Tm Molecule Research Articles

Prehospital tranexamic acid is associated with a dose-dependent decrease in syndecan-1 after trauma: A secondary analysis of a prospective randomized trial.

In the Study of Tranexamic Acid During Air and Ground Prehospital Transport (STAAMP) Trial, prehospital tranexamic acid (TXA) was associated with lower mortality in specific patient subgroups. The underlying mechanisms responsible for a TXA benefit remain incompletely characterized. We hypothesized that TXA may mitigate endothelial injury and sought to assess whether TXA was associated with decreased endothelial or tissue damage markers among all patients enrolled in the STAAMP Trial. We collected blood samples from STAAMP Trial patients and measured markers of endothelial function and tissue damage including syndecan-1, soluble thrombomodulin (sTM), and platelet endothelial cell adhesion molecule-1 (PECAM-1) at hospital admission (0 hours) and 12, 24, and 72 hours after admission. We compared these marker values for patients in each treatment group during the first 72 hours, and modeled the relationship between TXA and marker concentration using regression analysis to control for potential confounding factors. We analyzed samples from 766 patients: 383 placebo, 130 abbreviated dosing, 119 standard dosing, and 130 repeat dosing. Lower levels of syndecan-1, TM, and PECAM measured within the first 72 hours of hospital admission were associated with survival at 30 days (P < 0.001). At hospital admission, syndecan-1 was lower in the TXA group (28.30 [20.05, 42.75] vs. 33.50 [23.00, 54.00] P = 0.001) even after controlling for patient, injury, and prehospital factors (P = 0.001). For every 1 g increase in TXA administered over the first 8 hours of prehospital transport and hospital admission, there was a 4 ng/mL decrease in syndecan-1 at 12 hours controlling for patient, injury, and treatment factors (P = 0.03). Prehospital TXA was associated with decreased syndecan-1 at hospital admission. Syndecan-1 measured 12 hours after admission was inversely related to the dose of TXA received. Early pre- and in-hospital TXA may decrease endothelial glycocalyx damage or upregulate vascular repair mechanisms in a dose-dependent fashion. Level II, Secondary analysis of a prospective randomized trial.

Modulation of spin thermoelectric properties in transition metal porphyrin single-molecule spin caloritronic devices by Fano resonance

Abstract Single-molecule spin caloritronic devices, which convert the heat into electricity by using spin degree of freedom of electron, are attracting attention with the inspiration of finally shrinking electronic components by utilizing molecules as elementary blocks. In this work, one kind of molecular-scale spin caloritronic devices is theoretically proposed, which consists of a transition metal porphyrin (TM(Pr), TM=V, Mn, Fe, Co) molecule sandwiched between single-walled carbon nanotube (SWCNT) electrodes via carbon atomic chains (CACs). The corresponding spin transport properties are calculated. The results show there is robust Fano resonance in the transmission spectra around the Fermi energy for one spin component in each device, which is attributed to the quantum interference between continuous spin states originating from delocalized states over the pristine SWCNT-porphyrin device and “quasi-discrete” state mainly localized on the TM(Pr) molecule. Utilizing Fano resonance, spin-dependent Seebeck coefficients of the devices for one spin component can be strengthened. Interestingly, it is revealed the energy, spatial distributions, and parity of molecular orbitals of TM(Pr) molecules play an important role in the generation of Fano resonance, which is dependent on the kind of TM atom. Therefore, this further provides the possibility to optimize spin-dependent Seebeck coefficient by TM atoms. This work is helpful for the future design of spin caloritronic devices.

Theoretical design of single-molecule NOR and XNOR logic gates by using transition metal dibenzotetraaza[14]annulenes**Project supported by the National Natural Science Foundation of China (Grant Nos. 11874242, 21933002, and 11704230), China Postdoctoral Science Foundation (Grant No. 2017M612321), and the Taishan Scholar Project of Shandong Province of China.

