Fluorescence probe studies of the state of tropomyosin in reconstituted muscle thin filaments

The monomer fluorescence of N-(1-pyrenyl)maleimide-labeled tropomyosin bound to F-actin (PTm-actin) increases when myosin subfragment 1 (S1) binds to actin and is half complete when only approximately 1 S1 is bound to 7 actin subunits [Ishii, Y., & Lehrer, S. S. (1985) Biochemistry 24, 6631-6638]. Similar studies of the binding of S1 and S1-ADP to fully reconstituted thin filaments [PTm-actin-troponin (Tn)] are now reported. The pyrene monomer fluorescence change was half complete when approximately 0.5 S1/7 actin subunits and approximately 1.5 S1/7 actin subunits were bound in the presence and absence of Ca2+, respectively. In the presence of Mg2+-ADP, when S1 binding is weakened, the S1 binding profiles and fluorescence changes were sigmoidal, with the cooperative transitions occurring at lower [S1] in the presence of Ca2+ as first shown by Greene and Eisenberg for S1 binding [Greene, L., & Eisenberg, E. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2616-2620]. It was possible to fit both the binding and fluorescence data with the same parameters of a two-state (weak and strong S1 binding) cooperative binding model [Hill, T., Eisenberg, E., & Greene, L. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3186-3190] for each Ca2+ situation if the fluorescence change is interpreted as the fraction of tropomyosin (Tm) units in the strong S1 binding state. These data indicate that the fluorescence change is a direct measure of the S1-induced change of state of Tm in the fully reconstituted thin filament.(ABSTRACT TRUNCATED AT 250 WORDS)     

Excimer fluorescence of pyrenyliodoacetamide-labeled tropomyosin: a probe of the state of tropomyosin in reconstituted muscle thin filaments

Rabbit skeletal tropomyosin (Tm) specifically labeled at cysteine groups with N-(1-pyrenyl)-iodoacetamide (PIA) exhibits excimer fluorescence. The excimer fluorescence was sensitive to the local conformation of Tm, to actin binding, and, in reconstituted thin filaments, to the Tm state change induced by binding of myosin subfragment 1 (S1). The properties of PIATm were similar to previously studied pyrenylmaleimide-labeled Tm (PMTm) [Ishii, Y., & Lehrer, S.S. (1985) Biochemistry 24, 6631] except that S1 binding to actin-Tm increased the excimer fluorescence in contrast to the time-dependent decrease seen for PMTm. The fluorescence properties of PIATm are sensitive to the Tm chain-chain interaction via equilibria among pyrene configurations and nonfluorescent dimer as well as the monomer and excimer-forming configurations. The effect of bound troponin (Tn) on the excimer fluorescence of PIATm in the reconstituted systems was dependent on ionic strength with a slight Ca2+ dependence. S1 titrations in the absence and presence of Tn and Ca2+ indicated that the excimer fluorescence probes the state change of Tm from the weak S1 binding state to the strong S1 binding state which is facilitated by Ca2+ [Hill et al. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3186]. Binding of MgADP-S1 and MgAMPPNP-S1 produced the same total excimer fluorescence change as for nucleotide-free S1, showing that the strong S1 binding state of Tm-actin is independent of nucleotide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence properties of acrylodan-labeled tropomyosin and tropomyosin-actin: evidence for myosin subfragment 1 induced changes in geometry between tropomyosin and actin

The Cys groups of rabbit skeletal tropomyosin (Tm) and rabbit skeletal alpha alpha Tm were specifically labeled with acrylodan (AC). The probe on Tm is quite immobile yet exposed to solvent as indicated by its limiting polarization (P0 = 0.38) and fluorescence emission spectrum (lambda max = 520 nm) and its accessibility to solute quenching. Changes in the shape of the excitation spectrum with temperature correlated with the helix thermal pretransition and main transition without much spectral change of the emission spectrum. The probe environment of ACTm did not significantly change on binding to F-actin, but fluorescence energy transfer between tryptophan in actin and AC on Tm was indicated by a 15-20% increase in AC fluorescence and a few percent decrease in tryptophan fluorescence. This energy transfer increased when myosin subfragment 1 (S1) was bound to the ACTm-actin filament, in quantitative agreement with the postulated shift in state of Tm associated with the cooperative binding of S1 to actin (Hill et al., 1980). The increase in energy transfer shows that there is a change in the spatial relationship between Tm and actin associated with the S1-induced change in state of Tm.     

