Proteins involved in the regulation of muscle contraction

In this section the DSC studies on the proteins responsible for regulation of actin-myosin interaction during muscle contraction will be reviewed. Only the pro teins asso ciated with actin filaments will be considered here. These will include tropomyosin and troponin in skeletal and cardiac muscles or caldesmon and calponin in smooth muscles.

TROPOMYOSIN

Tropomyosin (Tm) is a coiled-coil actin-binding protein bound along the length of actin filament in both muscle and non-muscle cells. Tm molecules bind to themselves in a end-to-end manner, and form a continuous structure along the actin filament. The presence of Tm on actin filaments confers cooperativity on the interaction between myosin heads and actin. Together with the other regulatory proteins, troponin in striated skeletal and cardiac muscles or caldesmon in smooth muscle, it takes part in the Ca2+ regulation of muscle contraction. The Tm subunits consist of nearly 100% a-helix, and two polypeptide chains assemble into parallel and in-reg-ister coiled-coil dimers. Two isoforms (a and P) each containing 284 residues are expressed in smooth and skeletal muscles. Smooth muscle Tm consists of a 1:1 mixture of a and P chains which are predominantly assembled into the aP heterodimers. In skeletal muscle the a and P chains are present in the ratio of (3-4):1, and they can form all three possible dimers: aa, PP, and aP. Under physiological conditions, skeletal Tm is a mixture of aa-homodimers and aP-heterodimers.

Thermal unfolding of tropomyosin and effects of an interchain disulfide bond

Thermal denaturation of Tm homodimers from skeletal and smooth muscles has been investigated by DSC in detail. As shown by Potekhin and Privalov in 1982 [82] and then confirmed by other authors [83-85], the Tm molecule consists of cooperative blocks with different thermal stabilities. For example, both aa and PP homodimers of muscle Tm exhibited broad multistate thermal transitions composed of at least three individual transitions (or calorimetric domains) [84, 85]. In contrast, aP heterodimers showed a sharp transition composed of only two calorimetric domains [85]. Moreover, DSC studies on smooth muscle Tm clearly demonstrated formation of aP heterodimers during heating of a 1:1 mixture of aa and PP homodimers [85]. Each chain of skeletal Tm dimer (both a and P) contains a cysteine residue at position 190, and therefore SH-groups of Cys-190 can form a disulfide bond between two Cys-190 belonging to two chains of the Tm dimer. Formation of this interchain disulfide bond was shown to increase substantially the thermal stability of skeletal Tm [86].

The DSC profile of skeletal a-aTm is represented by two well distinguished peaks with maxima at ~43oC and ~54oC (Fig. 9A). When the experiment is performed in the absence of P-mercaptoethanol, the low-temperature peak completely disappears during the second heating of the sample. On the other hand, this peak increases during the second heating of the sample containing P-mercaptoethanol (Fig. 9A) [79]. These results are in good agreement with the DSC data of Williams and Swenson [87]. These authors showed that the low-temperature peak of Tm corresponds to the melting of C-terminal half of the Tm molecule with reduced SH-groups of Cys-190, whereas the high-temperature peak is composed of two overlapping thermal transitions. One of them corresponds to the N-terminal half of Tm, and another - to the oxidized C-terminal half of Tm with Cys-190 cross-linked by disulfide bond between two chains of the Tm dimer. We suppose that during the first heating of Tm in the presence of P-mercaptoethanol the SH-groups of Cys-190

Fig. 9 (A) - DSC profiles of the thermal denaturation of the complex of rabbit skeletal aa-tropomyosin (aa-Tm) with F-actin stabilized by phalloidin. For comparison, the thermal unfolding of aa-Tm in the absence of F-actin is also shown. Dashed line curves were obtained by a re-heating the corresponding samples represented by solid line curves. Conditions: 30 |j,M aa-Tm, 46 |j,M F-actin, 70 |j,M phalloidin in 20 mM Hepes, pH 7.3, 2 mM MgCl2, 1 mM P-mercaptoethanol. The vertical bar corresponds to 10 |j,W. Heating rate was 1 K/ min. (B) - Temperature dependence of dissociation of the complex of aa-Tm with F-actin. Conditions were the same as for DSC experiment in (A). 100% corresponds to the difference between light scattering of the aa-Tm-F-actin complex measured at 25oC and that of pure F-actin stabilized by phalloidin which was temperature independent within the temperature range used. A decrease in the light scattering intensity reflects dissociation of the aa-Tm-F-actin complex