The idea of replacing traditional silicon-based electronic components with the ones assembled by organic molecules to further scale down the electric circuits has been attracting extensive research focuses. Among the molecularly assembled components, the design of molecular logic gates with simple structure and high Boolean computing speed remains a great challenge. Here, by using the state-of-the-art nonequilibrium Green’s function theory in conjugation with first-principles method, the spin transport properties of single-molecule junctions comprised of two serially connected transition metal dibenzotetraaza[14]annulenes (TM(DBTAA), TM = Fe, Co) sandwiched between two single-walled carbon nanotube electrodes are theoretically investigated. The numerical results show a close dependence of the spin-resolved current-voltage characteristics on spin configurations between the left and right molecular kernels and the kind of TM atom in TM(DBTAA) molecule. By taking advantage of spin degree of freedom of electrons, NOR or XNOR Boolean logic gates can be realized in Fe(DBTAA) and Co(DBTAA) junctions depending on the definitions of input and output signals. This work proposes a new kind of molecular logic gates and hence is helpful for further miniaturization of the electric circuits.

Effects of two stabilizing substitutions, D137L and G126R, in the middle part of α-tropomyosin on the domain structure of its molecule

We applied differential scanning calorimetry (DSC) to investigate the effects of substitutions D137L and G126R (i.e. replacement of conserved non-canonical residues Asp137 and Gly126 by canonical ones) in the middle part of tropomyosin (Tm), as well as the combined one, D137L/G126R, on the thermal unfolding of Tm. Special approaches (e.g. combination of DSC with measurements of temperature dependences of pyrene excimer fluorescence) were applied to assign separate thermal transitions (calorimetric domains) on the DSC profiles to the certain parts of Tm molecule. The results show that substitutions D137L and G126R (and, especially, the combined one, D137L/G126R) may stabilize not only the middle region of Tm, but also the other parts of its molecule including N- and C-terminal parts. These results suggest that the stabilization of the Tm middle part can be transmitted along the coiled-coil length displaying long-range effects.

Structural and functional effects of two stabilizing substitutions, D137L and G126R, in the middle part of α‐tropomyosin molecule

Tropomyosin (Tm) is an α-helical coiled-coil protein that binds along the length of actin filament and plays an essential role in the regulation of muscle contraction. There are two highly conserved non-canonical residues in the middle part of the Tm molecule, Asp137 and Gly126, which are thought to impart conformational instability (flexibility) to this region of Tm which is considered crucial for its regulatory functions. It was shown previously that replacement of these residues by canonical ones (Leu substitution for Asp137 and Arg substitution for Gly126) results in stabilization of the coiled-coil in the middle of Tm and affects its regulatory function. Here we employed various methods to compare structural and functional features of Tm mutants carrying stabilizing substitutions Arg137Leu and Gly126Arg. Moreover, we for the first time analyzed the properties of Tm carrying both these substitutions within the same molecule. The results show that both substitutions similarly stabilize the Tm coiled-coil structure, and their combined action leads to further significant stabilization of the Tm molecule. This stabilization not only enhances maximal sliding velocity of regulated actin filaments in the in vitro motility assay at high Ca(2+) concentrations but also increases Ca(2+) sensitivity of the actin-myosin interaction underlying this sliding. We propose that the effects of these substitutions on the Ca(2+)-regulated actin-myosin interaction can be accounted for not only by decreased flexibility of actin-bound Tm but also by their influence on the interactions between the middle part of Tm and certain sites of the myosin head.