Effects of the amino-terminal regions of tropomyosin and troponin T on thin filament assembly.

Bacterially expressed alpha-tropomyosin lacks the amino-terminal acetylation present in muscle tropomyosin and binds poorly to actin (Hitchcock-DeGregori, S. E., and Heald, R. W. (1987) J. Biol. Chem. 262, 9730-9735). Using a linear lattice model, we determined the affinity (Ko) of unacetylated tropomyosin or troponin-unacetylated tropomyosin for an isolated site on the actin filament and the fold increase in affinity (y) when binding is to an adjacent site. The absence of tropomyosin acetylation decreased Ko 2 orders of magnitude in the absence of troponin. Tropomyosin acetylation also enhanced troponin-tropomyosin binding to actin, not by increasing cooperativity (y), but rather by increasing Ko. These results suggest that the amino-terminal region of tropomyosin is a crucial actin binding site. Troponin promoted unacetylated tropomyosin binding to actin, increasing Ko more than 1,000-fold. Troponin70-259, which lacks the troponin T peptide (1-69) spanning the overlap between adjacent tropomyosins, behaved similarly to intact troponin. Cooperative interactions between adjacent troponin-tropomyosin complexes remained strong despite the use of a nonpolymerizable tropomyosin and a troponin unable to bridge neighboring tropomyosins physically. The Ko for troponin70-259-unacetylated tropomyosin was 500-fold greater than for troponin159-259-unacetylated tropomyosin, indicating that troponin T residues 70-158 are critical for anchoring troponin-tropomyosin to F-actin. The mechanism of cooperative thin filament assembly is discussed.     

Tropomyosin and myosin subfragment 1 induce in thin muscle fiber filaments differing conformational changes in the C-terminal portion of the polypeptide chain of actin

Muscle fibres, free of myosin, troponin and tropomyosin, containing thin filaments reconstructed from G-actin and modified by fluorescent label 1,5-IAEDANS were used for polarized microfluorimetric studies of the effect of tropomyosin (TM) from smooth muscles, and of subfragment 1 (S1) from skeletal muscles on the structural state of F-actin. TM and S1 were shown to initiate different changes in polarized fluorescence of 1,5-IAEDANS of F-actin: TM increases, whereas S1 decreases fluorescent anisotropy. It was suggested that the structural state of F-actin may differ in the C-terminal of polypeptide chain of actin.     

Tropomodulin: a cytoskeletal protein that binds to the end of erythrocyte tropomyosin and inhibits tropomyosin binding to actin

Human erythrocytes contain a Mr 43,000 tropomyosin-binding protein that is unrelated to actin and that has been proposed to play a role in modulating the association of tropomyosin with spectrin-actin complexes based on its stoichiometry in the membrane skeleton of one Mr 43,000 monomer per short actin filament (Fowler, V. M. 1987. J. Biol. Chem. 262:12792-12800). Here, we describe an improved procedure to purify milligram quantities to 98% homogeneity and we show that this protein inhibits tropomyosin binding to actin by a novel mechanism. We have named this protein tropomodulin. Unlike other proteins that inhibit tropomyosin-actin interactions, tropomodulin itself does not bind to F-actin. EM of rotary-shadowed tropomodulin-tropomyosin complexes reveal that tropomodulin (14.5 +/- 2.4 nm [SD] in diameter) binds to one of the ends of the rod-like tropomyosin molecules (33 nm long). In agreement with this observation, Dixon plots of inhibition curves demonstrate that tropomodulin is a non-competitive inhibitor of tropomyosin binding to F-actin (Ki = 0.7 microM). Hill plots of the binding of the tropomodulin-tropomyosin complex to actin indicate that binding does not exhibit any positive cooperativity (n = 0.9), in contrast to tropomyosin (n = 1.9), and that the apparent affinity of the complex for actin is reduced 20-fold with respect to that of tropomyosin. These results suggest that binding of tropomodulin to tropomyosin may block the ability of tropomyosin to self-associate in a head-to-tail fashion along the actin filament, thereby weakening its binding to actin. Antibodies to tropomodulin cross-react strongly with striated muscle troponin I (but not with troponin T) as well as with a nontroponin Mr 43,000 polypeptide in muscle and in other nonerythroid cells and tissues, including brain, lens, neutrophils, and endothelial cells. Thus, erythrocyte tropomodulin may be one member of a family of tropomyosin-binding proteins that function to regulate tropomyosin-actin interactions in non-muscle cells and tissues.     

