Molecular Mechanisms of Pathologies of Skeletal and Cardiac Muscles Caused by Point Mutations in the Tropomyosin Genes.

The review is devoted to tropomyosin (Tpm) - actin-binding protein, which plays a crucial role in the regulation of contraction of skeletal and cardiac muscles. Special attention is paid to myopathies and cardiomyopathies - severe hereditary diseases of skeletal and cardiac muscles associated with point mutations in Tpm genes. The current views on the molecular mechanisms of these diseases and the effects of such mutations on the Tpm structure and functions are considered in detail. Besides, some part of the review is devoted to analysis of the properties of Tpm homodimers and heterodimers with myopathic substitutions of amino acid residues in only one of the two chains of the Tpm dimeric molecule.

Essential Light Chains of Myosin and Their Role in Functioning of the Myosin Motor.

This review summarizes current data on the structure and functions of myosin essential light chains (ELCs) and on their role in functioning of the myosin head as a molecular motor. The data on structural and functional features of the N-terminal extension of myosin ELC from skeletal and cardiac muscles are analyzed; the role of this extension in the ATP-dependent interaction of myosin heads with actin in the molecular mechanism of muscle contraction is discussed. The data on possible interactions of the ELC N-terminal extension with the myosin head motor domain in the myosin ATPase cycle are presented, including the results of the authors' studies that are in favor of such interactions.

Intermolecular Interactions of Myosin Subfragment 1 Induced by the N-Terminal Extension of Essential Light Chain 1.

We applied dynamic light scattering (DLS) to compare aggregation properties of two isoforms of myosin subfragment 1 (S1) containing different "essential" (or "alkali") light chains, A1 or A2, which differ by the presence of an N-terminal extension in A1. Upon mild heating (up to 40°C), which was not accompanied by thermal denaturation of the protein, we observed a significant growth in the hydrodynamic radius of the particles for S1(A1), from ~18 to ~600-700 nm, whereas the radius of S1(A2) remained unchanged and equal to ~18 nm. Similar difference between S1(A1) and S1(A2) was observed in the presence of ADP. In contrast, no differences were observed by DLS between these two S1 isoforms in their complexes S1-ADP-BeFx and S1-ADP-AlF4- which mimic the S1 ATPase intermediate states S1*-ATP and S1**-ADP-Pi. We propose that during the ATPase cycle the A1 N-terminal extension can interact with the motor domain of the same S1 molecule, and this can explain why S1(A1) and S1(A2) in S1-ADP-BeFx and S1-ADP-AlF4- complexes do not differ in their aggregation properties. In the absence of nucleotides (or in the presence of ADP), the A1 N-terminal extension can interact with actin, thus forming an additional actin-binding site on the myosin head. However, in the absence of actin, this extension seems to be unable to undergo intramolecular interaction, but it probably can interact with the motor domain of another S1 molecule. These intermolecular interactions of the A1 N-terminus can explain unusual aggregation properties of S1(A1).

Stabilization of the Central Part of Tropomyosin Molecule Alters the Ca2+-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.

Tropomyosin: double helix from the protein world.

This review concerns the structure and functions of tropomyosin (TM), an actin-binding protein that plays a key role in the regulation of muscle contraction. The TM molecule is a dimer of α-helices, which form a coiled-coil. Recent views on the TM structure are analyzed, and special attention is concentrated on those structural traits of the TM molecule that distinguish it from the other coiled-coil proteins. Modern data are presented on TM functional properties, such as its interaction with actin and ability to move on the surface of actin filaments, which underlies the regulation of the actin-myosin interaction upon contraction of skeletal and cardiac muscles. Also, part of the review is devoted to analysis of the effects of mutations in TM genes associated with muscle diseases (myopathies) on the structure and functions of TM.

Effects of Myosin "essential" light chain A1 on the aggregation properties of the Myosin head.

