A modulatory role for the troponin T tail domain in thin filament regulation

In striated muscle the force generating acto-myosin interaction is sterically regulated by the thin filament proteins tropomyosin and troponin (Tn), with the position of tropomyosin modulated by calcium binding to troponin. Troponin itself consists of three subunits, TnI, TnC, and TnT, widely characterized as being responsible for separate aspects of the regulatory process. TnI, the inhibitory unit is released from actin upon calcium binding to TnC, while TnT performs a structural role forming a globular head region with the regulatory TnI- TnC complex with a tail anchoring it within the thin filament. We have examined the properties of TnT and the TnT(1) tail fragment (residues 1-158) upon reconstituted actin-tropomyosin filaments. Their regulatory effects have been characterized in both myosin S1 ATPase and S1 kinetic and equilibrium binding experiments. We show that both inhibit the actin-tropomyosin-activated S1 ATPase with TnT(1) producing a greater inhibitory effect. The S1 binding data show that this inhibition is not caused by the formation of the blocked B-state but by significant stabilization of the closed C-state with a 10-fold reduction in the C- to M-state equilibrium, K(T), for TnT(1). This suggests TnT has a modulatory as well as structural role, providing an explanation for its large number of alternative isoforms.     

The regulation of myosin binding to actin filaments by Lethocerus troponin

Lethocerus indirect flight muscle has two isoforms of troponin C, TnC-F1 and F2, which are unusual in having only a single C-terminal calcium binding site (site IV, isoform F1) or one C-terminal and one N-terminal site (sites IV and II, isoform F2). We show here that thin filaments assembled from rabbit actin and Lethocerus tropomyosin (Tm) and troponin (Tn) regulate the binding of rabbit myosin to rabbit actin in much the same way as the mammalian regulatory proteins. The removal of calcium reduces the rate constant for S1 binding to regulated actin about threefold, independent of which TmTn is used. This is consistent with calcium removal causing the TmTn to occupy the B or blocked state to about 70% of the total. The mid point pCa for the switch differed for TnC-F1 and F2 (pCa 6.9 and 6.0, respectively) consistent with the reported calcium affinities for the two TnCs. Equilibrium titration of S1 binding to regulated actin filaments confirms calcium regulated binding of S1 to actin and shows that in the absence of calcium the three actin filaments (TnC-F1, TnC-F2 and mammalian control) are almost indistinguishable in terms of occupancy of the B and C states of the filament. In the presence of calcium TnC-F2 is very similar to the control with approximately 80% of the filament in the C-state and 10-15% in the fully on M-State while TnC-F1 has almost 50% in each of the C and M states. This higher occupancy of the M-state for TnC-F1, which occurs above pCa 6.9, is consistent with this isoform being involved in the calcium activation of stretch activation. However, it leaves unanswered how a C-terminal calcium binding site of TnC can activate the thin filament.     

Differences between Cardiac and Skeletal Troponin Interaction with the Thin Filament Probed by Troponin Exchange in Skeletal Myofibrils

