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).

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.

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.

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.

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.

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.

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.

Differential scanning calorimetric study of the complexes of modified myosin subfragment 1 with ADP and vanadate or beryllium fluoride.

The effects of various modifications of rabbit skeletal myosin subfragment 1 on the thermal denaturation of subfragment 1 in ternary complexes with Mg-ADP and orthovanadate (V1) or beryllium fluoride (BeFx) have been studied by differential scanning calorimetry. It has been shown that specific modifications of SH1 group of Cys-707 by different sulfhydryl reagents, trinitrophenylation of Lys-83, and reductive methylation of lysine residues promote the decomposition of the S1.ADP.Vi complex and change the character of structural transitions of the subfragment 1 molecule induced by the formation of this complex, but they have much less or no influence on subfragment 1 thermal stability in the S1.ADP.BeFx complex. Thus, the differential scanning calorimetric studies on modified subfragment 1 preparations reveal a significant difference between S1.ADP.Vi and S1.ADP.BeFx complexes. It is suggested that S1.ADP.Vi and S1.ADP.BeFx complexes represent structural analogues of different transition states of the ATPase cycle, namely the intermediate states S1**.ADP.Pi and S1*.ATP, respectively. It is also proposed that during formation of the S1.ADP.Vi complex the region containing both Cys-707 and Lys-83 plays an important role in the spread of conformational changes from the active site of subfragment 1 ATPase throughout the structure of the entire subfragment 1 molecule. In such a case, the effects of reductive methylation of lysine residues on the subfragment 1 structure in the S1.ADP.Vi complex are related to the modification of Lys-83.

[Calorimetric study of stable complexes of myosin subfragment I with adenosine diphosphate and phosphate analogs]. / Kalorimetricheskie issledovaniia stabil'nykh kompleksov subfragmenta 1 miozina s analogami adenozindifosfata i fosfata.

This short review is concerned with the application of the method of differential scanning calorimetry to study the conformational changes of isolated myosin head (myosin subfragment 1, S1) caused by the formation of the S1 complexes with Mg(2+)-ADP and P(i) analogues such as orthovanadate (V), aluminium fluoride (AIF4-) or beryllium fluoride (BeFx). These changes of the whole S1 molecule are reflected in a significant increase of S1 thermal stability and in a pronounced increase of the cooperativity of the thermal denaturation. Since the complexes S1-ADP-V, S1-ADP-AIF4- and S1-ADP-BeFx are stable analogues of the S1**-ADP-P(i) transition state of the S1-catalyzed ATP hydrolysis, it is concluded that DSC studies with these complexes offer a new and promising approach to investigate the structural changes which occur in the myosin head during Mg(2+)-ATPase reaction.

[The effect of modifying the lysine-83 residue on the thermal stability of myosin subfragment 1 and changes in it caused by nucleotide binding]. / Vliianie modifikatsii ostatka lizina-83 na termostabil'nost' subfragmenta 1 miozina i ee izmeneniia, vyzvaemye sviazyvaniem nukleotidov.

The effects of trinitrophenylation of lysyl residues of rabbit skeletal myosin subfragment 1 (S1) on thermal denaturation of S1 in the absence of nucleotides, in the presence of ADP and within S1 complexes with ADP and Pi analogues, orthovanadate (Vi) or beryllium fluoride (BeFx), have been studied by differential scanning calorimetry. It has been shown that lysyl trinitrophenylation significantly affects the thermal stability of S1, changes its domain structure, promotes the decomposition of S1.ADP.Vi and S1.ADP.BeFx complexes, and strongly prevents the structural changes in the S1 molecule induced by the formation of the S1.ADP.Vi complex without any effect on the thermal stability of S1 within S1.ADP and S1.ADP.BeFx complexes. It has been demonstrated that the effects of trinitrophenylation on the S1 structure are mainly due to specific modification of the epsilon-amino group of the Lys-83 residue.

Interaction of F-actin with phosphate analogues studied by differential scanning calorimetry.

The thermal unfolding of F-actin and the changes induced in it by the binding of phosphate analogues were studied by differential scanning calorimetry. It is shown that the conformation of actin is drastically altered by interaction with beryllium fluoride or aluminium fluoride, while the effects of vanadate and phosphate are negligible. The effect of beryllium fluoride on the F-actin structure, as reflected in a significant increase of the actin thermal stability, is much more pronounced in the presence of Mg2+ than in the case of F-actin polymerized by KCl or LiCl in the absence of Mg2+. It is concluded that differential scanning calorimetry is a very convenient method for probing the conformational changes in F-actin caused by the interaction with phosphate analogues.

Kinetics of interaction of myosin subfragment 1 isoforms with F-actin.