We compared the thermal aggregation properties of two isoforms of the isolated myosin head (myosin subfragment 1, S1) containing different "essential" (or "alkali") light chains, A1 or A2. Temperature dependencies for the aggregation of these two S1 isoforms, as measured by the increase in turbidity, were compared with the temperature dependencies of their thermal denaturation obtained from differential scanning calorimetry (DSC) experiments. At relatively high ionic strength (in the presence of 100 mM KCl) close to its physiological values in muscle fibers, we have found no appreciable difference between the two S1 isoforms in their thermally induced aggregation. Under these conditions, the aggregation of both S1 isoforms was independent of the protein concentration and resulted from their irreversible denaturation, which led to the cohesion of denatured S1 molecules. In contrast, a significant difference between these S1 isoforms was revealed in their aggregation measured at low ionic strength. Under these conditions, the aggregation of S1 containing a light chain A1 (but not A2) was strongly dependent on protein concentration, the increase of which (from 0.125 to 2.0 mg/ml) shifted the aggregation curve by ~10 degrees towards the lower temperatures. It has been concluded that the aggregation properties of this S1 isoform at low ionic strength is basically determined by intermolecular interactions of the N-terminal extension of the A1 light chain (which is absent in the A2 light chain) with other S1 molecules. These interactions seem to be independent of the S1 thermal denaturation, and they may take place even at low temperature.

Dissociative mechanism of F-actin thermal denaturation.

We have applied differential scanning calorimetry to investigate thermal unfolding of F-actin. It has been shown that the thermal stability of F-actin strongly depends on ADP concentration. The transition temperature, T(m), increases with increasing ADP concentration up to 1 mM. The T(m) value also depends on the concentration of F-actin: it increases by almost 3 degrees C as the F-actin concentration is increased from 0.5 to 2.0 mg/ml. Similar dependence of the T(m) value on protein concentration was demonstrated for F-actin stabilized by phalloidin, whereas it was much less pronounced in the presence of AlF4(-). However, T(m) was independent of protein concentration in the case of monomeric G-actin. The results suggest that at least two reversible stages precede irreversible thermal denaturation of F-actin; one of them is dissociation of ADP from actin subunits, and another is dissociation of subunits from the ends of actin filaments. The model explains why unfolding of F-actin depends on both ADP and protein concentration.

Actomyosin systems of biological motility.

Evolution of notions on the molecular mechanism of muscle contraction and other events based on the actin-myosin interaction, from the middle of XX century to the present time, is briefly reviewed, including recent views on the functioning of the myosin head as a "molecular motor". The results of structural and functional studies on the myosin head performed by the author and his colleagues using differential scanning calorimetry are also reviewed.

Effects of troponin on thermal unfolding of actin-bound tropomyosin.

Differential scanning calorimetry (DSC) was used to study the effect of troponin (Tn) and its isolated components on the thermal unfolding of skeletal muscle tropomyosin (Tm) bound to F-actin. It is shown that in the absence of actin the thermal unfolding of Tm is expressed in two well-distinguished thermal transitions with maxima at 42.8 and 53.8 degrees C. Interaction with F-actin affects the character of thermal unfolding of Tm leading to appearance of a new Tm transition with maximum at about 48 degrees C, but it has no influence on the thermal denaturation of F-actin stabilized by aluminum fluoride, which occurs within the temperature region above 70 degrees C. Addition of troponin leads to significant increase in the cooperativity and enthalpy of the thermal transition of the actin-bound Tm. The most pronounced effect of Tn was observed in the absence of calcium. To elucidate how troponin complex affects the properties of Tm, we studied the influence of its isolated components, troponin I (TnI) and troponin T (TnT), on the thermal unfolding of actin-bound Tm. Isolated TnT and TnI do not demonstrate cooperative thermal transitions on heating up to 100 degrees C. However, addition of TnI, and especially of TnT, to the F-actin-Tm complex significantly increased the cooperativity of the thermal unfolding of actin-bound tropomyosin.

Changes in the thermal unfolding of p-phenylenedimaleimide-modified myosin subfragment 1 induced by its 'weak' binding to F-actin.

Differential scanning calorimetry (DSC) was used to analyze the thermal unfolding of myosin subfragment 1 (S1) with the SH1 (Cys-707) and SH2 (Cys-697) groups cross-linked by N,N'-p-phenylenedimaleimide (pPDM-S1). It has been shown that F-actin affects the thermal unfolding of pPDM-S1 only at very low ionic strength, when some part of pPDM-S1 binds weakly to F-actin, but not at higher ionic strength (200 mM KCl). The weak binding of pPDM-S1 to F-actin shifted the thermal transition of pPDM-S1 by about 5 degrees C to a higher temperature. This actin-induced increase in thermal stability of pPDM-S1 was similar to that observed with 'strong' binding of unmodified S1 to F-actin. Our results show that actin-induced structural changes revealed by DSC in the myosin head occur not only upon strong binding but also on weak binding of the head to F-actin, thus suggesting that these changes may occur before the power-stroke and play an important role in the motor function of the head.

Charge changes in loop 2 affect the thermal unfolding of the myosin motor domain bound to F-actin.