Troponin (Tn) is the calcium-sensing protein of the thin filament. Although cardiac troponin (cTn) and skeletal troponin (sTn) accomplish the same function, their subunit interactions within Tn and with actin-tropomyosin are different. To further characterize these differences, myofibril ATPase activity as a function of pCa and labeled Tn exchange in rigor myofibrils was used to estimate Tn dissociation rates from the nonoverlap and overlap region as a function of pCa. Measurement of ATPase activity showed that skeletal myofibrils containing >96% cTn had a higher pCa 9 ATPase activity than, but similar pCa 4 activity to, sTn-containing myofibrils. Analysis of the pCa-ATPase activity relation showed that cTn myofibrils were more calcium sensitive but less cooperative (pCa₅₀ = 6.14, n_H = 1.46) than sTn myofibrils (pCa₅₀ = 5.90, n_H = 3.36). The time course of labeled Tn exchange at pCa 9 and 4 were quite different between cTn and sTn. The apparent cTn dissociation rates were ∼2-10-fold faster than sTn under all the conditions studied. The apparent dissociation rates for cTn were 5 × 10⁻³ min⁻¹, 150 × 10⁻³ min⁻¹, and 260 × 10⁻³ min⁻¹, whereas for sTn they were 0.6 × 10⁻³ min⁻¹, 88 × 10⁻³ min⁻¹, and 68 × 10⁻³ min⁻¹ for the nonoverlap region at pCa 9, nonoverlap region at pCa 4, and overlap region at pCa 4, respectively. Normalization of the apparent dissociation rates gives 1:30:50 for cTn compared with 1:150:110 for sTn (nonoverlap at pCa 9:nonoverlap at pCa 4:overlap at pCa 4) suggesting that calcium has a smaller influence, whereas strong cross-bridges have a larger influence on cTn dissociation compared with sTn. The higher cTn dissociation rate in the nonoverlap region and ATPase activity at pCa 9 suggest that it gives a less off or inactive thin filament. Analysis of the intensity ratio (after a short time of exchange) as a function of pCa showed that cTn had greater calcium sensitivity but lower cooperativity than sTn. In addition, the magnitude of the change in intensity ratio going from pCa 9 to 4 was less for cTn than sTn. These data suggest that the influence of calcium on cTn exchange is less than sTn even though calcium can activate ATPase activity to a similar extent in cTn compared with sTn myofibrils. This may be explained partially by cTn being less off or inactive at pCa 9. Modeling of the intensity profiles obtained after Tn exchange at pCa 5.8 suggest that the profiles are best explained by a model that includes a long-range cross-bridge effect that grades with distance from the rigor cross-bridge for both cTn and sTn.     

A kinetic model of troponin dissociation in relation to thin filament regulation in striated muscle

The apparent rate of troponin (Tn) dissociation from myofibrils has been used as a method to study thin filament regulation in striated muscle. The rate is dependent upon calcium and strong crossbridges and supports the three-state model for thin filament regulation. The dissociation rate of Tn is extremely low so it is not intuitively clear that such a slow process would probe thin filament regulation. We have investigated this issue by developing a simple kinetic model to explain the Tn dissociation rate measured by labeled Tn exchange in the myofibrils. Tn is composed of three interacting subunits, TnC, TnI and TnT. In our model, TnI’s regulatory domain switches from actin-tropomyosin to TnC followed by TnT dissociation from actin-tropomyosin. This TnI regulatory domain switching is linked to the transition of the thin filament from the blocked state to the closed state. It is calcium dependent and several orders of magnitude faster than TnT dissociation from actin-tropomyosin. By integrating the dimensionless rate equations of this model, we have computed the time course of each of the various components. In our numerical simulations, the rate constant for TnI switching from actin-tropomyosin to TnC was varied from 10 s^?1 to 1000 s^?1 to simulate the low calcium, blocked state to high calcium, closed state. The computed progress curves for labeled Tn exchange into the myofibrils and the derived intensity ratio between the non-overlap and overlap regions well explains the intensity ratio progress curves observed experimentally. These numerical simulations and experimental observations reveal that the apparent rate of Tn dissociation probes the blocked state to closed state equilibrium of the myofibrillar thin filament.     

The effect of partial extraction of troponin C on the elementary steps of the cross-bridge cycle in rabbit psoas muscle fibers.

The elementary steps of the cross-bridge cycle in which troponin C (TnC) was partially extracted were investigated by sinusoidal analysis in rabbit psoas muscle fibers. The effects of MgATP and phosphate on the rate constants of exponential processes were studied at 200 mM ionic strength, pCa 4.20, pH 7.00, and at 20 degrees C. The results were analyzed with the following cross-bridge scheme: [formula: see text] where A is actin, M is myosin, S is MgATP, D is MgADP, and P is phosphate (Pi). When TnC was extracted so that the average remaining tension was 11% (range 8-15%), K1 (MgATP association constant) increased to 7x, k2 (rate constant of cross-bridge detachment) increased to 1.55x, k-2 (reversal of detachment) decreased to 0.27x, and K2 (= k2/k-2: equilibrium constant of cross-bridge detachment) increased to 6.6x, k4 (rate constant of force generation) decreased to 0.4x, k-4 (reversal of force generation) increased to 2x, K4 (= k4/k-4) decreased to 0.17x, and K5 (Pi association constant) did not change. The activation factor alpha, which represents the fraction of cross-bridges participating in the cycling, decreased from 1 to 0.14 with TnC extraction. The fact that K1 increased with TnC extraction implies that the condition of the thin filament modifies the contour of the substrate binding site on the myosin head and is consistent with the Fenn effect. The fact that alpha decreased to 0.14 is consistent with the steric blocking mechanism (recruitment hypothesis) and indicates that some of the cross-bridges disappear from the active cycling pool. The fact that the equilibrium constants changed is consistent with the cooperative activation mechanism (graded activation hypothesis) among thin-filament regulatory units that consist of troponin (TnC, Tnl, TnT), tropomyosin, and seven actin molecules, and possibly include cross-bridges.     