Transmission of Stability Information through the N-domain of Tropomyosin Is Interrupted by a Stabilizing Mutation (A109L) in the Hydrophobic Core of the Stability Control Region (Residues 97–118)

Tropomyosin (Tm) is an actin-binding, thin filament, two-stranded α-helical coiled-coil critical for muscle contraction and cytoskeletal function. We made the first identification of a stability control region (SCR), residues 97-118, in the Tm sequence that controls overall protein stability but is not required for folding. We also showed that the individual α-helical strands of the coiled-coil are stabilized by Leu-110, whereas the hydrophobic core is destabilized in the SCR by Ala residues at three consecutive d positions. Our hypothesis is that the stabilization of the individual α-helices provides an optimum stability and allows functionally beneficial dynamic motion between the α-helices that is critical for the transmission of stabilizing information along the coiled-coil from the SCR. We prepared three recombinant (rat) Tm(1-131) proteins, including the wild type sequence, a destabilizing mutation L110A, and a stabilizing mutation A109L. These proteins were evaluated by circular dichroism (CD) and differential scanning calorimetry. The single mutation L110A destabilizes the entire Tm(1-131) molecule, showing that the effect of this mutation is transmitted 165 Å along the coiled-coil in the N-terminal direction. The single mutation A109L prevents the SCR from transmitting stabilizing information and separates the coiled-coil into two domains, one that is ∼9 °C more stable than wild type and one that is ∼16 °C less stable. We know of no other example of the substitution of a stabilizing Leu residue in a coiled-coil hydrophobic core position d that causes this dramatic effect. We demonstrate the importance of the SCR in controlling and transmitting the stability signal along this rodlike molecule.

Stabilization of the Central Part of Tropomyosin Molecule Alters the Ca 2+-sensitivity of Actin-Myosin Interaction

We show that the mutations D137L and G126R, which stabilize the central part of the tropomyosin (Tm) molecule, increase both the maximal sliding velocity of the regulated actin filaments in the in vitro motility assay at high Са(2+) concentrations and the Са(2+)-sensitivity of the actin-myosin interaction underlying this sliding. Based on an analysis of the recently published data on the structure of the actin-Tm-myosin complex, we suppose that the physiological effects of these mutations in Tm can be accounted for by their influence on the interactions between the central part of Tm and certain sites of the myosin head.

Periodicities designed in the tropomyosin sequence and structure define its functions

Tropomyosin is an actin binding protein that regulates actin filament dynamics and its interactions with actin binding proteins such as myosin, tropomodulin, formin, Arp2/3 and ADF-cofilin in most eukaryotic cells. Tropomyosin is the prototypical two-chained, α-helical coiled coil protein that associates end-to-end and binds to both sides of the actin filament. Each tropomyosin molecule spans four to seven actin monomers in the filament, depending on the size of the tropomyosin. Tropomyosins have a periodic heptad repeat sequence that is characteristic of coiled coil proteins as well as additional periodicities required for its interaction with the actin filament, where each periodic repeat interacts with one actin molecule. This review addresses the role of periodic features of the Tm molecule in carrying out its universal functions of binding to the actin filament and its regulation and the specific features that may determine the isoform specificity of tropomyosins.

A Periodic Pattern of Evolutionarily Conserved Basic and Acidic Residues Constitutes the Binding Interface of Actin-Tropomyosin

Actin filament cytoskeletal and muscle functions are regulated by actin binding proteins using a variety of mechanisms. A universal actin filament regulator is the protein tropomyosin, which binds end-to-end along the length of the filament. The actin-tropomyosin filament structure is unknown, but there are atomic models in different regulatory states based on electron microscopy reconstructions, computational modeling of actin-tropomyosin, and docking of atomic resolution structures of tropomyosin to actin filament models. Here, we have tested models of the actin-tropomyosin interface in the "closed state" where tropomyosin binds to actin in the absence of myosin or troponin. Using mutagenesis coupled with functional analyses, we determined residues of actin and tropomyosin required for complex formation. The sites of mutations in tropomyosin were based on an evolutionary analysis and revealed a pattern of basic and acidic residues in the first halves of the periodic repeats (periods) in tropomyosin. In periods P1, P4, and P6, basic residues are most important for actin affinity, in contrast to periods P2, P3, P5, and P7, where both basic and acidic residues or predominantly acidic residues contribute to actin affinity. Hydrophobic interactions were found to be relatively less important for actin binding. We mutated actin residues in subdomains 1 and 3 (Asp(25)-Glu(334)-Lys(326)-Lys(328)) that are poised to make electrostatic interactions with the residues in the repeating motif on tropomyosin in the models. Tropomyosin failed to bind mutant actin filaments. Our mutagenesis studies provide the first experimental support for the atomic models of the actin-tropomyosin interface.