The thermal unfolding of Dictyostelium discoideum myosin head fragments with alterations in the actin-binding surface loop 2 was studied by differential scanning calorimetry. Lengthening of loop 2 without concomitant charge changes led to decreases in the transition temperature of not more than 1.8 degrees C. Insertions with multiple positive or negative charges had a stronger destabilizing effect and led to reductions in the thermal transition temperature of up to 3.7 degrees C. In the presence of nucleotide, most mutants displayed similar or higher transition temperatures than M765. Only constructs M765(11/+6) and M765(20/+12) with long positively charged inserts showed transition temperatures that were more than 2 degrees C below the values measured for M765 in the presence of ADP, ADP-V(i), and ADP-BeF(3). Interaction with F-actin in the presence of ADP shifted the thermal transition of M765 by 6 degrees C, from 49.1 to 55.1 degrees C. The actin-induced increase in thermal stability varied between 1.2 and 9.1 degrees C and showed a strong correlation with the mutant constructs' affinity for actin. Our results show that length and charge changes in loop 2 do not significantly affect nucleotide-induced structural changes in the myosin motor domain, but they affect structural changes that occur when the motor domain is strongly bound to actin and affect the coupling between the actin- and nucleotide-binding sites.

Complexes of smooth muscle tropomyosin with F-actin studied by differential scanning calorimetry.

Differential scanning calorimetry (DSC) and light scattering were used to analyze the interaction of duck gizzard tropomyosin (tropomyosin) with rabbit skeletal-muscle F-actin. In the absence of F-actin, tropomyosin, represented mainly by heterodimers, unfolds at 41 degrees C with a sharp thermal transition. Interaction of tropomyosin heterodimers with F-actin causes a 2-6 degrees C shift in the tropomyosin thermal transition to higher temperature, depending on the tropomyosin/actin molar ratio and protein concentration. A pronounced shift of the tropomyosin thermal transition was observed only for tropomyosin heterodimers, and not for homodimers. The most pronounced effect was observed after complete saturation of F-actin with tropomyosin molecules, at tropomyosin/actin molar ratios > 1 : 7. Under these conditions, two well-separated peaks of tropomyosin were observed on the thermogram besides the peak of F-actin, the peak characteristic of free tropomyosin heterodimer, and the peak with a maximum at 45-47 degrees C corresponding to tropomyosin bound to F-actin. By measuring the temperature-dependence of light scattering, we found that thermal unfolding of tropomyosin is accompanied by its dissociation from F-actin. Thermal unfolding of tropomyosin is almost completely reversible, whereas F-actin denatures irreversibly. The addition of tropomyosin has no effect on thermal unfolding of F-actin, which denatures with a maximum at 64 degrees C in the absence and at 78 degrees C in the presence of a twofold molar excess of phalloidin. After the F-actin-tropomyosin complex had been heated to 90 degrees C and then cooled (i.e. after complete irreversible denaturation of F-actin), only the peak characteristic of free tropomyosin was observed on the thermogram during reheating, whereas the thermal transitions of F-actin and actin-bound tropomyosin completely disappeared. Therefore, the DSC method allows changes in thermal unfolding of tropomyosin resulting from its interaction with F-actin to be probed very precisely.

Interaction of bacterial flagellum filaments with skeletal muscle myosin.

Interaction of isolated bacterial flagellum filaments (BFF) and intact flagella from E. coli MS 1350 and B. brevis G.-B.p+ with rabbit skeletal myosin was studied. BFF were shown to coprecipitate with myosin (but not with isolated myosin rod) at low ionic strength, that is, under conditions of myosin aggregation. The data of electron microscopy indicate that filaments of intact bacterial flagella interact with isolated myosin heads (myosin subfragment 1, S1), and this interaction is fully prevented by addition of Mg2+ -ATP. Addition of BFF inhibited both K+ -EDTA- and Ca2+ -ATPase activity of skeletal muscle myosin, but had no effect on its Mg2+ -ATPase activity. Monomeric flagellin did not coprecipitate with myosin and had no effect on its ATPase activities. BFF were shown to compete with F-actin in myosin binding. It is concluded that BFF interact with myosin heads and affect their ATPase activity. Thus, BFF composed of a single protein flagellin are in many respects similar to actin filaments. Common origin of actin and flagellin may be a reason for this similarity.

Use of stable analogs of myosin ATPase intermediates for kinetic studies of the "weak" binding of myosin heads to F-actin.