Structural Basis for the Activation of Muscle Contraction by Troponin and Tropomyosin

The molecular regulation of striated muscle contraction couples the binding and dissociation of Ca²⁺ on troponin (Tn) to the movement of tropomyosin on actin filaments. In turn, this process exposes or blocks myosin binding sites on actin, thereby controlling myosin crossbridge dynamics and consequently muscle contraction. Using 3D electron microscopy, we recently provided structural evidence that a C-terminal extension of TnI is anchored on actin at low Ca²⁺ and competes with tropomyosin for a common site to drive tropomyosin to the B-state location, a constrained, relaxing position on actin that inhibits myosin-crossbridge association. Here, we show that release of this constraint at high Ca²⁺ allows a second segment of troponin, probably representing parts of TnT or the troponin core domain, to promote tropomyosin movement on actin to the Ca²⁺-induced C-state location. With tropomyosin stabilized in this position, myosin binding interactions can begin. Tropomyosin appears to oscillate to a higher degree between respective B- and C-state positions on troponin-free filaments than on fully regulated filaments, suggesting that tropomyosin positioning in both states is troponin-dependent. By biasing tropomyosin to either of these two positions, troponin appears to have two distinct structural functions; in relaxed muscles at low Ca²⁺, troponin operates as an inhibitor, while in activated muscles at high Ca²⁺, it acts as a promoter to initiate contraction.     

Influence of ADP on cross-bridge-dependent activation of myofibrillar thin filaments

Contraction of skeletal muscle is regulated by calcium at the level of the thin filament via troponin and tropomyosin. Studies have indicated that strong cross-bridge binding is also involved in activation of the thin filament. To further test this, myofibrils were incubated with a wide range of fluorescent myosin subfragment 1(fS1) at pCa 9 or pCa 4 with or without ADP. Sarcomere fluorescence intensity and the fluorescence intensity ratio (non-overlap region/overlap region) were measured to determine the amount and location of bound fS1 in the myofibril. There was lower sarcomere fluorescence intensity with ADP compared to without ADP for both calcium levels. Similar data were obtained from biochemical measures of bound fS1, validating the fluorescence microscopy measurements. The intensity ratio, which is related to activation of the thin filament, increased with increasing [fS1] with or without ADP. At pCa 9, the fluorescence intensity ratio was constant until 80-160 nM fS1 without ADP conditions, then it went up dramatically and finally attained saturation. The dramatic shift of the ratio demonstrated the cooperative character of strong cross-bridge binding, and this was not observed at high calcium. A similar pattern was observed with ADP in that the ratio was right-shifted with respect to total [fS1]. Saturation was obtained with both the fluorescence intensity and ratio data. Plots of intensity ratio as a function of normalized sarcomere intensity (bound fS1) showed little difference between with and without ADP. This suggests that the amount of strongly bound fS1, not fS1 state (with or without ADP) is related to activation of the thin filament.     

Role of Ca2+ and cross-bridges in skeletal muscle thin filament activation probed with Ca2+ sensitizers

Thin filament regulation of contraction is thought to involve the binding of two activating ligands: Ca2+ and strongly bound cross-bridges. The specific cross-bridge states required to promote thin filament activation have not been identified. This study examines the relationship between cross-bridge cycling and thin filament activation by comparing the results of kinetic experiments using the Ca2+ sensitizers caffeine and bepridil. In single skinned rat soleus fibers, 30 mM caffeine produced a leftward shift in the tension-pCa relation from 6.03 +/- 0.03 to 6.51 +/- 0.03 pCa units and lowered the maximum tension to 0.60 +/- 0.01 of the control tension. In addition, the rate of tension redevelopment (ktr) was decreased from 3.51 +/- 0.12 s-1 to 2.70 +/- 0.19 s-1, and Vmax decreased from 1.24 +/- 0.07 to 0.64 +/- 0.02 M.L./s. Bepridil produced a similar shift in the tension-pCa curves but had no effect on the kinetics. Thus bepridil increases the Ca2+ sensitivity through direct effects on TnC, whereas caffeine has significant effects on the cross-bridge interaction. Interestingly, caffeine also produced a significant increase in stiffness under relaxing conditions (pCa 9.0), indicating that caffeine induces some strongly bound cross-bridges, even in the absence of Ca2+. The results are interpreted in terms of a model integrating cross-bridge cycling with a three-state thin-filament activation model. Significantly, strongly bound, non-tension-producing cross-bridges were essential to modeling of complete activation of the thin filament.     