Role of caldesmon in the Ca2+ regulation of smooth muscle thin filaments: evidence for a cooperative switching mechanism

Smooth muscle thin filaments are made up of actin, tropomyosin, caldesmon, and a Ca(2+)-binding protein and their interaction with myosin is Ca(2+)-regulated. We suggested that Ca(2+) regulation by caldesmon and Ca(2+)-calmodulin is achieved by controlling the state of thin filament through a cooperative-allosteric mechanism homologous to troponin-tropomyosin in striated muscles. In the present work, we have tested this hypothesis. We monitored directly the thin filament transition between the ON and OFF state using the excimer fluorescence of pyrene iodoacetamide (PIA)-labeled smooth muscle alphaalpha-tropomyosin homodimers. In steady state fluorescence measurements, myosin subfragment 1 (S1) cooperatively switches the thin filaments to the ON state, and this is exhibited as an increase in the excimer fluorescence. In contrast, caldesmon decreases the excimer fluorescence, indicating a switch of the thin filament to the OFF state. Addition of Ca(2+)-calmodulin increases the excimer fluorescence, indicating a switch of the thin filament to the ON state. The excimer fluorescence was also used to monitor the kinetics of the ON-OFF transition in a stopped-flow apparatus. When ATP induces S1 dissociation from actin-PIA-tropomyosin, the transition to the OFF state is delayed until all S1 molecules are dissociated actin. In contrast, caldesmon switches the thin filament to the OFF state in a cooperative way, and no lag is displayed in the time course of the caldesmon-induced fluorescence decrease. We have also studied caldesmon and Ca(2+)-calmodulin-caldesmon binding to actin-tropomyosin in the ON and OFF states. The results are used to discuss both caldesmon inhibition and Ca(2+)-calmodulin-caldesmon activation of actin-tropomyosin.     

Role of tropomyosin in smooth muscle contraction: effect of tropomyosin binding to actin on actin activation of myosin ATPase

The binding of gizzard tropomyosin to gizzard F-actin is highly dependent on free Mg2+ concentration. At 2 mM free Mg2+, a concentration at which actin-activated ATPase activity was shown to be Ca2+ sensitive, a molar ratio of 1:3 (tropomyosin:actin monomer) is required to saturate the F-actin with tropomyosin to the stoichiometric ratio of 1 mol of tropomyosin to 7 mol of actin monomer. Increasing the Mg2+ could decrease the amount of tropomyosin required for saturating the F-actin filament to the stoichiometric level. Analysis of the binding of smooth muscle tropomyosin to smooth muscle actin by the use of Scatchard plots indicates that the binding exhibits strong positive cooperativity at all Mg2+ concentrations. Calcium has no effect on the binding of tropomyosin to actin, irrespective of the free Mg2+ concentration. However, maximal activation of the smooth muscle actomyosin ATPase in low free Mg2+ requires the presence of Ca2+ and stoichiometric binding of tropomyosin to actin. The lack of effect of Ca2+ on the binding of tropomyosin to actin shows that the activation of actomyosin ATPase by Ca2+ in the presence of tropomyosin is not due to a calcium-mediated binding of tropomyosin to actin.     