Conserved Noncanonical Residue Gly-126 Confers Instability to the Middle Part of the Tropomyosin Molecule

Tropomyosin (Tm) is a two-stranded α-helical coiled-coil protein with a well established role in regulation of actin cytoskeleton and muscle contraction. It is believed that many Tm functions are enabled by its flexibility whose nature has not been completely understood. We hypothesized that the well conserved non-canonical residue Gly-126 causes local destabilization of Tm. To test this, we substituted Gly-126 in skeletal muscle α-Tm either with an Ala residue, which should stabilize the Tm α-helix, or with an Arg residue, which is expected to stabilize both α-helix and coiled-coil structure of Tm. We have shown that both mutations dramatically reduce the rate of Tm proteolysis by trypsin at Asp-133. Differential scanning calorimetry was used for detailed investigation of thermal unfolding of the Tm mutants, both free in solution and bound to F-actin. It was shown that a significant part of wild type Tm unfolds in a non-cooperative manner at low temperature, and both mutations confer cooperativity to this part of the Tm molecule. The size of the flexible middle part of Tm is estimated to be 60-70 amino acid residues, about a quarter of the Tm molecule. Thus, our results show that flexibility is unevenly distributed in the Tm molecule and achieves the highest extent in its middle part. We conclude that the highly conserved Gly-126, acting in concert with the previously identified non-canonical Asp-137, destabilizes the middle part of Tm, resulting in a more flexible region that is important for Tm function.

Alternatively spliced N-terminal exons in tropomyosin isoforms do not act as autonomous targeting signals

Tropomyosin (Tm) polymerises head-to-tail to form a continuous polymer located in the major groove of the actin filament. Multiple Tm isoforms are generated by alternative splicing of four genes, and individual isoforms show specific localisation patterns in many cell types, and can have differing effects on the actin cytoskeleton. Fluorescently-tagged Tm isoforms and mutants were expressed in C2C12 cells to investigate the mechanisms of alternative localisation of high molecular weight (HMW) and low molecular weight (LMW) Tms. Fluorescently-tagged Tm constructs show similar localisation to endogenous Tms as observed by antibodies, with the HMW Tm3 relatively diminished at the periphery of cells compared to LMW isoforms Tm5b or Tm5NM1. Tm3 and Tm5b only differ in their N-terminal exons, but these N-terminal exons do not independently direct localisation within the cell, as chimeric mutants Tm3-Tm5NM1 and Tm5b-Tm5NM1 show an increased peripheral localisation similar to Tm5NM1. The lower abundance of Tm3 at the periphery of the cell is not a result of different protein dynamics, as Tm3 and Tm5b show similar recovery after photobleaching. The relative exclusion of Tm3 from the periphery of cells does, however, require interaction with the actin filament, as mutants with truncations at either the N-terminus or the C-terminus are unable to localise to actin stress fibres, and are present in the most peripheral regions of the cell. We conclude that it is the entire Tm molecule which is the unit of sorting, and that the alternatively spliced N-terminal exons do not act as autonomous targeting signals.

