and absence of Ca(2+) by measuring the extent of acrylamide quenching of fluorescence and lifetime-resolved anisotropy. In the troponin-tropomyosin (Tn-Tm) complex, the AEDANS labels on both Cys-96 and Cys-117 were less accessible to solvent and less flexible in the presence of Ca(2+), reflecting closer interactions with TnC under these conditions. In reconstituted thin filaments, the environment of the AEDANS on Cys-96 was not greatly affected by Ca(2+), while the AEDANS on Cys-117 was more accessible but significantly less flexible as it moved away from actin and interacted strongly with TnC in the presence of Ca(2+). We used fluorescence resonance energy transfer (FRET) to measure distances between AEDANS on TnI Cys-96 or Cys-117 and 4-?[(dimethylamino)phenyl]azo?phenyl-4'-maleimide (DABmal) on actin Cys-374 in reconstituted thin filaments. In the absence of Ca(2+), the mean distances were 40.2 A for Cys-96 and 35.2 A for Cys-117. In the presence of Ca(2+), Cys-96 moved away from actin Cys-374 by approximately 3.6 A, while Cys-117 moved away by approximately 8 A. This suggests the existence of a flexible "hinge" region near the middle of TnI, allowing amino acid residues in the N-terminal half of TnI to interact with TnC in a Ca(2+)-independent manner, while the C-terminal half of TnI binds to actin in the absence of Ca(2+) or to TnC in the presence of Ca(2+). This is the first report to demonstrate structural movement of the inhibitory region of TnI in the thin filament.     

Ca(2+)- and S1-induced movement of troponin T on reconstituted skeletal muscle thin filaments observed by fluorescence energy transfer spectroscopy

Troponin T (TnT) is an essential component of troponin (Tn) for the Ca(2+)-regulation of vertebrate striated muscle contraction. TnT consists of an extended NH(2)-terminal domain that interacts with tropomyosin (Tm) and a globular COOH-terminal domain that interacts with Tm, troponin I (TnI), and troponin C (TnC). We have generated two mutants of a rabbit skeletal beta-TnT 25-kDa fragment (59-266) that have a unique cysteine at position 60 (N-terminal region) or 250 (C-terminal region). To understand the spatial rearrangement of TnT on the thin filament in response to Ca(2+) binding to TnC, we measured distances from Cys-60 and Cys-250 of TnT to Gln-41 and Cys-374 of F-actin on the reconstituted thin filament by using fluorescence resonance energy transfer (FRET). The distances from Cys-60 and Cys-250 of TnT to Gln-41 of F-actin were 39.5 and 30.0 A, respectively in the absence of Ca(2+), and increased by 2.6 and 5.8 A, respectively upon binding of Ca(2+) to TnC. The rigor binding of myosin subfragment 1 (S1) further increased these distances by 4 and 5 A respectively, when the thin filaments were fully decorated with S1. This indicates that not only the C-terminal but also the N-terminal region of TnT showed the Ca(2+)- and S1-induced movement, and the C-terminal region moved more than N-terminal region. In the absence of Ca(2+), the rigor S1 binding also increased the distances to the same extent as the presence of Ca(2+) when the thin filaments were fully decorated with S1. The addition of ATP completely reversed the changes in FRET induced by rigor S1 binding both in the presence and absence of Ca(2+). However, plots of the extent of S1-induced conformational change vs. molar ratio of S1 to actin showed hyperbolic curve in the presence of Ca(2+) but sigmoidal curve in the absence of Ca(2+). FRET measurement of the distances from Cys-60 and Cys-250 of TnT to Cys-374 of actin showed almost the same results as the case of Gln-41 of actin. The present FRET measurements demonstrated that not only TnI but also TnT change their positions on the thin filament corresponding to three states of thin filaments (relaxed, Ca(2+)-induced or closed, and S1-induced or open states).     