The ends of tropomyosin are major determinants of actin affinity and myosin subfragment 1-induced binding to F-actin in the open state

Tropomyosin (TM) is thought to exist in equilibrium between two states on F-actin, closed and open [Geeves, M. A., and Lehrer, S. S. (1994) Biophys. J. 67, 273-282]. Myosin shifts the equilibrium to the open state in which myosin binds strongly and develops force. Tropomyosin isoforms, that primarily differ in their N- and C-terminal sequences, have different equilibria between the closed and open states. The aim of the research is to understand how the alternate ends of TM affect cooperative actin binding and the relationship between actin affinity and the cooperativity with which myosin S1 promotes binding of TM to actin in the open state. A series of rat alpha-tropomyosin variants was expressed in Escherichia coli that are identical except for the ends, which are encoded by exons 1a or 1b and exons 9a, 9c or 9d. Both the N- and C-terminal sequences, and the particular combination within a TM molecule, determine actin affinity. Compared to tropomyosins with an exon 1a-encoded N-terminus, found in long isoforms, the exon 1b-encoded sequence, expressed in 247-residue nonmuscle tropomyosins, increases actin affinity in tropomyosins expressing 9a or 9d but has little effect with 9c, a brain-specific exon. The relative actin affinities, in decreasing order, are 1b9d > 1b9a > acetylated 1a9a > 1a9d > 1a9a > or = 1a9c congruent with 1b9c. Myosin S1 greatly increases the affinity of all tropomyosin variants for actin. In this, the actin affinity is the primary factor in the cooperativity with which myosin S1 induces TM binding to actin in the open state; generally, the higher the actin affinity, the lower the occupancy by myosin required to saturate the actin with tropomyosin: 1b9d >1a9d> 1b9a > or = acetylated 1a9a > 1a9a > 1a9c congruent with 1b9c.     

Interaction between caldesmon and tropomyosin in the presence and absence of smooth muscle actin

Cysteine residues of caldesmon were labeled with the fluorescent reagent N-(1-pyrenyl)maleimide. The number of sulfhydryl (SH) groups in caldesmon was around 3.5 on the basis of reactivity to 5,5'-dithiobis(2-nitrobenzoate); 80% of the SH groups were labeled with pyrene. The fluorescence spectrum from pyrene-caldesmon showed the presence of excited monomer and dimer (excimer). As the ionic strength increased, excimer fluorescence decreased, disappearing at salt concentrations higher than around 50 mM. The labeling of caldesmon with pyrene did not affect its ability to inhibit actin activation of heavy meromyosin Mg-ATPase and the release of this inhibition in the presence of Ca2+-calmodulin. Tropomyosin induced a change in the fluorescence spectrum of pyrene-caldesmon, indicating a conformational change associated with the interaction between caldesmon and tropomyosin. The affinity of caldesmon to tropomyosin was dependent on ionic strength. The binding constant was 5 x 10(6) M-1 in low salt, and the affinity was 20-fold less at ionic strengths close to physiological conditions. In the presence of actin, the affinity of caldesmon to tropomyosin was increased 5-fold. The addition of tropomyosin also changed the fluorescence spectrum of pyrene-caldesmon bound to actin filaments. The change in the conformation of tropomyosin, caused by the interaction between caldesmon and tropomyosin, was studied with pyrene-labeled tropomyosin. Fluorescence change was evident when unlabeled caldesmon was added to pyrene-tropomyosin bound to actin. These data suggest that the interaction between caldesmon and tropomyosin on the actin filament is associated with conformational changes on these thin filament associated proteins. These conformational changes may modulate the ability of thin filament to interact with myosin heads.     