Effect of rigor and cycling cross-bridges on the structure of troponin C and on the Ca2+ affinity of the Ca2+-specific regulatory sites in skinned rabbit psoas fibers

Intrinsic troponin C (TnC) was extracted from small bundles of rabbit psoas fibers and replaced with TnC labeled with dansylaziridine (5-dimethylaminonaphthalene-1-sulfonyl). The flourescence of incorporated dansylaziridine-labeled TnC was enhanced by the binding of Ca2+ to the Ca2+-specific (regulatory) sites of TnC and was measured simultaneously with force (Zot, H.G., Güth, K., and Potter, J.D. (1986) J. Biol. Chem. 261, 15883-15890). Various myosin cross-bridge states also altered the fluorescence of dansylaziridine-labeled TnC in the filament, with cycling cross-bridges having a greater effect than rigor cross-bridges; and in both cases, there was an additional effect of Ca2+. The paired fluorescence and tension data were used to calculate the apparent Ca2+ affinity of the regulatory sites in the thin filament and were shown to increase at least 10-fold during muscle activation presumably due to the interaction of cycling cross-bridges with the thin filament. The cross-bridge state responsible for this enhanced Ca2+ affinity was shown to be the myosin-ADP state present only when cross-bridges are cycling. The steepness of the pCa force curves (where pCa represents the -log of the free Ca2+ concentration) obtained in the presence of ATP at short and long sarcomere lengths was the same, suggesting that cooperative interactions between adjacent troponin-tropomyosin units may spread along much of the actin filament when cross-bridges are attached to it. In contrast to the cycling cross-bridges, rigor bridges only increased the Ca2+ affinity of the regulatory sites 2-fold. Taken together, the results presented here indicate a strong coupling between the Ca2+ regulatory sites and cross-bridge interactions with the thin filament.     

Spectroscopic and ITC study of the conformational change upon Ca2+-binding in TnC C-lobe and TnI peptide complex from Akazara scallop striated muscle

Akazara scallop (Chlamys nipponensis akazara) troponin C (TnC) of striated adductor muscle binds only one Ca²⁺ ion at the C-terminal EF-hand motif (Site IV), but it works as the Ca²⁺-dependent regulator in adductor muscle contraction. In addition, the scallop troponin (Tn) has been thought to regulate muscle contraction via activating mechanisms that involve the region spanning from the TnC C-lobe (C-lobe) binding site to the inhibitory region of the TnI, and no alternative binding of the TnI C-terminal region to TnC because of no similarity between second TnC-binding regions of vertebrate and the scallop TnIs. To clarify the Ca²⁺-regulatory mechanism of muscle contraction by scallop Tn, we have analyzed the Ca²⁺-binding properties of the complex of TnC C-lobe and TnI peptide, and their interaction using isothermal titration microcalorimetry, nuclear magnetic resonance, circular dichroism, and gel filtration chromatography. The results showed that single Ca²⁺-binding to the Site IV leads to a structural transition not only in Site IV but also Site III through the structural network in the C-lobe of scallop TnC. We therefore assumed that the effect of Ca²⁺-binding must lead to a change in the interaction mode between the C-lobe of TnC and the TnI peptide. The change should be the first event of the transmission of Ca²⁺ signal to TnI in Tn ternary complex.     