Engineered troponin C constructs correct disease-related cardiac myofilament calcium sensitivity

Aberrant myofilament Ca(2+) sensitivity is commonly observed with multiple cardiac diseases, especially familial cardiomyopathies. Although the etiology of the cardiomyopathies remains unclear, improving cardiac muscle Ca(2+) sensitivity through either pharmacological or genetic approaches shows promise of alleviating the disease-related symptoms. Due to its central role as the Ca(2+) sensor for cardiac muscle contraction, troponin C (TnC) stands out as an obvious and versatile target to reset disease-associated myofilament Ca(2+) sensitivity back to normal. To test the hypothesis that aberrant myofilament Ca(2+) sensitivity and its related function can be corrected through rationally engineered TnC constructs, three thin filament protein modifications representing different proteins (troponin I or troponin T), modifications (missense mutation, deletion, or truncation), and disease subtypes (familial or acquired) were studied. A fluorescent TnC was utilized to measure Ca(2+) binding to TnC in the physiologically relevant biochemical model system of reconstituted thin filaments. Consistent with the pathophysiology, the restrictive cardiomyopathy mutation, troponin I R192H, and ischemia-induced truncation of troponin I (residues 1-192) increased the Ca(2+) sensitivity of TnC on the thin filament, whereas the dilated cardiomyopathy mutation, troponin T ΔK210, decreased the Ca(2+) sensitivity of TnC on the thin filament. Rationally engineered TnC constructs corrected the abnormal Ca(2+) sensitivities of the thin filament, reconstituted actomyosin ATPase activity, and force generation in skinned trabeculae. Thus, the present study provides a novel and versatile therapeutic strategy to restore diseased cardiac muscle Ca(2+) sensitivity.     

Conformational changes induced in troponin I by interaction with troponin T and actin/tropomyosin.

Troponin I (TnI) is the inhibitory component of the striated muscle Ca2+ regulatory protein troponin (Tn). The other two components of Tn are troponin C (TnC), the Ca2+-binding component, and troponin T (TnT), the tropomyosin-binding component. We have used limited chymotryptic digestion to probe the local conformation of TnI in the free state, the binary TnC*TnI complex, the ternary TnC*. TnI*TnT (Tn) complex, and in the reconstituted Tn*tropomyosin*F-actin filament. The digestion of TnI alone or in the TnC*TnI complex produced initially two major fragments via a cleavage of the peptide bond between Phe100 and Asp101 in the so-called inhibitory region. In the ternary Tn complex cleavage occurred at a new site between Leu140 and Lys141. In the absence of Ca2+ this was followed by digestion of the 1-140 fragment at Leu122 and Met116. In the reconstituted thin filament the same fragments as in the case of the ternary complex were produced, but the rate of digestion was slower in the absence than in the presence of Ca2+. These results indicate firstly that in both free TnI and TnI complexed with TnC there is an exposed and flexible site in the inhibitory region. Secondly, TnT affects the conformation of TnI in the inhibitory region and also in the region that contains the 140-141 bond. Thirdly, the 140-141 region of TnI is likely to interact with actin in the reconstituted thin filament when Ca2+ is absent. These findings are discussed in terms of the role of TnI in the mechanism of thin filament regulation, and in light of our previous results [Y. Luo, J.-L. Wu, J. Gergely, T. Tao, Biochemistry 36 (1997) 13449-13454] on the global conformation of TnI.     

The mobility of troponin C and troponin I in muscle

In vertebrate skeletal muscle, contraction is initiated by the elevation of the intracellular Ca²⁺ concentration. The binding of Ca²⁺ to TnC induces a series of conformational changes which ultimately release the inhibition of the actomyosin ATPase activity by Tnl. In this study we have characterized the dynamic behavior of TnC and Tnl in solution, as well as in reconstituted fibers, using EPR and ST-EPR spectroscopy. Cys98 of TnC and Cys133 of Tnl were specifically labeled with malemide spin label (MSL) and indane dione nitroxide spin label (InVSL). In solution, the labeled TnC and Tnl exhibited fast nanosecond motion. MSL-TnC is sensitive to cation binding to the high affinity sites (τ_r increases from 1.5 to 3.7 ns), InVSL-TnC s sensitive to the replacement of Mg²⁺ by Ca²⁺ at these sites (τ_r increase from 1.7 to 6 ns). Upon reconstitution into fibers, the nanosecond mobility is reduced by interactions with other proteins. TnC and Tnl both exhibited microsecond anisotropic motion in fibers similar to that of the actin monomers within the filament. The microsecond motion of TnC was found to be modulated by the binding of Ca²⁺ and by cross-bridge attachment, but this was not the case for the global mobility of Tnl. © 1997 John Wiley & Sons, Ltd.     