It is known that ternary complexes of myosin subfragment 1 (S1) with ADP and the Pi analogs beryllium fluoride (BeFx) and aluminum fluoride (AlF4-) are stable analogs of the myosin ATPase intermediates M* x ATP and M** x ADP x Pi, respectively. Using kinetic approaches, we compared the rate of formation of the complexes S1 x ADP x BeFx and S1 x ADP x AlF4- in the absence and in the presence of F-actin, as well as of the interaction of these complexes with F-actin. We show that in the absence of F-actin the formation of S1 x ADP x BeFx occurs much faster (3-4 min) than that of S1 x ADP x AlF4- (hours). The formation of these complexes in the presence of F-actin led to dissociation of S1 from F-actin, this process being monitored by a decrease in light scattering. The light scattering decrease of the acto-S1 complex occurred much faster after addition of BeFx (during 1 min) than after addition of AlF4- (more than 20 min). In both cases the light scattering of the acto-S1 complex decreased by 40-50%, but it remained much higher than that of F-actin measured in the absence of S1. The interaction of the S1 x ADP x BeFx and S1 x ADP x AlF4- complexes with F-actin was studied by the stopped-flow technique with high time resolution (no more than 0.6 sec after mixing of S1 with F-actin). We found that the binding of S1 x ADP x BeFx or S1 x ADP x AlF4- to F-actin is accompanied by a fast increase in light scattering, but it does not affect the fluorescence of a pyrene label specifically attached to F-actin. We conclude from these data that within this time range a "weak" binding of the S1 x ADP x BeFx and S1 x ADP x AlF4- complexes to F-actin occurs without the subsequent transition of the "weak" binding state to the "strong" binding state. Comparison of the light scattering kinetic curves shows that S1 x ADP x AlF4- binds to F-actin faster than S1 x ADP x BeFx does: the second-order rate constants for the "weak" binding to F-actin are (62.8 +/- 1.8) x 10(6) M-1 x sec-1 in the case of S1 x ADP x AlF4- and (22.6 +/- 0.4) x 10(6) M-1 x sec-1 in the case of S1 x ADP x BeFx. We conclude that the stable ternary complexes S1 x ADP x BeFx and S1 x ADP x AlF4- can be successfully used for kinetic studies of the "weak" binding of the myosin heads to F-actin.

Effects of SH1 and SH2 modifications on myosin: similarities and differences.

The properties of myosin modified at the SH2 group (Cys-697) were studied and compared with the previously reported properties of myosin modified at the SH1 group (Cys-707). 4-[N-[(iodoacetoxy)ethyl]-N methylamino]-7-nitrobenz-2-oxa-1, 3-diazole (IANBD) was used for selective modification of the SH2 group on myosin. SH2-labeled heavy meromyosin (SH2-HMM), similar to SH1-labeled HMM (SH1-HMM), did not propel actin filaments in the in vitro motility assays. SH1- and SH2-HMM produced similar amounts of load in the mixtures with unmodified HMM; the sliding speed of actin filaments gradually decreased with an increase in the fraction of either one of the modified HMMs in the mixture. In analogy to SH1-labeled myosin subfragment 1 (SH1-S1), SH2-labeled S1 (SH2-S1) activated regulated actin in the in vitro motility assays. SH2 modification inhibited Mg-ATPase of S1 and its activation by actin. The weak binding of S1 to actin was unaffected whereas the strong binding was weakened by SH2 modification. Overall, our results demonstrate similar behavior of SH1- and SH2-modified myosin heads in the in vitro motility assays despite some differences in their enzymatic properties. The effects of these modifications are ascribed to the location of the SH1-SH2 helix relative to other functional centers of S1.

Calorimetric studies of the thermal unfolding of smooth muscle myosin fragments and their complexes with ADP and phosphate analogs.

The thermal unfolding of turkey gizzard smooth muscle myosin subfragment 1 (S1) and heavy meromyosin (HMM) in the absence of added nucleotides, in the presence of ADP, and in S1 or HMM ternary complexes with ADP and Pi analogs, orthovanadate (Vi), beryllium fluoride (BeFx), or aluminum fluoride (AlF4-), have been studied by differential scanning calorimetry (DSC). It has been shown that the formation of these ternary complexes causes significant structural changes in S1 or in the heads of HMM which are reflected in a pronounced increase of the protein thermal stability. The effect of BeFx was less distinct than that of AlF4- or Vi. Phosphorylation of regulatory light chains (RLC) in S1 or in HMM had practically no influence on these effects. In general, the changes caused by various Pi analogs in smooth muscle S1 or HMM were similar to those observed earlier with skeletal muscle S1 devoid of RLC. It is concluded that RLC and their phosphorylation do not significantly affect the character of structural changes induced in motor domains of the HMM heads by the formation of ternary complexes HMM--ADP--Vi, HMM--ADP--AlF4-, and HMM--ADP--BeFx--stable analogs of the intermediate states of the HMM ATPase reaction, HMM.ADP.Pi and HMM. ATP.

