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

Nikolaeva, Olga; Орлов, В. Н.; Dedova, Irina; Drachev, V. A.; Levitsky, Dmitrii I.

doi:10.1080/15216549600201253

Cited by 13 publications

(19 citation statements)

References 13 publications

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“…This method was successfully used for probing the structural changes that occur in the myosin head due to its strong binding to F‐actin in the presence of ADP. It was shown that the binding of skeletal S1 to F‐actin significantly increased the thermal stability of S1 [11,12]. A very similar effect was observed by DSC with recombinant Dictyostelium discoideum myosin head fragment corresponding to the globular motor portion of the head [13].…”

Section: Introductionsupporting

confidence: 60%

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Changes in the thermal unfolding of p‐phenylenedimaleimide‐modified myosin subfragment 1 induced by its ‘weak’ binding to F‐actin

et al. 2001

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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,NP P-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 5³C 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. ß

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Section: Introductionsupporting

confidence: 60%

“…DSC experiments were performed on a DASM‐4M differential scanning microcalorimeter (Institute for Biological Instrumentation, Pushchino, Russia) as described earlier [11–13,18,20]. All measurements were carried out in 15 mM HEPES, pH 7.3, containing 2 mM MgCl 2 , 0.5 mM ADP, and twice‐diluted G buffer, at a scanning rate of 1°C/min.…”

Section: Methodsmentioning

confidence: 99%

Changes in the thermal unfolding of p‐phenylenedimaleimide‐modified myosin subfragment 1 induced by its ‘weak’ binding to F‐actin

et al. 2001

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“…2A). In this respect, the tropomyosin–F‐actin complex is quite different from the complex of F‐actin with myosin subfragment 1 (S1) earlier studied by DSC [38]. Interaction of S1 with F‐actin resulted in a significant shift of the S1 thermal transition to higher temperature, but no changes in the transition temperature of actin‐bound S1 were observed at various S1/actin molar ratios ranging from 1 : 10 to 2 : 1.…”

Section: Discussionmentioning

confidence: 79%

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

Levitsky

Rostkova

Орлов

et al. 2000

European Journal of Biochemistry

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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 8C with a sharp thermal transition. Interaction of tropomyosin heterodimers with F-actin causes a 2±6 8C 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 8C 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 8C in the absence and at 78 8C in the presence of a twofold molar excess of phalloidin. After the F-actin±tropomyosin complex had been heated to 90 8C 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.Keywords: differential scanning calorimetry; F-actin; thermal unfolding; tropomyosin.Tropomyosin is a coiled-coil actin-binding protein bound along the length of actin filament in all types of muscle [1,2]. One tropomyosin molecule spans the length of seven actin monomers in the filament. Tropomyosin molecules bind to themselves in a end-to-end manner [3,4], and form a continuous structure along the actin filament. The presence of tropomyosin on actin filaments confers co-operativity on the interaction between myosin heads and actin [5±7]. Together with the other regulatory proteins, troponin in striated skeletal muscle or caldesmon in smooth muscle, it takes part in the Ca 21 regulation of muscle contraction [8,9]. Recent structural studies on skeletal muscle thin filaments suggest that tropomyosin can occupy three different positions on the actin surface, depending on the presence or absence of troponin, myosin, and Ca 21 [10]. These results are consistent with independently proposed models of the regulation of actomyosin interaction by tropomyos...

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“…Moreover, DSC was also successfully used for probing the structural changes that occur in the myosin head due to its strong binding to F‐actin in the presence of ADP. It was shown that the binding of skeletal S1 to F‐actin significantly increased the thermal stability of S1 [18,19]. A very similar effect was observed by DSC with recombinant D. discoideum myosin head fragment corresponding to the globular motor portion of the head [20].…”

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confidence: 72%

Differential scanning calorimetric study of myosin subfragment 1 with tryptic cleavage at the N‐terminal region of the heavy chain

Nikolaeva

Орлов

Bobkov

et al. 2002

European Journal of Biochemistry

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The thermal unfolding of myosin subfragment 1 (S1) cleaved by trypsin was studied by differential scanning calorimetry. In the absence of nucleotides, trypsin splits the S1 heavy chain into three fragments (25, 50, and 20 kDa). This cleavage has no appreciable influence on the thermal unfolding of S1 examined in the presence of ADP, in the ternary complexes of S1 with ADP and phosphate analogs, such as orthovanadate (V i ) or beryllium fluoride (BeF x ), and in the presence of F-actin. In the presence of ATP and in the complexes S1AEADPAEV i or S1AEADPAEBeF x , trypsin produces two additional cleavages in the S1 heavy chain: a faster cleavage in the N-terminal region between Arg23 and Ile24, and a slower cleavage at the 50 kDa fragment. It has been shown that the N-terminal cleavage strongly decreases the thermal stability of S1 by shifting the maximum of its thermal transition by about 7°C to a lower temperature, from 50°C to 42.4°C, whereas the cleavage at both these sites causes dramatic destabilization of the S1 molecule leading to total loss of its thermal transition. Our results show that S1 with ATP-induced N-terminal cleavage is able, like uncleaved S1, to undergo global structural changes in forming the stable ternary complexes with ADP and P i analogs (V i , BeF x ). These changes are reflected in a pronounced increase of S1 thermal stability. However, S1 cleaved by trypsin in the N-terminal region is unable, unlike S1, to undergo structural changes induced by interaction with F-actin that are expressed in a 4-5°C shift of the S1 thermal transition to higher temperature. Thus, the cleavage between Arg23 and Ile24 does not significantly affect nucleotide-induced structural changes in the S1, but it prevents structural changes that occur when S1 is bound to F-actin. The results suggest that the N-terminal region of the S1 heavy chain plays an important role in structural stabilization of the entire motor domain of the myosin head, and a long-distance communication pathway may exist between this region and the actin-binding sites.Keywords: myosin subfragment 1; thermal unfolding; differential scanning calorimetry.Cyclic association-dissociation of actin and myosin coupled with ATP hydrolysis by myosin ATPase is the most essential process of muscle contraction. The globular head of myosin, called subfragment 1 or S1, where both the nucleotide-and actin-binding sites of the molecule are located, is responsible for the generation of force during contraction. The function of the myosin head as a Ômole-cular motorÕ is explained by significant conformational changes, which occur in the head during ATPase reaction and alter the character of actin-myosin interaction [1,2]. Thus the description of nucleotide-and actin-induced structural changes in the myosin head is essential for the understanding of the motor mechanism.Among a variety of methods employed, the method of differential scanning calorimetry (DSC) is especially useful for probing global structural changes that occur in the myosin head due to interactio...

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

Cited by 13 publications

References 13 publications

Changes in the thermal unfolding of p‐phenylenedimaleimide‐modified myosin subfragment 1 induced by its ‘weak’ binding to F‐actin

Changes in the thermal unfolding of p‐phenylenedimaleimide‐modified myosin subfragment 1 induced by its ‘weak’ binding to F‐actin

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

Differential scanning calorimetric study of myosin subfragment 1 with tryptic cleavage at the N‐terminal region of the heavy chain

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