Annealing of gelsolin-severed actin fragments by tropomyosin in the presence of Ca2+. Potentiation of the annealing process by caldesmon

Previous results from our laboratory have shown that 1) cultured rat cells contain two classes of tropomyosin (TM), one (high Mr TMs) with higher Mr values and greater affinity for actin than the other (low Mr TMs); 2) presaturation of F-actin with high Mr TMs, but not with low Mr TMs, inhibits both actin-severing and actin binding activities of gelsolin; and 3) nonmuscle caldesmon not only enhances the inhibitory effects of high Mr TMs but also makes low Mr TMs capable of inhibiting the severing activity of gelsolin (Ishikawa, R., Yamashiro, S., and Matsumura, F. (1989) J. Biol. Chem. 264, 7490-7497). These results suggest that gelsolin has much lower affinity for F-actin-TM-caldesmon complexes than for pure F-actin. We have therefore examined whether addition of TM and/or caldesmon to gelsolin-severed actin filaments can make gelsolin dissociate from barbed ends of actin filaments, resulting in annealing of short actin filaments into long ones. Flow birefringence and electron microscopic studies have suggested that high Mr TMs slowly and partially anneal gelsolin-severed actin fragments in 3 h, whereas low Mr TMs have no effects. Nonmuscle caldesmon greatly potentiates the effects of high Mr TMs and accelerates the process to 20 min, whereas nonmuscle caldesmon alone shows no effects. Furthermore, nonmuscle caldesmon makes low Mr TMs capable of reversing gelsolin-severing action. Actin binding assay has shown that gelsolin (or a gelsolin-actin complex) is dissociated from these annealed actin filaments. Smooth muscle TM and smooth muscle caldesmon also appear to anneal gelsolin-severed actin fragments as do high Mr TMs and nonmuscle caldesmon. Calmodulin decreases the potentiation effects of caldesmon as calmodulin inhibits actin binding of caldesmon. These results suggest that tropomyosin and caldesmon may regulate both capping and severing activities of gelsolin.     

The inhibitory region of troponin-I alters the ability of F-actin to interact with different segments of myosin.

Peptides corresponding to the N-terminus of skeletal myosin light chain 1 (rsMLC1 1-37) and the short loop of human cardiac beta-myosin (hcM398-414) have been shown to interact with skeletal F-actin by NMR and fluorescence measurements. Skeletal tropomyosin strengthens the binding of the myosin peptides to actin but does not interact with the peptides. The binding of peptides corresponding to the inhibitory region of cardiac troponin I (e.g. hcTnI128-153) to F-actin to form a 1 : 1 molar complex is also strengthened in the presence of tropomyosin. In the presence of inhibitory peptide at relatively lower concentrations the myosin peptides and a troponin I peptide C-terminal to the inhibitory region, rcTnI161-181, all dissociate from F-actin. Structural and fluorescence evidence indicate that the troponin I inhibitory region and the myosin peptides do not bind in an identical manner to F-actin. It is concluded that the binding of the inhibitory region of troponin I to F-actin produces a conformational change in the actin monomer with the result that interaction at different locations of F-actin is impeded. These observations are interpreted to indicate that a major conformational change occurs in actin on binding to troponin I that is fundamental to the regulatory process in muscle. The data are discussed in the context of tropomyosin's ability to stabilize the actin filament and facilitate the transmission of the conformational change to actin monomers not in direct contact with troponin I.     

Caldesmon freezes the structure of actin filaments during the actomyosin ATPase cycle

Hybrid contractile apparatus was reconstituted in skeletal muscle ghost fibers by incorporation of skeletal muscle myosin subfragment 1 (S1), smooth muscle tropomyosin and caldesmon. The spatial orientation of FITC-phalloidin-labeled actin and IAEDANS-labeled S1 during sequential steps of the acto-S1 ATPase cycle was studied by measurement of polarized fluorescence in the absence or presence of nucleotides conditioning the binding affinity of both proteins. In the fibers devoid of caldesmon addition of nucleotides evoked unidirectional synchronous changes in the orientation of the fluorescent probes attached to F-actin or S1. The results support the suggestion on the multistep rotation of the cross-bridge (myosin head and actin monomers) during the ATPase cycle. The maximal cross-bridge rotation by 7 degrees relative to the fiber axis and the increase in its rigidity by 30% were observed at transition between A**.M**.ADP.Pi (weak binding) and A--.M--.ADP (strong binding) states. When caldesmon was present in the fibers (OFF-state of the thin filament) the unidirectional changes in the orientation of actin monomers and S1 were uncoupled. The tilting of the myosin head and of the actin monomer decreased by 29% and 90%, respectively. It is suggested that in the "closed" position caldesmon "freezes" the actin filament structure and induces the transition of the intermediate state of actomyosin towards the weak-binding states, thereby inhibiting the ATPase activity of the actomyosin.     