I DSC I

I DSC I

Fig. 9 (A) - DSC profiles of the thermal denaturation of the complex of rabbit skeletal aa-tropomyosin (aa-Tm) with F-actin stabilized by phalloidin. For comparison, the thermal unfolding of aa-Tm in the absence of F-actin is also shown. Dashed line curves were obtained by a re-heating the corresponding samples represented by solid line curves. Conditions: 30 |j,M aa-Tm, 46 |j,M F-actin, 70 |j,M phalloidin in 20 mM Hepes, pH 7.3, 2 mM MgCl2, 1 mM P-mercaptoethanol. The vertical bar corresponds to 10 |j,W. Heating rate was 1 K/ min. (B) - Temperature dependence of dissociation of the complex of aa-Tm with F-actin. Conditions were the same as for DSC experiment in (A). 100% corresponds to the difference between light scattering of the aa-Tm-F-actin complex measured at 25oC and that of pure F-actin stabilized by phalloidin which was temperature independent within the temperature range used. A decrease in the light scattering intensity reflects dissociation of the aa-Tm-F-actin complex

20 30 40 50 60 70 80 90 Temperature (t'J

20 30 40 50 60 70 80 90 Temperature (t'J

become reduced, and therefore we observe the increase of the low-temperature peak and decrease of the high-temperature peak during the second heating of Tm [79] (Fig. 9A). Thus, the state of the SH-group of Cys-190 (reduced or oxidized with formation of disulfide bond between two chains of Tm dimer) plays a very important role in the thermal unfolding of Tm, as it determines the thermal stability of the C-terminal half of Tm molecule.

Complexes of tropomyosin with F-actin

Current views of the regulation of actomyosin interaction in striated muscle suggest that Tm can occupy three different positions or states on actin (B-state -'blocked' or Ca2+-free, C-state - 'closed' or Ca2+-induced, and M-state - 'open' or myosin-induced), depending on the presence or absence of troponin, myosin, and Ca2+ [88]. Caldesmom-induced movements of Tm between two different positions along F-actin were shown also for smooth muscle [89]. Thus, the movements of actin-bound Tm are believed to play a crucial role in the regulation of muscle contraction. According to recent model of Tm functions, actin-bound Tm can be considered as a continuous flexible filament whose dynamic properties are modulated by external influences such as the presence of troponin and actin-bound myosin [90]. Protein flexibility may correlate with thermal instability and therefore DSC studies of thermal unfolding of actin-bound Tm may provide valuable information on the dynamic properties of Tm in its different states on the surface of actin filament.

Recently DSC was used to study the specific interaction of smooth muscle Tm with actin filaments [78]. The results demonstrated that the interaction of Tm with F-actin produces a pronounced increase in the thermal stability of Tm (shift of the Tm thermal transition to higher temperature by 2-6oC depending on the Tm/actin molar ratio), but it has no effect on the thermal unfolding of phalloidin-stabilized F-actin, which denatures at a much higher temperature than Tm. A pronounced shift of the Tm thermal transition was observed only for heterodimers of Tm, and not for homodimers. It has also been found, by measuring the temperature dependence of light scattering, that thermal unfolding of Tm is accompanied by its dissociation from F-actin [78]. Thus, the use of DSC in combination with other methods offers a new and promising approach for structural characterization of actin-bound Tm.

Very recently we have applied this approach to studies on skeletal Tm. The character of the thermal unfolding of Tm is noticeably changed if it is bound to F-actin. This is expressed in an appearance of a new highly coop erative thermal transition with maximum at ~47oC (Fig. 9A). After heating of the complex Tm-F-actin to 90oC (i. e. after complete irreversible denaturation of actin) and following cooling, only peaks at 42.5 and 53.5 oC corresponding to the thermal unfolding of free Tm were observed on heat sorption curve during reheating (dotted line curves on Fig. 9A). Thus, we conclude that the appearance of the peak at ~47oC in the presence of F-actin reflects the thermal unfolding of actin-bound Tm. A very good correlation is found between the maximum temperature of this actin-induced thermal transition revealed by DSC (Fig. 9A) and the temperature of dissociation of the Tm-F-actin complex, i. e. the temperature at which a 50% decrease in light scattering occurs (Fig. 9B). This leads to the conclusion that actin-induced changes in the thermal unfolding of Tm are associated with dissociation of Tm from F-actin. It is also important to note that the appearance of new actin-induced transition at ~47oC is accompanied by disappearance of the low-temperature peak of Tm, with no changes in the high-temperature peak (Fig. 9A).