Reciprocal coupling between troponin C and myosin crossbridge attachment

The attachment of cycling myosin crossbridges to actin and the resultant muscle contraction are regulated in skeletal muscle by the binding of Ca2+ to the amino-terminal, regulatory sites of the troponin C (TnC) subunit of the thin filament protein troponin. Conversely, the attachment of crossbridges to actin has been shown to alter the affinity of TnC for Ca2+. In this study, fluorescently labeled TnC incorporated into reconstituted thin filaments was used to investigate the relationship between crossbridge attachment to actin and structural changes in the amino-terminal region of TnC. Fluorescence intensity changes were measured under the following conditions: saturating [Ca2+] in the absence of crossbridges, rigor crossbridge attachment in the presence and absence of Ca2+, and cycling crossbridge attachment. The percent of heavy meromyosin crossbridges associated with the thin filaments under these conditions was also determined. The results show that, in addition to the binding of Ca2+ to TnC, the attachment of both rigor and cycling crossbridges to actin alters the structure of TnC near the regulatory, Ca2+-specific sites of the molecule. A differential coupling between weakly versus strongly bound crossbridge states and TnC structure was detected, suggesting a possible differential regulation of these states by conformational changes in TnC. These findings illustrate a reciprocal coupling, via thin filament protein interactions, between structural changes in TnC and the attachment of myosin crossbridges to actin, such that each can influence the other, and indicate that TnC is not simply an on-off switch but may exist in a number of different conformations.     

Ca(2+)-dependent, myosin subfragment 1-induced proximity changes between actin and the inhibitory region of troponin I.

In order to help understand the spatial rearrangements of thin filament proteins during the regulation of muscle contraction, we used fluorescence resonance energy transfer (FRET) to measure Ca(2+)-dependent, myosin-induced changes in distances and fluorescence energy transfer efficiencies between actin and the inhibitory region of troponin I (TnI). We labeled the single Cys-117 of a mutant TnI with N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine (IAEDANS) and Cys-374 of actin with 4-dimethylaminophenylazophenyl-4'-maleimide (DABmal). These fluorescent probes were used as donor and acceptor, respectively, for the FRET measurements. We reconstituted a troponin-tropomyosin (Tn-Tm) complex which contained the AEDANS-labeled mutant TnI, together with natural troponin T (TnT), troponin C (TnC) and tropomyosin (Tm) from rabbit fast skeletal muscle. Fluorescence titration of the AEDANS-labeled Tn-Tm complex with DABmal-labeled actin, in the presence and absence of Ca(2+), resulted in proportional, linear increases in energy transfer efficiency up to a 7:1 molar excess of actin over Tn-Tm. The distance between AEDANS on TnI Cys-117 and DABmal on actin Cys-374 increased from 37.9 A to 44.1 A when Ca(2+) bound to the regulatory sites of TnC. Titration of reconstituted thin filaments, containing AEDANS-labeled Tn-Tm and DABmal-labeled actin, with myosin subfragment 1 (S1) decreased the energy transfer efficiency, in both the presence and absence of Ca(2+). The maximum decrease occurred at well below stoichiometric levels of S1 binding to actin, showing a cooperative effect of S1 on the state of the thin filaments. S1:actin molar ratios of approximately 0.1 in the presence of Ca(2+), and approximately 0.3 in the absence of Ca(2+), were sufficient to cause a 50% reduction in normalized transfer efficiency. The distance between AEDANS on TnI Cys-117 and DABmal on actin Cys-374 increased by approximately 7 A in the presence of Ca(2+) and by approximately 2 A in the absence of Ca(2+) when S1 bound to actin. Our results suggest that TnI's interaction with actin inhibits actomyosin ATPase activity by modulating the equilibria among active and inactive states of the thin filament. Structural rearrangements caused by myosin S1 binding to the thin filament, as detected by FRET measurements, are consistent with the cooperative behavior of the thin filament proteins.     