Thermally induced chain exchange of smooth muscle tropomyosin dimers studied by differential scanning calorimetry.

The thermal unfolding of duck gizzard tropomyosin dimers, alphabeta, alphaalpha, and betabeta, and of a 1:1 mixture of alphaalpha and betabeta homodimers was studied by differential scanning calorimetry (DSC). Both alphaalpha and betabeta homodimers demonstrated a broad thermal transition with maxima at 37.4 degrees C and 44.6 degrees C, respectively. However, a sharp cooperative thermal transition at 41.5 degrees C characteristic for alphabeta heterodimer appeared on the thermogram of the mixture of homodimers. The appearance of this transition was prevented by disulfide cross-linking of polypeptide chains in the homodimers. Thus, DSC studies clearly demonstrate formation of tropomyosin heterodimers during heating of the mixture of homodimers and in agreement with earlier published reports indicate thermally induced chain exchange between tropomyosin dimers.

Differential scanning calorimetric studies on myosin and actin.

This review is concerned with the application of the method of differential scanning calorimetry (DSC) to structural and functional studies of myosin and actin--the main two proteins of muscles and many other systems of biological motility. The domain organization of these proteins as revealed by DSC is considered. Data are presented on the conformational changes which occur in the myosin head and in F-actin due to the formation of the ternary complexes with ADP and Pi analogs (such as orthovanadate, beryllium fluoride, or aluminum fluoride). Recent data on the application of DSC to studies on the interaction of F-actin with myosin heads and with tropomyosin are also considered. It is concluded that DSC offers a new and promising approach to probe the structural changes which occur in the myosin head and in F-actin during ATP hydrolysis and due to interaction of these proteins with each other.

Differential scanning calorimetric study of the thermal unfolding of the motor domain fragments of Dictyostelium discoideum myosin II.

The thermal unfolding of two recombinant fragments of the head of Dictyostelium discoideum myosin II was studied by differential scanning calorimetry. These fragments M754 and M761 correspond to the globular motor portion of the myosin head that contains ATP- and actin-binding sites but lacks the light chain binding domain. Our results show that M754 is less thermostable than M761: the maximum of the thermal transition occurred at 41.7 degrees C for M754 and at 45.6 degrees C for M761, and the calorimetric enthalpy value determined for M754 (677 kJ/mol) was about half of that for M761 (1417 kJ/mol). This indicates that the region containing residues 755-761 plays a very important role in the structural stabilization of the entire globular motor part of the myosin head. ADP binding induces structural changes in both myosin fragments which are reflected in a 2-3.5 degrees C shift of the thermal transitions to higher temperature. The formation of stable ternary complexes of these myosin fragments with ADP and phosphate analogues such as orthovanadate, beryllium fluoride or aluminium fluoride causes additional structural changes which are reflected in a pronounced increase of thermal stability. The effect of beryllium fluoride was less distinct than that of aluminium fluoride or orthovanadate. In general, the changes caused by various phosphate analogues were similar to those observed with skeletal myosin subfragment 1. Thus, structural changes revealed by differential scanning calorimetry in the myosin head, that are due to the formation of stable ternary complexes with ADP and Pi analogues, occur mainly in the globular motor portion of the head.

Interaction of myosin subfragment 1 with F-actin studied by differential scanning calorimetry.

The thermal unfolding of the myosin subfragment 1 (S1) and of filamentous actin (F-actin) in their strong complex obtained in the presence of ADP was studied by differential scanning calorimetry (DSC). It is shown that in the acto-S1 complexes S1 and F-actin melt separately, and thermal transitions of each protein can be easily followed. Interaction of S1 with F-actin significantly increases S1 thermal stability and also affects the thermal stability of F-actin. Although S1 unfolds at much lower temperature than F-actin, the molecules of S1 remain bound to F-actin even after full denaturation. Under these conditions S1 may induce cross-linking between actin filaments. It is concluded that DSC studies on the acto-S1 complexes offer a new and promising approach to investigate the structural changes which occur in the myosin head and in F-actin due to their interaction.