These results allow us to propose the following mechanism of the thermal unfolding of actin-bound Tm. F-actin protects the actin-bound Tm from thermal dena-turation, which only occurs upon dissociation of Tm from F-actin. Therefore the new cooperative transition at ~47oC, that appears only in the presence of actin, reflects thermal denaturation of those parts of Tm which have to denature, in the absence of actin, at temperatures lower than the temperature of dissociation (mainly the low-temperature peak of free Tm, i. e. the C-terminal half of Tm with reduced SH-groups of Cys-190). These parts of Tm denature, in the presence of F-actin, within a very narrow temperature range. All other parts of Tm (the N-terminal half and the C-terminal half with Cys-190 cross-linked by disulfide bonds between two chains of Tm), which melt at higher temperature than that of dissociation, denature independently of the presence of actin after dissociation of Tm from F-actin.

We have also applied the DSC approach to characterize the thermal unfolding of actin-bound Tm in its different states on the surface of actin filaments. The results of our experiments indicate that the transition of smooth muscle Tm to the M-state induced by the binding of myosin to actin is accompanied by significant increase in the thermal stability of actin-bound Tm: the maximum of its thermal transition shifts by 5-6oC to higher temperature without appreciable changes in the cooperativity of the transition. On the other hand, transition of skeletal Tm to the B-state induced by addition of troponin in the absence of Ca2+ leads to significant increase in the cooperativity of the thermal unfolding of actin-bound Tm [79].

TROPONIN

Troponin (Tn) is a protein complex composed of three subunits interacting with one another: the Ca2+-binding troponin C (TnC), the inhibitory troponin I (TnI) which inhibits the ATPase activity of actomy osin, and the troponin T (TnT) responsible for Tn binding to Tm. In the absence of Ca2+, TnC interacts weakly with the other two Tn components, while TnI and TnT bind strongly to Tm and fix it in the 'blocked' B-state on the actin filament. Binding of Ca + to TnC strengthens the interaction between components of the Tn complex and weakens their interaction with Tm, leading to transition of actin-bound Tm from B-state into C-state. Due to this ability, Tn plays a key role in Ca2+-regulation of striated (skeletal and cardiac) muscle contraction.

DSC studies on isolated components of the Tn complex have shown that both TnI and TnT demonstrate no cooperative thermal transitions during heating up to

100oC [79, 91]. Calorimetric effects of these two proteins were observed only indirectly, when they affected the thermal unfolding of actin-bound Tm [79] (see pages 150-151). TnC is the only component of the Tn complex that denatures with a cooperative transition [92, 93]. In 1980 Tsalkova and Privalov described the thermal unfolding of TnC [92]. They have shown that in the presence of divalent cations the DSC curve of TnC is represented by two well-separated thermal transitions corresponding to two domains in the TnC molecule, onle of them containing Ca2+-specific binding sites, and another - Ca2+-Mg2+-binding sites. Binding of Ca2+ to Ca2+-specific sites was shown to increase dramatically the thermal stability of the domain containing these sites [92]. Thus, DSC studies reveled global structural changes which occur in TnC due to specific binding of Ca2+ and play an important role in the functioning of TnC as a regulatory protein.

CALDESMON AND CALPONIN

Smooth muscle contraction is regulated primarily by the phosphorylation of myosin regulatory light chains by a Ca2+-calmodulin-dependent myosin light chain kinase (MLCK). Two smooth muscle actin binding proteins, caldesmon and calponin, may also be involved in regulation of smooth muscle contraction and act as a secondary control of the contraction. Both proteins inhibit the actin-activated myosin ATPase activity and the movement of actin filaments in in vitro motility assays. Caldesmon alters the position of Tm on actin [89] but in a manner that is different from the effect of Tn in skeletal muscle.

Our DSC experiments showed that both caldesmon and calponin do not demonstrate cooperative thermal transitions on heating up to 100oC. Furthermore, we have not found any effects of these proteins on the thermal unfolding of F-actin and actin-bound Tm. The only thermal effect of calponin observed till now by DSC was its influence on the phase transitions of phospholipid vesicles [94]. This fact indicated that calponin may interact with phospholipids and by this means anchor actin filaments to the cellular membrane.

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