Tropomyosin Flexibility Evaluated by Electron Microscopy Image Analysis

Movement of tropomyosin (Tm) on thin filaments in response to Ca2+-binding to troponin and myosin binding to actin is an inherent feature of muscle regulation. As part of this process, the cable-like mechanical properties of the Tm coiled-coil are thought to underlie cooperative on and off switching of contraction. In principle, movement of Tm over the flat surface of actin may not require significant molecular flexibility. However, local perturbations caused, for example, by myosin binding on actin may necessitate some plasticity of the Tm molecule. In contrast, any large-scale Tm flexibility might dampen its cooperative movement. In the current study, we directly assessed the flexibility of Tm by examining EM images of both rotary shadowed and negatively stained molecules. Single Tm molecules in both image sets showed no obvious signs of sharp bending or kinks, and displayed contours close to those predicted from a high-resolution 3D model of Tm (Lorenz et al., 1995). Short multimeric strings of end-to-end bonded Tm were commonly observed in the rotary shadowed images. These showed no pronounced bending or joints at the intermolecular junctions. The persistence length of Tm was calculated to be over twice the length of the molecule based on these EM images. The data as a whole imply that Tm molecules exhibit an intrinsic stiffness sufficient to contribute to cooperativity on thin filaments and thus are consistent with models of muscle regulation (e.g. Lehrer & Geeves, 1998; Lehman et al., 2000). A tropomyosin mutant in which the coiled-coil interface was destabilized by introduction of Ala clusters in the 2nd and 3rd periodic repeats (Y60A-L64A-L106A; Singh & Hitchcock-DeGregori, unpublished) was also examined by EM. No obvious extra bending was observed, although the calculated persistence length was significantly shortened, showing the sensitivity of the technique.

Fluorescence Resonance Energy Transfer between Residues on Troponin and Tropomyosin in the Reconstituted Thin Filament: Modeling the Troponin–Tropomyosin Complex

Troponin (Tn), in association with tropomyosin (Tm), plays a central role in the calcium regulation of striated muscle contraction. Fluorescence resonance energy transfer (FRET) between probes attached to the Tn subunits (TnC, TnI, TnT) and to Tm was measured to study the spatial relationship between Tn and Tm on the thin filament. We generated single-cysteine mutants of rabbit skeletal muscle α-Tm, TnI and the β-TnT 25-kDa fragment. The energy donor was attached to a single-cysteine residue at position 60, 73, 127, 159, 200 or 250 on TnT, at 98 on TnC and at 1, 9, 133 or 181 on TnI, while the energy acceptor was located at 13, 146, 160, 174, 190, 209, 230, 271 or 279 on Tm. FRET analysis showed a distinct Ca 2+-induced conformational change of the Tm–Tn complex and revealed that TnT60 and TnT73 were closer to Tm13 than Tm279, indicating that the elongated N-terminal region of TnT extends beyond the beginning of the next Tm molecule on the actin filament. Using the atomic coordinates of the crystal structures of Tm and the Tn core domain, we searched for the disposition and orientation of these structures by minimizing the deviations of the calculated FRET efficiencies from the observed FRET efficiencies in order to construct atomic models of the Tn–Tm complex with and without bound Ca 2+. In the best-fit models, the Tn core domain is located on residues 160–200 of Tm, with the arrowhead-shaped I-T arm tilting toward the C-terminus of Tm. The angle between the Tm axis and the long axis of TnC is ∼ 75° and ∼ 85° with and without bound Ca 2+, respectively. The models indicate that the long axis of TnC is perpendicular to the thin filament without bound Ca 2+, and that TnC and the I-T arm tilt toward the filament axis and rotate around the Tm axis by ∼ 20° upon Ca 2+ binding.