The interaction of the myosin subfragment-1 (S-1) isoforms containing different alkali light chains A1 and A2 with F-actin has been studied using the stopped-flow technique and sedimentation in an analytical ultracentrifuge. The data obtained suggest a different mode of attachment of the S-1 isoforms to F-actin filaments.

Effects of nucleotide binding on thermal transitions and domain structure of myosin subfragment 1.

The thermal unfolding and domain structure of myosin subfragment 1 (S1) from rabbit skeletal muscles and their changes induced by nucleotide binding were studied by differential scanning calorimetry. The binding of ADP to S1 practically does not influence the position of the thermal transition (maximum at 47.2 degrees C), while the binding of the non-hydrolysable analogue of ATP, adenosine 5'-[beta, gamma-imido]triphosphate (AdoPP[NH]P) to S1, or trapping of ADP in S1 by orthovanadate (Vi), shift the maximum of the heat adsorption curve for S1 up to 53.2 and 56.1 degrees C, respectively. Such an increase of S1 thermostability in the complexes S1-AdoPP[NH]P and S1-ADP-Vi is confirmed by results of turbidity and tryptophan fluorescence measurements. The total heat adsorption curves for S1 and its complexes with nucleotides were decomposed into elementary peaks corresponding to the melting of structural domains in the S1 molecule. Quantitative analysis of the data shows that the domain structure of S1 in the complexes S1-AdoPP[NH]P and S1-ADP-Vi is similar and differs radically from that of nucleotide-free S1 and S1 in the S1-ADP complex. These data are the first direct evidence that the S1 molecule can be in two main conformations which may correspond to different states during the ATP hydrolysis: one of them corresponds to nucleotide-free S1 and to the complex S1-ADP, and the other corresponds to the intermediate complexes S1-ATP and S1-ADP-Pi. Surprisingly it turned out that the domain structure of S1 with ADP trapped by p-phenylene-N, N'-dimaleimide (pPDM) thiol cross-linking almost does not differ from that of the nucleotide-free S1. This means that pPDM-cross-linked S1 in contrast to S1-AdoPP[NH]P and S1-ADP-Vi can not be considered a structural analogue of the intermediate complexes S1-ATP and S1-ADP-Pi.

[Interaction of isoforms of subfragment-1 of myosin, containing fluorescently labelled alkaline light chains, with muscle fiber actin]. / Vzaimodeistvie izoform subfragmenta-1 miozina, soderzhashchikh fluorestsentno mechennye shchelochnye legkie tsepi, s aktinom myshechnykh volokon.

Using polarization microfluorimetry, the interaction of myosin subfragment 1 (S1) isoforms containing alkali light chains A1 and A2 respectively (S1(A1) and S1(A2] with F-actin of single glycerinated rabbit skeletal muscle fibers was studied. The alkali light chains of S1 were substituted by reassociation for A1 or A2 chains modified by a fluorescent label (1.5-IAEDANS) at the single SH-group located in the C-terminus. It was found that in S1(A1) bound to muscle fiber F-actin the mobility of the fluorescent label is lower than in S1(A2). At the same time the S1(A1) and S1(A2) interaction with F-actin induces similar changes in polarized fluorescence of rhodamine linked to falloidine which, in turn, is specifically bound to F-actin. It is concluded that the both S1 isoforms bind to F-actin and produce similar effects on the conformational state of actin filaments in muscle fibers. Local differences between S1(A1) and S1(A2) seem to be due to the interaction of the N-terminus of A1 within S1(A1) with the C-terminal region of actin.

The effect of alkali light chains on the thermal stability of myosin subfragment 1.

Thermal denaturation of myosin subfragment 1 (S1) isoforms from rabbit skeletal muscle containing the different alkali light chains A1 and A2 [S1(A1) and S1(A2), respectively] were studied by various methods. Turbidity measurements showed that thermally induced (heating rate 1 degrees C min-1) aggregation of S1(A1) occurs at lower temperatures than that of S1(A2). However, the temperature dependences of the tryptophan fluorescence spectrum and that for ATPase inactivation were the same for S1(A1) and S1(A2). Thermal denaturation of the S1 isoforms was also studied by differential scanning microcalorimetry with the 'successive annealing' method. Three independently melting cooperative regions (domains) were revealed in the molecules of both isoforms. Heat sorption curves for the S1 isoforms were different only for the most thermolabile domain, which had a maximum at 36 degrees C for S1(A1) and at 40.5 degrees C for S1(A2). Two other peaks had maxima at 46-47 degrees C and 50-51 degrees C for both isoforms. It is proposed that alkali light chains A1 and A2 differently affect the conformation of the most thermolabile domain, which probably does not contain trytophan residues and does not take part directly in the formation of the active site of the S1 ATPase.