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

Nikolaeva, Olga; Орлов, В. Н.; Bobkov, Andrey A.; Levitsky, Dmitrii I.

doi:10.1046/j.1432-1033.2002.03279.x

Cited by 17 publications

(15 citation statements)

References 46 publications

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“…DSC experiments were performed on a DASM-4M differential scanning microcalorimeter (Institute for Biological Instrumentation, Pushchino, Russia) as described earlier [30–32]. Samples containing S1 isoforms (1.2–1.9 mg/mL) were heated with constant rate of 1 °C/min from 15 °C to 85 °C in 20 mM Hepes-KOH (pH 7.3) containing 1 mM MgCl 2 in the presence or absence of 100 mM KCl.…”

Section: Methodsmentioning

confidence: 99%

Thermal Denaturation and Aggregation of Myosin Subfragment 1 Isoforms with Different Essential Light Chains

Markov

Zubov

Nikolaeva

et al. 2010

IJMS

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We compared thermally induced denaturation and aggregation of two isoforms of the isolated myosin head (myosin subfragment 1, S1) containing different “essential” (or “alkali”) light chains, A1 or A2. We applied differential scanning calorimetry (DSC) to investigate the domain structure of these two S1 isoforms. For this purpose, a special calorimetric approach was developed to analyze the DSC profiles of irreversibly denaturing multidomain proteins. Using this approach, we revealed two calorimetric domains in the S1 molecule, the more thermostable domain denaturing in two steps. Comparing the DSC data with temperature dependences of intrinsic fluorescence parameters and S1 ATPase inactivation, we have identified these two calorimetric domains as motor domain and regulatory domain of the myosin head, the motor domain being more thermostable. Some difference between the two S1 isoforms was only revealed by DSC in thermal denaturation of the regulatory domain. We also applied dynamic light scattering (DLS) to analyze the aggregation of S1 isoforms induced by their thermal denaturation. We have found no appreciable difference between these S1 isoforms in their aggregation properties under ionic strength conditions close to those in the muscle fiber (in the presence of 100 mM KCl). Under these conditions kinetics of this process was independent of protein concentration, and the aggregation rate was limited by irreversible denaturation of the S1 motor domain.

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Section: Methodsmentioning

confidence: 99%

Thermal Denaturation and Aggregation of Myosin Subfragment 1 Isoforms with Different Essential Light Chains

Markov

Zubov

Nikolaeva

et al. 2010

IJMS

Self Cite

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“…They further suggested that the N-terminal region is only indirectly coupled to the active site but that it is sensitive to direct interactions with the lever arm in the strongly bound states of the ATPase cycle (30,31). Contacts between the N terminus and heavy chain residues 750 -760 in the converter region (D. discoideum numbering) have been implicated by differential scanning calorimetry as important for the structural integrity and stability of the entire motor domain (32). Following tryptic cleavage of the skeletal muscle myosin heavy chain between Arg 23 and Ile 24 , Levitsky and co-workers (32) observed a reduction of the thermal transition from 49 to 42°C, whereas nucleotide binding was unaffected.…”

Section: Nucleotidementioning

confidence: 99%

“…1, B and C). Additional structural stability in this region is maintained by hydrophobic interactions formed between residues Phe 25 -Lys 32 and residues 760 -765 on an adjacent helix that emerges from the converter region (Fig. 1A).…”

Section: Nucleotidementioning

confidence: 99%

Functional Characterization of the N-terminal Region of Myosin-2

Fujita‐Becker

Tsiavaliaris

Ohkura

et al. 2006

Journal of Biological Chemistry

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All class 2 myosins contain an N-terminal extension of ϳ80 residues that includes an Src homology 3 (SH3)-like subdomain. To explore the functional importance of this region, which is also present in most other myosin classes, we generated truncated constructs of Dictyostelium discoideum myosin-2. Truncation at position 80 resulted in the complete loss of myosin-2 function in vivo. Actin affinity was more than 80-fold, and the rate of ADP release ϳ40-fold decreased in this mutant. In contrast, a myosin construct that lacks only the SH3-like subdomain, corresponding to residues 33-79, displayed much smaller functional defects. In complementation experiments with myosin-2 null cells, this construct rescued myosin-2-dependent processes such as cytokinesis, fruiting body formation, and sporogenesis. An 8-fold reduction in motile activity and changes of similar extent in the affinity for ADP and filamentous actin indicate the importance of the SH3-like subdomain for correct communication between the functional regions within the myosin motor domain and suggest that local perturbations in this region can play a role in modulating myosin-2 motor activity.Members of the myosin superfamily of actin-based motors act in a variety of cellular functions such as muscle contraction, cell and organelle movement, membrane trafficking, and signal transduction. Although myosin motor domains show a high degree of sequence conservation, the individual myosin classes are clearly defined by differences in the head structure (1). Extensive biochemical investigations of the myosin ATPase cycle together with structural information of the motor domain and electron microscopy of the actomyosin complex have led to detailed molecular models of the nucleotide-dependent actomyosin interaction (2, 3). However, the exact functional roles of several regions of the myosin motor domain remain to be elucidated. In particular, the role of a protruding, six-stranded, antiparallel, ␤-barrel subdomain with similarities to the SH3 2 domains, which appears to be present in most myosins and comprises ϳ50 amino acids of the heavy chain, is largely unknown. The structure of this subdomain has been solved for class 2, 5, and 6 myosins (4 -7). The crystal structure of the nucleotide-free smooth muscle myosin motor domain with essential light chain (ELC) bound, shows the ELC to be in contact with the N-terminal domain of the heavy chain. However, in the presence of MgADP⅐AlF4 Ϫ the ELC is rotated up to 70°f rom the position in nucleotide-free myosin subfragment 1 (S1) and forms a contact with loop 1 (8).Sequence alignments of the N-terminal region of myosins from different classes reveal that this region varies greatly in length and amino acid composition among the individual members. Class 1 myosins completely lack the N-terminal region corresponding to the first 79 residues of myosin-2 (9). In the case of class 2 myosins, residues 1-33 form an extended structure crossing the interface between the motor domain and the neck region (Fig. 1). Residues from this s...

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“…Thermal denaturation studies on S1(A1) and S1(A2) were carried out by means of DSC on a DASM–4M differential scanning microcalorimeter (Institute for Biological Instrumentation, Russian Academy of Sciences (RAS), Pushchino, Russia) as described earlier [21, 23, 24]. Samples containing S1 isoforms (1.5 mg/ml) were heated at a 1 o C/min rate from 1 5 °C to 7 5 ° C in a 20 mM Hepes–KOH (pH 7.3) containing 1 m M MgCl 2 in the presence or absence of 100 mM KCl.…”

Section: Methodsmentioning

confidence: 99%

Effects of Myosin "Essential" Light Chain A1 on the Aggregation Properties of the Myosin Head

2010

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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.

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

Cited by 17 publications

References 46 publications

Thermal Denaturation and Aggregation of Myosin Subfragment 1 Isoforms with Different Essential Light Chains

Thermal Denaturation and Aggregation of Myosin Subfragment 1 Isoforms with Different Essential Light Chains

Functional Characterization of the N-terminal Region of Myosin-2

Effects of Myosin "Essential" Light Chain A1 on the Aggregation Properties of the Myosin Head

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