Tropomyosin in the groove? Molecular insights into an inherited myopathy

Tropomyosin in the groove? Molecular insights into an inherited myopathy

Different Effects of Trifluoroethanol and Glycerol on the Stability of Tropomyosin Helices and the Head-to-Tail Complex

Tropomyosin (Tm) is a dimeric coiled-coil protein, composed of 284 amino acids (410 Å), that forms linear homopolymers through head-to-tail interactions at low ionic strength. The head-to-tail complex involves the overlap of approximately nine N-terminal residues of one molecule with nine C-terminal residues of another Tm molecule. In this study, we investigate the influence of 2,2,2-trifluoroethanol (TFE) and glycerol on the stability of recombinant Tm fragments (ASTm 1–142, Tm 143–284(5OHW269)) and of the dimeric head-to-tail complex formed by the association of these two fragments. The C-terminal fragment (Tm 143–284(5OHW269)) contains a 5-hydroxytryptophan (5OHW) probe at position 269 whose fluorescence is sensitive to the head-to-tail interaction and allows us to accompany titrations of Tm 143–284(5OHW269) with ASTm 1–142 to calculate the dissociation constant ( K d) and the interaction energy at TFE and glycerol concentrations between 0% and 15%. We observe that TFE, but not glycerol, reduces the stability of the head-to-tail complex. Thermal denaturation experiments also showed that the head-to-tail complex increases the overall conformational stability of the Tm fragments. Urea and thermal denaturation assays demonstrated that both TFE and glycerol increase the stability of the isolated N- and C-terminal fragments; however, only TFE caused a significant reduction in the cooperativity of unfolding these fragments. Our results show that these two cosolvents stabilize the structures of individual Tm fragments in different manners and that these differences may be related to their opposing effects on head-to-tail complex formation.

The Second Half of the Fourth Period of Tropomyosin Is a Key Region for Ca2+-Dependent Regulation of Striated Muscle Thin Filaments

Rabbit skeletal muscle alpha-tropomyosin (Tm), a 284-residue dimeric coiled-coil protein, spans seven actin monomers and contains seven quasiequivalent periods. X-ray analysis of cocrystals of Tm and troponin (Tn) placed the Tn core domain near residues 150-180 of Tm. To identify the Ca(2+)-sensitive Tn interaction site on Tm, we generated three Tm mutants to compare the consequences of sequence substitution inside and outside of the Tn core domain-binding region. Residues 152-165 and 156-162 in the second half of period 4 were replaced by corresponding residues 33-46 and 37-43 in the second half of period 1, respectively (termed mTm152-165 and mTm156-162, respectively), and residues 134-147 in the first half of period 4 were replaced with residues 15-28 in the first half of period 1 (mTm134-147). Recombinant Tms designed with an additional tripeptide, Ala-Ala-Ser, at the N-terminus were expressed in Escherichia coli. Both mTm152-165 and mTm156-162 suppressed the actin-activated myosin subfragment-1 Mg(2+)-ATPase rate regardless of whether Ca(2+) and Tn were present. On the other hand, mTm134-147 retained the normal Ca(2+)-sensitive regulation, although the actin binding of mTm alone was significantly impaired. Differential scanning calorimetry showed that the sequence substitution in the second half of period 4 affected the thermal stability of the complete Tm molecule and also the actin-induced stabilization. These results suggest that the second half of period 4 of Tm is a key region for inducing conformational changes of the regulated thin filament required for its fully activated state.

Thermal unfolding of smooth muscle and nonmuscle tropomyosin α‐homodimers with alternatively spliced exons

We used differential scanning calorimetry (DSC) and circular dichroism (CD) to investigate thermal unfolding of recombinant fibroblast isoforms of alpha-tropomyosin (Tm) in comparison with that of smooth muscle Tm. These two nonmuscle Tm isoforms 5a and 5b differ internally only by exons 6b/6a, and they both differ from smooth muscle Tm by the N-terminal exon 1b which replaces the muscle-specific exons 1a and 2a. We show that the presence of exon 1b dramatically decreases the measurable calorimetric enthalpy of the thermal unfolding of Tm observed with DSC, although it has no influence on the alpha-helix content of Tm or on the end-to-end interaction between Tm dimers. The results suggest that a significant part of the molecule of fibroblast Tm (but not smooth muscle Tm) unfolds noncooperatively, with the enthalpy no longer visible in the cooperative thermal transitions measured. On the other hand, both DSC and CD studies show that replacement of muscle exons 1a and 2a by nonmuscle exon 1b not only increases the thermal stability of the N-terminal part of Tm, but also significantly stabilizes Tm by shifting the major thermal transition of Tm to higher temperature. Replacement of exon 6b by exon 6a leads to additional increase in the alpha-Tm thermal stability. Thus, our data show for the first time a significant difference in the thermal unfolding between muscle and nonmuscle alpha-Tm isoforms, and indicate that replacement of alternatively spliced exons alters the stability of the entire Tm molecule.