Kinetic analysis of the interactions between troponin C (TnC) and troponin I (TnI) binding peptides: evidence for separate binding sites for the 'structural' N-terminus and the 'regulatory' C-terminus of TnI on TnC

The Ca(2+)/Mg(2+)-dependent interactions between TnC and TnI play a critical role in regulating the 'on' and 'off' states of muscle contraction as well as maintaining the structural integrity of the troponin complex in the off state. In the present study, we have investigated the binding interactions between the N-terminus of TnI (residues 1-40 of skeletal TnI) and skeletal TnC in the presence of Ca(2+) ions, Mg(2+) ions and in the presence of the C-terminal regulatory region peptides: TnI(96-115), TnI(96-131) and TnI(96-139). Our results show the N-terminus of TnI can bind to TnC with high affinity in the presence of Ca(2+) or Mg(2+) ions with apparent equilibrium dissociation constants of K(d(Ca(2+) ) ) = 48 nM and K(d(Mg(2+) ) ) = 29 nM. The apparent association and dissociation rate constants for the interactions were, k(on) = 4.8 x 10(5) M (-1) s(-1), 3.4 x 10(5) M (-1) s(-1) and k(off) = 2.3 x 10(-2) s(-1), 1.0 x 10(-2) s(-1) for TnC(Ca(2+)) and TnC(Mg(2+)) states, respectively. Competition studies between each of the TnI regions and TnC showed that both TnI regions can bind simultaneously to TnC while native gel electrophoresis and SEC confirmed the formation of stable ternary complexes between TnI(96-139) (or TnI(96-131)) and TnC-TnI(1-40). Further analysis of the binding interactions in the ternary complex showed the binding of the TnI regulatory region to TnC was critically dependent upon the presence of both TnC binding sites (i.e. TnI(96-115) and TnI(116-131)) and the presence of Ca(2+). Furthermore, the presence of TnI(1-40) slightly weakened the affinity of the regulatory peptides for TnC. Taken together, these results support the model for TnI-TnC interaction where the N-terminus of TnI remains bound to the C-domain of TnC in the presence of high and low Ca(2+) levels while the TnI regulatory region (residues 96-139) switches in its binding interactions between the actin-tropomyosin thin filament and its own sites on the N- and C-domain of TnC at high Ca(2+) levels, thus regulating muscle contraction.     

Cooperative effects of rigor and cycling cross-bridges on calcium binding to troponin C

The effects of rigor and cycling cross-bridges on distributions of calcium (Ca) bound within sarcomeres of rabbit psoas muscle fibers were compared using electron probe x-ray microanalysis. Calcium in the overlap region of rigor fibers, after correction for that bound to thick filaments, was significantly higher than in the I-band at all pCa levels tested between 6.9 and 4.8, but the difference was greatest at pCa 6.9. With addition of MgATP, differences were significant at high levels of activation (pCa 5.6 and 4.9); near and below the threshold for activation, Ca was the same in I-band and overlap regions. Comparison of Ca and mass profiles at the A-I junction showed elevation of Ca extending 55-110 nm (up to three regulatory units) into the I-band. Extraction of TnC-reduced I-band and overlap Ca in rigor fibers at pCa 5.6 to the same levels found in unextracted fibers at pCa 8.9, suggesting that variations reported here reflect changes in Ca bound to troponin C (TnC). Taken together, these observations provide evidence for near-neighbor cooperative effects of both rigor and cycling cross-bridges on Ca(2+) binding to TnC.     

The effects of force inhibition by sodium vanadate on cross-bridge binding, force redevelopment, and Ca2+ activation in cardiac muscle

Strongly bound, force-generating myosin cross-bridges play an important role as allosteric activators of cardiac thin filaments. Sodium vanadate (Vi) is a phosphate analog that inhibits force by preventing cross-bridge transition into force-producing states. This study characterizes the mechanical state of cross-bridges with bound Vi as a tool to examine the contribution of cross-bridges to cardiac contractile activation. The K(i) of force inhibition by Vi was approximately 40 microM. Sinusoidal stiffness was inhibited with Vi, although to a lesser extent than force. We used chord stiffness measurements to monitor Vi-induced changes in cross-bridge attachment/detachment kinetics at saturating [Ca(2+)]. Vi decreased chord stiffness at the fastest rates of stretch, whereas at slow rates chord stiffness actually increased. This suggests a shift in cross-bridge population toward low force states with very slow attachment/detachment kinetics. Low angle x-ray diffraction measurements indicate that with Vi cross-bridge mass shifted away from thin filaments, implying decreased cross-bridge/thin filament interaction. The combined x-ray and mechanical data suggest at least two cross-bridge populations with Vi; one characteristic of normal cycling cross-bridges, and a population of weak-binding cross-bridges with bound Vi and slow attachment/detachment kinetics. The Ca(2+) sensitivity of force (pCa(50)) and force redevelopment kinetics (k(TR)) were measured to study the effects of Vi on contractile activation. When maximal force was inhibited by 40% with Vi pCa(50) decreased, but greater force inhibition at higher [Vi] did not further alter pCa(50). In contrast, the Ca(2+) sensitivity of k(TR) was unaffected by Vi. Interestingly, when force was inhibited by Vi k(TR) increased at submaximal levels of Ca(2+)-activated force. Additionally, k(TR) is faster at saturating Ca(2+) at [Vi] that inhibit force by > approximately 70%. The effects of Vi on k(TR) imply that k(TR) is determined not only by the intrinsic properties of the cross-bridge cycle, but also by cross-bridge contribution to thin filament activation.     