Kinetic analysis of F-actin depolymerization in the presence of platelet gelsolin and gelsolin-actin complexes

Platelet gelsolin (G), a 90,000-mol-wt protein, binds tightly to actin (A) and calcium at low ionic strength to form a 1:2:2 complex, GA2Ca2 (Bryan, J., and M. Kurth, 1984, J. Biol. Chem. 259:7480-7487). Chromatography of actin and gelsolin mixtures in EGTA-containing solutions isolates a stable binary complex, GA1Ca1 (Kurth, M., and J. Bryan, 1984, J. Biol. Chem. 259:7473-7479). The effects of platelet gelsolin and the binary gelsolin-actin complex on the depolymerization kinetics of rabbit skeletal muscle actin were studied by diluting pyrenyl F-actin into gelsolin or complex-containing buffers; a decrease in fluorescence represents disassembly of filaments. Dilution of F- actin to below the critical concentration required for filament assembly gave a biphasic depolymerization curve with both fast and slow components. Dilution into buffers containing gelsolin, as GCa2, increased the rate of depolymerization and gave a first order decay. The rate of decrease in fluorescence was found to be gelsolin concentration dependent. Electron microscopy of samples taken shortly after dilution into GCa2 showed a marked reduction in filament length consistent with filament severing and an increase in the number of ends. Conversely, occupancy of the EGTA-stable actin-binding site by an actin monomer eliminated the severing activity. Dilution of F-actin into the gelsolin-actin complex, either as GA1Ca1 or GA1Ca2, resulted in a decrease in the rate of depolymerization that was consistent with filament end capping. This result indicates that the EGTA-stable binding site is required and must be unoccupied for filament severing to occur. The effectiveness of gelsolin, GCa2, in causing filament depolymerization was dependent upon the ionic conditions: in KCI, actin filaments appeared to be more stable and less susceptible to gelsolin, whereas in Mg2+, actin filaments were more easily fragmented. Finally, a comparison of the number of kinetically active ends generated when filaments were diluted into gelsolin versus the number formed when gelsolin can function as a nucleation site suggests that gelsolin may sever more than once. The data are consistent with a mechanism where gelsolin, with both actin-binding sites unoccupied, can sever but not cap F-actin. Occupancy of the EGTA-stable binding site yields a gelsolin-actin complex that can no longer sever filaments, but can cap filament ends.     

Modulation of actin-bundling activity of 55-kDa protein by multiple isoforms of tropomyosin

Cultured rat cells contain five isoforms of tropomyosin (Matsumura, F., Yamashiro-Matsumura, S., and Lin, J.J.-C. (1983) J. Biol. Chem. 258, 6636-6644). To explore the roles of the multiple tropomyosin isoforms in the microfilament organization of cultured cells, we have examined effects of tropomyosins on the bundling activity of the 55-kDa protein recently purified from HeLa cells (Yamashiro-Matsumura, S., and Matsumura, F. (1985) J. Biol. Chem. 260, 5087-5097). Maximum bundling of F-actin was observed at a molar ratio of 55-kDa protein to actin higher than 1:8. None of the isoforms of cultured rat cell tropomyosin significantly altered the F-actin-bundling activity of 55-kDa protein at this ratio, whereas skeletal muscle tropomyosin inhibited the bundling activity to about 50%. Also, cultured cell tropomyosins did not inhibit binding of 55-kDa protein to actin, whereas skeletal muscle tropomyosin inhibited it by 50%. The effect of 55-kDa protein on the binding of tropomyosin to actin varied with the isoform type of tropomyosin. Most (80%) of the tropomyosins with low Mr values (Mr 32,400 or 32,000) were caused to dissociate from actin by 55-kDa protein, but only 20% of tropomyosins with high Mr values (Mr 40,000 or 36,500) was dissociated from actin in these conditions. Immunofluorescence has shown that, while tropomyosin was localized in stress fibers, 55-kDa protein was found in microspikes as well as stress fibers, both of which are known to contain bundles of microfilaments. Therefore, we suggest that 55-kDa protein together with the multiple tropomyosin isoforms may regulate the formation of two types of actin-filament bundles, bundles containing tropomyosin and those without tropomyosin.     