Functional Consequences of Hypertrophic and Dilated Cardiomyopathy-causing Mutations in α-Tropomyosin

To study the functional consequences of various cardiomyopathic mutations in human cardiac alpha-tropomyosin (Tm), a method of depletion/reconstitution of native Tm and troponin (Tn) complex (Tm-Tn) in cardiac myofibril preparations has been developed. The endogenous Tm-Tn complex was selectively removed from myofibrils and replaced with recombinant wild-type or mutant proteins. Successful depletion and reconstitution steps were verified by SDS-gel electrophoresis and by the loss and regain of Ca2+-dependent regulation of ATPase activity. Five Tm mutations were chosen for this study: the hypertrophic cardiomyopathy (HCM) mutations E62Q, E180G, and L185R and the dilated cardiomyopathy (DCM) mutations E40K and E54K. Through the use of this new depletion/reconstitution method, the functional consequences of these mutations were determined utilizing myofibrillar ATPase measurements. The results of our studies showed that 1) depletion of >80% of Tm-Tn from myofibrils resulted in a complete loss of the Ca2+-regulated ATPase activity and a significant loss in the maximal ATPase activity, 2) reconstitution of exogenous wild-type Tm-Tn resulted in complete regain in the calcium regulation and in the maximal ATPase activity, and 3) all HCM-associated Tm mutations increased the Ca2+ sensitivity of ATPase activity and all had decreased abilities to inhibit ATPase activity. In contrast, the DCM-associated mutations both decreased the Ca2+ sensitivity of ATPase activity and had no effect on the inhibition of ATPase activity. These findings have demonstrated that the mutations which cause HCM and DCM disrupt discrete mechanisms, which may culminate in the distinct cardiomyopathic phenotypes.

Two-site attachment of troponin to pyrene-labeled tropomyosin.

The interaction of troponin (a complex of troponin T, troponin I, and troponin C) with tropomyosin (Tm) was studied using the fluorescence of pyrene-labeled Tm. Fluorescence titrations showed that an increase in salt concentration not only decreased the binding constant of troponin to Tm but also decreased the magnitude of the total pyrene fluorescence change. This indicates that the interaction of the part of troponin (TnT2) with the middle of Tm near Cys-190 is weakened at high ionic strength, while the other part (TnT1) maintains its binding with the C-terminal region of Tm. The temperature dependence of the pyrene excimer fluorescence of Tm showed that troponin stabilized the whole Tm molecule with much less effect on the stability of the middle region where the TnT2 binds. TnT1, the N-terminal chymotryptic fragment of troponin T (TnT), stabilized Tm in a similar manner to troponin by inhibiting dissociation of Tm chains in the main unfolding transition. Thus, these data indicate a loose interaction of the TnT2 part of troponin with the region of Tm near Cys-190 and a tight interaction of the TnT1 part with the C-terminal region of Tm. TnT alone interacted more strongly with Tm near Cys-190 than when in the troponin complex. This was indicated by the following observations in the Tm.TnT complex compared to the Tm-troponin complex: (i) a more hydrophobic environment of pyrene at Cys-190 of Tm; (ii) stabilization against the local unfolding of Tm near Cys-190; (iii) more fluorescence resonance energy transfer between Tm and TnT. These studies indicate a loose and flexible interaction of troponin with the middle region of Tm which may be important in Ca2+ regulation by troponin.Tm.