Troponin C modulates the activation of thin filaments by rigor cross-bridges.

Extraction of troponin C (TnC) from skinned muscle fibers reduces maximum Ca2+ and rigor cross-bridge (RXB)-activated tensions and reduces cooperativity between neighboring regulatory units (one troponin-tropomyosin complex and the seven associated actins) of thin filaments. This suggests that TnC has a determining role in RXB, as well as in Ca(2+)-dependent activation processes. To investigate this possibility further, we replaced fast TnC (fTnC) of rabbit psoas fibers with either CaM[3,4TnC] or cardiac TnC (cTnC) and compared the effects of these substitutions on Ca2+ and RXB activation of tension. CaM[3,4TnC] substitution has the same effect on Ca(2+)- and RXB-activated tensions; they are reduced 50%, and cooperativity between regulatory units is reduced 40%. cTnC substitution also reduces the maximum Ca(2+)-activated tension and cooperativity. But with RXB activation the effects on tension and cooperativity are opposite; cTnC substitution potentiates tension but reduces cooperativity. We considered whether tension potentiation could be explained by increased activation by cycling cross-bridges (CXBs), but the concerted transition formalism predicts fibers will fail to relax in high substrate and high pCa when CXBs are activator ligands. It predicts resting tension, which is not observed in either control or cTnC-substituted fibers. Rather, it appears that cTnC facilitates RXB activation of fast fibers more effectively than fTnC. The order of RXB-activated tension facilitation is cTnC > fTnC > CaM[3,4TnC] > empty TnC-binding sites. Comparison of the structures of fTnC, CaM[3,4TnC], and cTnC indicates that the critical region for this property lies in the central helix or N-terminal domain, including EF hand motifs 1 and 2.     

The Rates of Ca2+ Dissociation and Cross-bridge Detachment from Ventricular Myofibrils as Reported by a Fluorescent Cardiac Troponin C

The rate-limiting step of cardiac muscle relaxation has been proposed to reside in the myofilament. Both the rates of cross-bridge detachment and Ca(2+) dissociation from troponin C (TnC) have been hypothesized to rate-limit myofilament inactivation. In this study we used a fluorescent TnC to measure both the rate of Ca(2+) dissociation from TnC and the rate of cross-bridge detachment from several different species of ventricular myofibrils. The fluorescently labeled TnC was sensitive to both Ca(2+) dissociation and cross-bridge detachment at low Ca(2+) (presence of EGTA), allowing for a direct comparison between the two proposed rates of myofilament inactivation. Unlike Ca(2+) dissociation from TnC, cross-bridge detachment varied in myofibrils from different species and was rate-limited by ADP release. At subphysiological temperatures (<20 °C), the rate of Ca(2+) dissociation from TnC was faster than the rate of cross-bridge detachment in the presence of ADP. These results support the hypothesis that cross-bridge detachment rate-limits relaxation. However, Ca(2+) dissociation from TnC was not as temperature-sensitive as cross-bridge detachment. At a near physiological temperature (35 °C) and ADP, the rate of cross-bridge detachment may actually be faster than the rate of Ca(2+) dissociation. This provides evidence that there may not be a simple, single rate-limiting step of myofilament inactivation.     