Thin filament regulation and ionic interactions between the N-terminal region in actin and troponin

The N-terminal region in actin has been shown to interact with both myosin and troponin (Tn) during the cross-bridge cycle and in regulation. To study the role of this region in regulation, we used yeast actin mutants with increased and decreased numbers of acidic residues. The mutants included D24A/D25A, with Asp(24) and Asp(25) replaced with alanines; DNEQ, with the substitution of Asp(2) and Glu(4) with their amide analogs; and 4Ac, with Glu(3) and Asp(4) inserted in lieu of Ser(3). In the in vitro motility assay, using reconstituted regulated thin filaments, the sliding speeds of DNEQ, D24A/D25A, and 4Ac were similar at all pCa values. Thus, Ca(2+)-sensitivity of the thin filaments and the inhibitory function of TnI appear to be insensitive to changes in charge (+/-2) at the N-terminus of actin, suggesting little, if any, role of that actin region in regulation. A Ca(2+)-independent conformational change in that region was detected upon troponin binding to actin-Tm via an increase in the fluorescence of a pyrene probe attached to another yeast actin mutant that we used (Cys(1)).     

Binding of troponin I and the troponin I-troponin C complex to actin-tropomyosin. Dissociation by myosin subfragment 1

Troponin I (TnI) is the component of the troponin complex, TnI, TnC, TnT, that is responsible for inhibition of actomyosin ATPase activity. Using the fluorescence of pyrene-labeled tropomyosin (Tm), we probed the interaction of TnI and TnIC with Tm on the reconstituted muscle thin filament. The results indicate that TnI and TnIC(-Ca(2+)) bind specifically and strongly to actin-Tm with a stoichiometry of 1 TnI or 1 TnIC/1 Tm/7 actin, in agreement with previous results. The binding of myosin heads (S1) to actin-Tm at low levels of saturation caused TnI and TnIC to dissociate from actin-Tm. These results are interpreted in terms of the S1-binding state allosteric-cooperative model of the actin-Tm thin filament, closed/open. Thus, TnI and TnIC(-Ca(2+)) bind to the closed state of actin-Tm and their binding is greatly weakened in the S1-induced open state, indicating that they act as allosteric inhibitors. The fluorescence change and the stoichiometry indicate that the TnI-binding site is composed of regions from both actin and Tm probably in the vicinity of Cys 190.     

Time-resolved fluorescence study of the single tryptophans of engineered skeletal muscle troponin C.

The regulatory domain of troponin C (TnC) from chicken skeletal muscle was studied using genetically generated mutants which contained a single tryptophan at positions 22, 52, and 90. The quantum yields of Trp-22 are 0.33 and 0.25 in the presence of Mg2+ (2-Mg state) and Ca2+ (4-Ca state), respectively. The large quantum yield of the 2-Mg state is due to a relatively small nonradiative decay rate and consistent with the emission peak at 331 nm. The intensity decay of this state is monoexponential with a single lifetime of 5.65 ns, independent of wavelength. In the 4-Ca state, the decay is biexponential with the mean of the two lifetimes increasing from 4.54 to 4.92 ns across the emission band. The decay-associated spectrum of the short lifetime is red-shifted by 19 nm relative to the steady-state spectrum. The decay of Trp-52 is biexponential in the 2-Mg state and triexponential in the 4-Ca state. The decay of Trp-90 requires three exponential terms for a satisfactory fit, but can be fitted with two exponential terms in the 4-Ca state. The lower quantum yields (< 0.15) of these two tryptophans are due to a combination of smaller radiative and larger nonradiative decay rates. The results from Trp-22 suggest a homogeneous ground-state indole ring in the absence of bound Ca2+ at the regulatory sites and a ground-state heterogeneity induced by activator Ca2+. The Ca(2+)-induced environmental changes of Trp-52 and Trp-90 deviate from those predicted by a modeled structure of the 4-Ca state. The anisotropy decays of all three tryptophans show two rotational correlation times. The long correlation times (phi 1 = 8.1-8.3 ns) derived from Trp-22 and Trp-90 suggest an asymmetric hydrodynamic shape. TnC becomes more asymmetric upon binding activator Ca2+ (phi 1 = 10.1-11.6 ns). The values of phi 1 obtained from Trp-52 are 3-4 ns shorter than those from Trp-22 and Trp-90, and these reduced correlation times may be related to the mobility of the residue and/or local segmental flexibility.     

