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

Golitsina, Nina L.; Bobkov, Andrey A.; Dedova, Irina; Павлов, Д. А.; Nikolaeva, Olga; Орлов, В. Н.; Levitsky, Dmitrii I.

doi:10.1007/bf00123363

Cited by 17 publications

(12 citation statements)

References 39 publications

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“…3B). These results are very similar to those obtained earlier with uncleaved S1 [15,16,36]. Thus, the ternary complexes of Nt‐S1 with ADP and P i analogs are as stable as the complexes obtained with control uncleaved S1 as they do not significantly decompose a few days after removal of excess reagents.…”

Section: The Ternary Complexes Of T‐s1 and Nt‐s1 With Adp And Phosphasupporting

confidence: 89%

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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Section: The Ternary Complexes Of T‐s1 and Nt‐s1 With Adp And Phosphasupporting

confidence: 89%

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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“…Spin‐labelling affects the ATPase activity of myosin [46] and the unfolding of S1 [47]. In contrast to experiments performed on spin‐labelled S1, our measurements, which were performed on TCSL fibers did not exhibit significant alterations in melting temperatures and transition enthalpies.…”

Section: Discussionmentioning

confidence: 99%

Nucleotide binding induces global and local structural changes of myosin head in muscle fibres

Lőrinczy¹,

Belágyi

2001

European Journal of Biochemistry

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Thermal stability and internal dynamics of myosin heads in fiber bundles from rabbit psoas muscle has been studied by electron paramagnetic resonance (EPR) spectroscopy and differential scanning calorimetry (DSC). Using ADP, ATP and orthovanadate (V i ), three intermediate states of the ATP hydrolysis cycle were simulated in glycerinated muscle fibers. DSC transitions contained three overlapping endotherms in each state. Deconvolution showed that the transition temperature of 58.4 8C was almost independent of the intermediate state of myosin, while nucleotide binding shifted the melting temperatures of 54.0 and 62.3 8C, and changed the enthalpies. These changes suggest global rearrangements of the internal structure in myosin head. In the presence of ADP and ADP plus V i , the conventional EPR spectra showed changes in the ordering of the probe molecules, suggesting local conformational and motional changes in the internal structure of myosin heads. Saturation transfer EPR measurements reported increased rotational mobility of spin labels in the presence of ATP plus orthovanadate corresponding to a weakly binding state of myosin to actin.Keywords: DSC; EPR; thermal unfolding; myosin; orthovanadate.Muscle is a chemo-mechanical machine that converts chemical energy into work and heat. The energy source is the myosin-catalyzed ATP hydrolysis, and its rate is affected by actin. In the presence of MgATP the energy released from hydrolysis produces conformational changes in myosin [1][2][3][4] and/or in actin [5,6] which can be manifested as an internal rotation of myosin head while bounds to actin. In the ATP hydrolysis pathway the AM : ADP : P i complex (where A denotes actin and M represents myosin) plays an important role in the power-stroke of the cross bridge cycle; therefore the main goal of the recent efforts is the investigation of the energetics of the actomyosin ATPase in the different intermediates of the contractile cycle [7 -10]. The X-ray diffraction experiments derive the precise structure of subfragment-1 [11,12]. A better understanding of the interrelation between the chemical events observed in protein solutions and the mechanical activity on muscle fibers requires experiments on supramolecular complexes where stabilizing forces may modulate the hydrolysis process. Therefore, experiments were extended to study the fiber system prepared from psoas muscle of rabbit in rigor, strongly binding and weakly binding states of myosin to actin [13 -15].Recent studies suggest that domain movements in the myosin head play a decisive role in the energy transduction process of the muscle contraction. This process is a multistep process that produces several conformational states of myosin. It is generally accepted that myosin head consists of two large domains, the motor and regulatory domain. The former contains the nucleotide and actin binding regions; the essential and the regulatory light chains are located on the long a helical part of the regulatory domain [3,16]. Extensive studies using different technique...

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“…The conformation of S1 alone and in complexes with nucleotides was also altered by SH1 modification [110]. These data show that the SH1-SH2 helix is a functionally important site on S1, but the mechanism by which this helix is involved in the force generation cycle is still unknown.…”

Section: Cross Linking Of Sh1-sh2mentioning

confidence: 84%

Fluorescence labeling and computational analysis of the strut of myosin’s 50 kDa cleft

Gawalapu¹,

Root²

2006

Archives of Biochemistry and Biophysics

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Gawalapu, Ravi Kumar. Fluorescence labeling and computational analysis of the strut of myosin's 50 kDa cleft. Doctor of Philosophy (Biochemistry), August 2007, 148 pp., 3 tables, 60 illustrations, 120 references, 120 titles.In order to understand the structural changes in myosin S1, fluorescence polarization and computational dynamics simulations were used. Dynamics simulations on the S1 motor domain indicated that significant flexibility was present throughout the molecular model. The constrained opening versus closing of the 50 kDa cleft appeared to induce opposite directions of movement in the lever arm. A sequence called the "strut" which traverses the 50 kDa cleft and may play an important role in positioning the actomyosin binding interface during actin binding is thought to be intimately linked to distant structural changes in the myosin's nucleotide cleft and neck regions. To study the dynamics of the strut region, a method of fluorescent labeling of the strut was discovered using the dye CY3. CY3 served as a hydrophobic tag for purification by hydrophobic interaction chromatography which enabled the separation of labeled and unlabeled species of S1 including a fraction labeled specifically at the strut sequence. The high specificity of labeling was verified by proteolytic digestions, gel electrophoresis, and mass spectroscopy.Analysis of the labeled S1 by collisional quenching, fluorescence polarization, and actinactivated ATPase activity were consistent with predictions from structural models of the probe's location. Although the fluorescent intensity of the CY3 was insensitive to actin binding, its fluorescence polarization was notably affected. Intriguingly, the mobility of the probe increases upon S1 binding to actin suggesting that the CY3 becomes displaced from interactions with the surface of S1 and is consistent with a structural change in the strut due to cleft motions. Labeling the strut reduced the affinity of S1 for actin but did not prevent actin-activated ATPase activity which makes it a potentially useful probe of the actomyosin interface. The different conformations of myosin S1 indicated that the strut is not as flexible as several other key regions of myosin as determined by the application of force constraints to elastic portions of the myosin

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

Cited by 17 publications

References 39 publications

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

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

Nucleotide binding induces global and local structural changes of myosin head in muscle fibres

Fluorescence labeling and computational analysis of the strut of myosin’s 50 kDa cleft

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