Kinetic analysis of the interactions between troponin C and the C-terminal troponin I regulatory region and validation of a new peptide delivery/capture system used for surface plasmon resonance

Using surface plasmon resonance (SPR)-based biosensor analysis and fluorescence spectroscopy, the apparent kinetic constants, k(on) and k(off), and equilibrium dissociation constant, K(d), have been determined for the binding interaction between rabbit skeletal troponin C (TnC) and rabbit skeletal troponin I (TnI) regulatory region peptides: TnI(96-115), TnI(96-131) and TnI(96-139). To carry out SPR analysis, a new peptide delivery/capture system was utilized in which the TnI peptides were conjugated to the E-coil strand of a de novo designed heterodimeric coiled-coil domain. The TnI peptide conjugates were then captured via dimerization to the opposite strand (K-coil), which was immobilized on the biosensor surface. TnC was then injected over the biosensor surface for quantitative binding analysis. For fluorescence spectroscopy analysis, the environmentally sensitive fluoroprobe 5-((((2-iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid (1,5-IAEDANS) was covalently linked to Cys98 of TnC and free TnI peptides were added. SPR analysis yielded equilibrium dissociation constants for TnC (plus Ca(2+)) binding to the C-terminal TnI regulatory peptides TnI(96-131) and TnI(96-139) of 89nM and 58nM, respectively. The apparent association and dissociation rate constants for each interaction were k(on)=2.3x10(5)M(-1)s(-1), 2.0x10(5)M(-1)s(-1) and k(off)=2.0x10(-2)s(-1), 1.2x10(-2)s(-1) for TnI(96-131) and TnI(96-139) peptides, respectively. These results were consistent with those obtained by fluorescence spectroscopy analysis: K(d) being equal to 130nM and 56nM for TnC-TnI(96-131) and TnC-TnI(96-139), respectively. Interestingly, although the inhibitory region peptide (TnI(96-115)) was observed to bind with an affinity similar to that of TnI(96-131) by fluorescence analysis (K(d)=380nM), its binding was not detected by SPR. Subsequent investigations examining salt effects suggested that the binding mechanism for the inhibitory region peptide is best characterized by an electrostatically driven fast on-rate ( approximately 1x10(8) to 1x10(9)M(-1)s(-1)) and a fast off-rate ( approximately 1x10(2)s(-1)). Taken together, the determination of these kinetic rate constants permits a clearer view of the interactions between the TnC and TnI proteins of the troponin complex.     

亲和层析法分离人心脏特异性肌钙蛋白Ⅰ(CSTnⅠ)

在Ca~(2+)存在下,肌钙蛋白C(TnC)和肌钙蛋白Ⅰ(TnⅠ)可于高浓度的尿素溶液中形成稳定的复合体,而该复合体又能被EGTA解离。并且TnC和TnⅠ的Ca~(2+)依赖性结合反应没有种间或者肌肉类型间的特异性。将提纯的兔快肌TnC和活化的琼脂糖凝胶进行偶联,制备TnC-Sepharose 4B亲和层析柱,用来从人心肌中直接提纯心脏特异性肌钙蛋白Ⅰ(CSTnⅠ)。SDS-PAGE测得其分子量为29kD左右。对其氨基酸组成亦进行了分析。     

Conformational change of skeletal muscle troponin

The fluorescence titration curve of skeletal muscle troponin containing TnI with 2-[4'-iodoacetamido)anilino)naphthalene-6-sulfonic acid-labeled Cys-48 and/or Cys-64 was composed of two transition curves. One transition occurred at the pCa region higher than 8.0, and the other between pCa 8.0 and 6.0. The transition at the lower pCa region had a midpoint of pCa 6.85, and the midpoint did not depend on Mg2+. The time course of the fluorescence change subsequent to the rapid pCa-jump of the solution was biphasic. The fast phase was due to the transition at the lower pCa region, and the rate constant of the process was characteristic of the conformational change of the protein induced by Ca2+ binding to the low affinity Ca2+-binding sites of TnC. The slow phase was from the transition at the higher pCa region, and its rate constant was characteristic of the conformational change of the protein induced by Ca2+ binding to the high affinity Ca2+-binding sites of TnC. Therefore we can conclude that the fluorescence probe bound to Cys-48 and/or Cys-64 of TnI detects the conformational change of the Tn complex induced by Ca2+ binding to both the low and high affinity Ca2+-binding sites of TnC. The fluorescence probe bound to Cys-133 of TnI or Met residues of TnT detected the conformational change of the Tn complex induced by Ca2+ binding to the low affinity Ca2+-binding sites of TnC.     

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)).