Calcium structural transition of human cardiac troponin C in reconstituted muscle fibres as studied by site-directed spin labelling

The in situ structure of human cardiac troponin C (hcTnC) has been studied with site-directed, spin labelling, electron paramagnetic resonance (SDSL-EPR). Analysis of the in situ structures of hcTnC is essential for elucidating the molecular mechanism behind its Ca(2+)-sensitive regulation. We prepared two hcTnC mutants (C35S and C84S) containing one native cysteine residue (84 and 35, respectively) for spin labelling. The mutants were labelled with a methane thiosulfonate spin label (MTSSL) and the TnC was reconstituted into permeabilized muscle fibres. The mobility of Cys84-MTSSL changed markedly after addition of Ca2+, while that of the Cys35 residue did not change in the monomer state or in fibres. The rotational correlation time of Cys84-MTSSL decreased from 32ns to 13ns upon Ca(2+)-binding in the monomer state, whereas in fibres the spectrum of Cys84-MTSSL was resolved into mobile (16ns) and immobile (35ns) components and the addition of Ca2+ increased the immobile component. Moreover, the accessibility of Cys84-MTSSL to molecular oxygen increased slightly in the presence of Ca2+. These data suggest that Cys35 remains in the same location regardless of the addition of Ca2+, whereas Cys84 is located at the position that interacts with B and C helices of hcTnC and interacts with troponin I (TnI) at high concentrations of Ca2+. We determined the distances between Cys35 and Cys84 by measuring pulsed electron-electron double resonance spectra. The distances were 26.0 angstroms and 27.2 angstroms in the monomer state and in fibres, respectively, and the addition of Ca2+ decreased the distance to 23.2 angstroms in fibres but only slightly in the monomer state, showing that Ca2+ binding to the N-domain of hcTnC induced a larger structural change in muscle fibres than in the monomer state.     

Fluorescence spectroscopic analysis of the proximity changes between the central helix of troponin C and the C-terminus of troponin T from chicken skeletal muscle

Recent structural studies of the troponin (Tn) core complex have shown that the regulatory head containing the N-lobe of TnC is connected to the IT arm by a flexible linker of TnC. The IT arm is a long coiled-coil formed by alpha-helices of TnI and TnT, plus the C-lobe of TnC. The TnT is thought to play a pivotal role in the linking of Ca(2+) -triggered conformational changes in thin filament regulatory proteins to the activation of cross-bridge cycling. However, a functional domain at the C-terminus of TnT is missing from the Tn core complex. In this study, we intended to determine the proximity relationship between the central helix of TnC and the TnT C-terminus in the binary and the ternary complex with and without Ca2+ by using pyrene excimer fluorescence spectroscopy and fluorescence resonance energy transfer. Chicken fast skeletal TnC contains a Cys102 at the E helix, while TnT has a Cys264 at its C-terminus. These two cysteines were specifically labeled with sulfhydryl-reactive fluorescence probes. The measured distance in the binary complex was about 19 Angstroms and slightly increased when they formed the ternary complex with TnI (20 Angstroms). Upon Ca2+ binding the distance was not affected in the binary complex but increased by approximately 4 Angstroms in the ternary complex. These results suggest that TnI plays an essential role in the Ca(2+) -mediated change in the spatial relationship between the C-lobe of TnC and the C-terminus of TnT.     

Force-calcium relationship depends on myosin heavy chain and troponin isoforms in rat diaphragm muscle fibers.