Caldesmon restricts the movement of both C- and N-termini of tropomyosin on F-actin in ghost fibers during the actomyosin ATPase cycle

New data on the movements of tropomyosin singly labeled at alpha- or beta-chain during the ATP hydrolysis cycle in reconstituted ghost fibers have been obtained by using the polarized fluorescence technique which allowed us following the azimuthal movements of tropomyosin on actin filaments. Pronounced structural changes in tropomyosin evoked by myosin heads suggested the "rolling" of the tropomyosin molecule on F-actin surface during the ATP hydrolysis cycle. The movements of actin-bound tropomyosin correlated to the strength of S1 to actin binding. Weak binding of myosin to actin led to an increase in the affinity of the tropomyosin N-terminus to actin with simultaneous decrease in the affinity of the C-terminus. On the contrary, strong binding of myosin to actin resulted in the opposite changes of the affinity to actin of both ends of the tropomyosin molecule. Caldesmon inhibited the "rolling" of tropomyosin on the surface of the thin filament during the ATP hydrolysis cycle, drastically decreased the affinity of the whole tropomyosin molecule to actin, and "freezed" tropomyosin in the position characteristic of the weak binding of myosin to actin.     

The effect of phosphorylation of myosin light chains on the structural state of tropomyosin in thin filaments, decorated with heavy meromyosin

The structural state of tropomyosin (TM) modified by 5-(iodoacetamidoethyl)-aminonaphthalene-1-sulfonate (1.5-IAEDANS) upon F-actin decoration with myosin subfragment 1 (S1) and heavy meromyosin (HMM) in glycerinated myosin- and troponin-free muscle fibers was studied. HMM preparations contained native phosphorylated myosin light chains, while S1 preparations did not. The changes in the polarized fluorescence of 1.5-IAEDANS-TM during the F-actin interaction with S1 were independent of light chains phosphorylation and Ca2+ concentration, but were dependent on these factors during the F-actin interaction with HMM. The binding of myosin heads to F-actin is supposed to initiate conformational changes in TM which are accompanied by changes in the flexibility and molecular arrangement of TM. In the presence of light chains, the structural changes in TM depend on light chains phosphorylation and Ca2+ concentration. The conformational changes in TM seem to be responsible for the mechanisms of coupling of the myosin and tropomyosin modulation system during the actin-myosin interaction in skeletal muscles.     

Differential mobility of skeletal and cardiac tropomyosin on the surface of F-actin.