The present study examined Ca(2+) sensitivity of diaphragm muscle (Dia(m)) fibers expressing different myosin heavy chain (MHC) isoforms. We hypothesized that Dia(m) fibers expressing the MHC(slow) isoform have greater Ca(2+) sensitivity than fibers expressing fast MHC isoforms and that this fiber-type difference in Ca(2+) sensitivity reflects the isoform composition of the troponin (Tn) complex (TnC, TnT, and TnI). Studies were performed in single Triton-X-permeabilized Dia(m) fibers. The Ca(2+) concentration at which 50% maximal force was generated (pCa(50)) was determined for each fiber. SDS-PAGE and Western analyses were used to determine the MHC and Tn isoform composition of single fibers. The pCa(50) for Dia(m) fibers expressing MHC(slow) was significantly greater than that of fibers expressing fast MHC isoforms, and this greater Ca(2+) sensitivity was associated with expression of slow isoforms of the Tn complex. However, some Dia(m) fibers expressing MHC(slow) contained the fast TnC isoform. These results suggest that the combination of TnT, TnI, and TnC isoforms may determine Ca(2+) sensitivity in Dia(m) fibers.     

A disulfide crosslink between Cys98 of troponin-C and Cys133 of troponin-I abolishes the activity of rabbit skeletal troponin.

Various thio-reactive bifunctional crosslinkers as well as 5,5'-dithiobis(2-nitrobenzoate)-mediated disulfide bond formation were used to crosslink troponin-C and troponin-I, the Ca(2+)-binding and inhibitory subunits of troponin, respectively. In all cases, substantial crosslinking was obtained when the reactions were carried out in the absence of Ca2+. No disulfide crosslinking occurred if either Cys98 of TnC, or Cys133 of TnI were blocked, indicating that these thiols are involved in the crosslinking. Troponin containing the disulfide crosslink is no longer capable of regulating actomyosin ATPase activity in a Ca(2+)-dependent manner. Our results suggest that the relative movement between the Cys98 region of TnC and the Cys133 region of TnI is required for the Ca(2+)-regulatory process in skeletal muscle.     

Orientational information of troponin C within the thin filaments obtained by neutron fiber diffraction

In striated muscles contraction is regulated by the thin filament-based proteins, troponin consisting of three subunits (TnC, TnI, and TnT), and tropomyosin. Knowledge of in situ structures of these proteins is indispensable for elucidating this Ca(2+)-sensitive regulatory mechanism. We employed neutron scattering to investigate the structure of TnC within the thin filament, and found that TnC assumes extended dumbbell-like structures and moves toward the filament axis by binding of Ca(2+). Here, in order to obtain more detailed in situ structural information of TnC, neutron fiber diffraction measurements were performed. Sols of native thin filaments and the thin filaments containing deuterated TnC were prepared in (2)H(2)O. The oriented samples were obtained by placing these sols sealed in quartz capillaries with a diameter of 3 mm in a magnetic field of 18 Tesla. Neutron fiber diffraction patterns were obtained from these oriented samples in the absence and presence of Ca(2+). The patterns obtained showed strong equatorial diffraction due to the thin filaments, 59 A and 51 A layer-lines due to actin, and meridional reflections due to Tn-complex. Analysis of the meridional reflections due to Tn-complex with aid of model calculation showed that the angle between the thin filament axis and the long axis of TnC was estimated to be 67(+/-7) degrees and 49(+/-17) degrees , in the absence and presence of Ca(2+), respectively, suggesting that TnC, which assumes orientations rather perpendicular to the filament axis in the absence of Ca(2+), tilts toward the filament axis and the orientational and positional disorder increases by binding Ca(2+). It also showed that the relative position of the TnC moved by about 22 A by binding Ca(2+), and this apparent movement was concomitant with the movements of other Tn-subunits. This implies that by binding Ca(2+), significant structural rearrangements of Tn-subunits occur.     