Polarized phosphorescence from the triplet probe erythrosin-5-iodoacetamide attached to sulfhydryls in rabbit skeletal and cardiac muscle tropomyosin (Tm) was used to measure the microsecond rotational dynamics of these tropomyosins in a complex with F-actin. The steady-state phosphorescence anisotropy of skeletal tropomyosin on F-actin was 0.025 +/- 0.005 at 20 degrees C; the comparable anisotropy for cardiac tropomyosin was 0.010 +/- 0. 003. Measurements of the anisotropy as a function of temperature and solution viscosity (modulated by addition of glycerol) indicated that both skeletal and cardiac tropomyosin undergo complex rotational motions on the surface of F-actin. Models assuming either long axis rotation of a rigid rod or torsional twisting of a flexible rod adequately fit these data; both analyses indicated that cardiac Tm is more mobile than skeletal Tm and that the increased mobility on the surface of F-actin reflected either the rotational motion of a smaller physical unit or the torsional twisting of a less rigid molecule. The binding of myosin heads (S1) to the Tm-F-actin complexes increased the anisotropy to 0.049 +/- 0.004 for skeletal and 0.054 +/- 0.007 for cardiac tropomyosin. The titration of the skeletal tropomyosin-F-actin complex by S1 showed a break at an S1/actin ratio of 0.14; this complex had an anisotropy of 0.040 +/- 0.007, suggesting that one bound head effectively restricted the motion of each skeletal tropomyosin. A similar titration with cardiac tropomyosin reached a plateau at an S1/actin ratio of 0.4, suggesting that 2-3 myosin heads are required to immobilize cardiac Tm. Surface mobility is predicted by structural models of the interaction of tropomyosin with the actin filament while the decrease in tropomyosin mobility upon S1 binding is consistent with current theories for the proposed role of myosin binding in the mechanism of tropomyosin-based regulation of muscle contraction.     

Fluorescence from pyrene-labeled native and reconstituted chicken gizzard tropomyosins

Sulfhydryl groups at Cys-36 on the beta chain and at Cys-190 on the gamma chain of chicken gizzard tropomyosin were reacted with the pyrene-containing sulfhydryl-specific reagents N-(1-pyrenyl)iodoacetamide and N-(1-pyrenyl)maleimide. Tropomyosin prepared and labeled under nondenaturing conditions displayed significant pyrene monomer emission but low levels of pyrene excimer fluorescence. In contrast, tropomyosin subjected to denaturation and renaturation prior to labeling, or labeled in the denatured state prior to renaturation, displayed considerable excimer emission. Furthermore, labeling of isolated beta or gamma chains in denaturant, followed by reconstitution, gave separate samples of beta beta- and gamma gamma-tropomyosin that exhibited even greater pyrene excimer to monomer emission ratios. As pyrene excimers can form only when an excited pyrene is immediately adjacent to a ground state pyrene, i.e., when the labeled Cys residues on the two chains in a tropomyosin coiled coil share the same cross section, these results support conclusions based upon chemical crosslinking studies [C. Sanders, L. D. Burtnick, and L. B. Smillie (1986) J. Biol. Chem. 261, 12774-12778] that native gizzard tropomyosin exists predominantly as a beta gamma-heterodimer. In addition, the low degree of labeling of native gizzard tropomyosin and the differences in degrees of labeling of beta beta- and gamma gamma-tropomyosins in the absence of denaturants reflect on the accessibilities of the sulfhydryl groups in these tropomyosin isoforms. Circular dichroism measurements indicate that the labeled proteins form stable coiled coil structures that have thermal stabilities comparable to that of the native protein.     

Effect of ethanol on tropomyosin in solution and in reconstituted thin filaments

The effects of ethanol at concentrations below 10% on the conformation of tropomyosin, its end-to-end polymerization, its binding to F-actin, and its effects on actomyosin ATPase activity were studied. Ethanol stabilized the tropomyosin conformation by shifting the helix thermal unfolding profile to higher temperatures, and increased the end-to-end polymerization of tropomyosin. Ethanol-induced changes in the excimer fluorescence of pyrene-tropomyosin indicated that its conformation was stabilized by ethanol both free and bound to F-actin. Effects of tropomyosin and tropomyosin-troponin on actomyosin ATPase activity were measured under conditions for which tropomyosin binding to F-actin increases the activity. Under conditions for which the binding of tropomyosin to F-actin is optimum, in the presence of tropomyosin, the actomyosin ATPase activity decreased as the ethanol concentration increased, further indicating that ethanol induces a structural change in the tropomyosin-F-actin complex. Under conditions for which the binding of tropomyosin to F-actin is weak (low salt or high temperature), addition of ethanol increased the ATPase activity due to increased binding of tropomyosin to F-actin. Thus, ethanol appears to modify actomyosin ATPase activity by increasing the binding of tropomyosin to F-actin and affecting the structure of tropomyosin in the tropomyosin-F-actin filament.