Structural transition of the inhibitory region of troponin I within the regulated cardiac thin filament

Contraction and relaxation of cardiac muscle are regulated by the inhibitory and regulatory regions of troponin I (cTnI). Our previous FRET studies showed that the inhibitory region of cTnI in isolated troponin experiences a structural transition from a beta-turn/coil motif to an extended conformation upon Ca(2+) activation. During the relaxation process, the kinetics of the reversal of this conformation is coupled to the closing of the Ca(2+)-induced open conformation of the N-domain of troponin C (cTnC) and an interaction between cTnC and cTnI in their interface. We have since extended the structural kinetic study of the inhibitory region to fully regulated thin filament. Single-tryptophan and single-cysteine mutant cTnI(L129W/S151C) was labeled with 1,5-IAEDANS at Cys151, and the tryptophan-AEDANS pair served as a donor-acceptor pair. Labeled cTnI mutant was used to prepare regulated thin filaments. Ca(2+)-induced conformational changes in the segment of Trp129-Cys151 of cTnI were monitored by FRET sensitized acceptor (AEDANS) emission in Ca(2+) titration and stopped-flow measurements. Control experiments suggested energy transfer from endogenous tryptophan residues of actin and myosin S1 to AEDANS attached to Cys151 of cTnI was very small and Ca(2+) independent. The present results show that the rate of Ca(2+)-induced structural transition and Ca(2+) sensitivity of the inhibitory region of cTnI were modified by (1) thin filament formation, (2) the presence of strongly bound S1, and (3) PKA phosphorylation of the N-terminus of cTnI. Ca(2+) sensitivity was not significantly changed by the presence of cTm and actin. However, the cTn-cTm interaction decreased the cooperativity and kinetics of the structural transition within cTnI, while actin filaments elicited opposite effects. The strongly bound S1 significantly increased the Ca(2+) sensitivity and slowed down the kinetics of structural transition. In contrast, PKA phosphorylation of cTnI decreased the Ca(2+) sensitivity and accelerated the structural transition rate of the inhibitory region of cTnI on thin filaments. These results support the idea of a feedback mechanism by strong cross-bridge interaction with actin and provide insights on the molecular basis for the fine tuning of cardiac function by beta-adrenergic stimulation.     

Structural basis for Ca2+-regulated muscle relaxation at interaction sites of troponin with actin and tropomyosin

Troponin and tropomyosin on actin filaments constitute a Ca2+-sensitive switch that regulates the contraction of vertebrate striated muscle through a series of conformational changes within the actin-based thin filament. Troponin consists of three subunits: an inhibitory subunit (TnI), a Ca2+-binding subunit (TnC), and a tropomyosin-binding subunit (TnT). Ca2+-binding to TnC is believed to weaken interactions between troponin and actin, and triggers a large conformational change of the troponin complex. However, the atomic details of the actin-binding sites of troponin have not been determined. Ternary troponin complexes have been reconstituted from recombinant chicken skeletal TnI, TnC, and TnT2 (the C-terminal region of TnT), among which only TnI was uniformly labelled with 15N and/or 13C. By applying NMR spectroscopy, the solution structures of a "mobile" actin-binding domain (approximately 6.1 kDa) in the troponin ternary complex (approximately 52 kDa) were determined. The mobile domain appears to tumble independently of the core domain of troponin. Ca2+-induced changes in the chemical shift and line shape suggested that its tumbling was more restricted at high Ca2+ concentrations. The atomic details of interactions between actin and the mobile domain of troponin were defined by docking the mobile domain into the cryo-electron microscopy (cryo-EM) density map of thin filament at low [Ca2+]. This allowed the determination of the 3D position of residue 133 of TnI, which has been an important landmark to incorporate the available information. This enabled unique docking of the entire globular head region of troponin into the thin filament cryo-EM map at a low Ca2+ concentration. The resultant atomic model suggests that troponin interacted electrostatically with actin and caused the shift of tropomyosin to achieve muscle relaxation. An important feature is that the coiled-coil region of troponin pushed tropomyosin at a low Ca2+ concentration. Moreover, the relationship between myosin and the mobile domain on actin filaments suggests that the latter works as a fail-safe latch.