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Dmitrii I. Levitsky - One of the best experts on this subject based on the ideXlab platform.
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intermolecular interactions of Myosin Subfragment 1 induced by the n terminal extension of essential light chain 1
Biochemistry, 2017Co-Authors: Olga P. Nikolaeva, Dmitrii I. Levitsky, Daria S LogvinovaAbstract: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).
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thermal denaturation and aggregation of Myosin Subfragment 1 isoforms with different essential light chains
International Journal of Molecular Sciences, 2010Co-Authors: Denis I. Markov, Olga P. Nikolaeva, Eugene O Zubov, B I Kurganov, Dmitrii I. LevitskyAbstract: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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nucleotide induced and actin induced structural changes in sh1 sh2 modified Myosin Subfragment 1
Journal of Muscle Research and Cell Motility, 2007Co-Authors: Lubov Shakirova, Dmitrii I. Levitsky, Valeria V Mikhailova, Elena Siletskaya, V P TimofeevAbstract:We compared the structural properties of Myosin Subfragment 1 (S1) modified at both reactive SH-groups, SH1 (Cys707) and SH2 (Cys697), with the properties of unmodified S1 and SH1-modified S1. It is shown using differential scanning calorimetry (DSC) that SH1 modification has no noticeable influence on the changes in S1 thermal unfolding induced by the formation of S1 ternary complexes with ADP and Pi analogs (Vi, AlF4−, and BeFx). These changes, however, normally expressed in a significant increase of S1 thermal stability, are almost fully prevented by modification of both SH1 and SH2. In contrast, SH2 modification had no effect on the changes induced by the formation of the ternary complexes S1-ADP-Vi, S1-ADP-AlF4−, and S1-ADP-BeFx in EPR spectra of S1 spin-labeled at SH1 group. Interaction of S1 with F-actin substantially increased the thermal stability of S1; a similar effect was observed by DSC with both SH1- and SH1-SH2-modified S1. Overall, our results demonstrate that modification of both reactive SH-groups on S1 has no influence on the actin-induced changes of S1 and on the local nucleotide-induced conformational changes in the SH1 group region, but strongly prevents the global nucleotide-induced structural changes in the entire S1 molecule. The results suggest that modification of SH1 and SH2 impairs the spread of nucleotide-induced conformational changes from the ATPase site throughout the structure of the entire S1 molecule, thus disturbing a coupling between the motor and regulatory domains in the Myosin head.
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changes in the thermal unfolding of p phenylenedimaleimide modified Myosin Subfragment 1 induced by its weak binding to f actin
FEBS Letters, 2001Co-Authors: Olga V Kaspieva, Olga P. Nikolaeva, Dmitrii I. Levitsky, Victor N. Orlov, V. A. Drachev, Michael A PonomarevAbstract: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°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.
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differential scanning calorimetric study of the complexes of modified Myosin Subfragment 1 with adp and vanadate or beryllium fluoride
Journal of Muscle Research and Cell Motility, 1996Co-Authors: Nina L Golitsina, Olga P. Nikolaeva, Victor N. Orlov, Irina Dedova, Andrey A Bobkov, D A Pavlov, Dmitrii I. LevitskyAbstract:The effects of various modifications of rabbit skeletal Myosin Subfragment 1 on thermal denaturation of Subfragment 1 in ternary complexes with Mg-ADP and orthovanadate (Vi) 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.
Emil Reisler - One of the best experts on this subject based on the ideXlab platform.
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Is SH1-SH2-cross-linked Myosin Subfragment 1 a structural analog of the weakly-bound state of Myosin
2016Co-Authors: Andrey A Bobkov, Emil ReislerAbstract:ABSTRACT Myosin Subfragment 1 (S1) with SH1 (Cys707) and SH2 (Cys697) groups cross-linked by p-phenylenedimaleimide (pPDM-S1) is thought to be an analog of the weakly bound states of Myosin bound to actin. The structural properties of pPDM-S1 were compared in this study to those of S1zADPzBeFx and S1zADPzAlF4 2, i.e., the established structural analogs of the Myosin weakly bound states. To distinguish between the conformational effects of SH1-SH2 cross-linking and those due to their monofunctional modification, we used S1 with the SH1 and SH2 groups labeled with N-phenylmaleimide (NPM-S1) as a control in our experiments. The state of the nucleotide pocket was probed using a hydrophobic fluorescent dye, 3-[4-(3-phenyl-2-pyrazolin-1-yl)benzene-1-sulfonylamido]phenylboronic acid (PPBA). Differential scanning calorimetry (DSC) was used to study the thermal stability of S1. By both methods the conformational state of pPDM-S1 was different from that of unmodified S1 in the S1zADPzBeFx and S1zADPzAlF4 2 complexes and closer to that of nucleotide-free S1. Moreover, BeFx and AlF4 2 binding failed to induce conformational changes in pPDM-S1 similar to those observed in unmodified S1. Surprisingly, when pPDM cross-linking was performed on S1zADPzBeFx complex, ADPzBeFx protected to some extent the nucleotide pocket of S1 from the effects of pPDM modification. NPM-S1 behaved similarly to pPDM-S1 in our experiments
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Solution properties of full length and truncated forms of Myosin Subfragment 1 from Dictyostelium discoideum
Journal of Muscle Research & Cell Motility, 2001Co-Authors: Juan Reynoso, Andras Muhlrad, Andrey Bobkov, Emil ReislerAbstract:The atomic structures for several Myosin head isoforms in different nucleotide states have been determined in recent years. The comparison of these structures is complicated by the use of Myosin Subfragment 1 (S1) constructs of different length in different studies. Several atomic structures of the S1 nucleotide complex were obtained using Dictyostelium discoideum S1dC, a genetically truncated form of S1 lacking the light chain binding domain (LCBD) and both light chains. The goal of the present study has been to assess the effects of such a truncation on the solution properties of S1 and in particular, on its active site, actin binding site and the converter region. The nucleotide and actin binding properties, CD spectra and the reactivities of Lys-84 (corresponds to the ‘reactive lysine’, Lys-83 in rabbit skeletal S1) and Cys-678 (corresponds to the ‘SH2-group’, Cys-697 in rabbit S1) were compared for the full length (flS1) and the truncated (S1dC) forms of Dictyostelium S1. The two forms showed similar nucleotide binding properties. However, S1dC had a lower structural stability and a significantly higher K _m value for actin-activated ATPase as compared to flS1. Differences were found also in the near-UV CD spectrum between flS1 and S1dC. SH2 reactivity in S1dC appeared to be greatly inhibited compared with that in flS1. The modification of Lys-84 caused a greater increase in the MgATPase activity in S1dC than in flS1. ADP inhibited this activation for both S1dC and flS1. Taken together our results identify both truncation-caused differences between S1dC and flS1, as well as isoform-related differences between skeletal and Dictyostelium S1.
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is sh1 sh2 cross linked Myosin Subfragment 1 a structural analog of the weakly bound state of Myosin
Biophysical Journal, 2000Co-Authors: Andrey A Bobkov, Emil ReislerAbstract:Myosin Subfragment 1 (S1) with SH1 (Cys(707)) and SH2 (Cys(697)) groups cross-linked by p-phenylenedimaleimide (pPDM-S1) is thought to be an analog of the weakly bound states of Myosin bound to actin. The structural properties of pPDM-S1 were compared in this study to those of S1.ADP.BeF(x) and S1.ADP.AlF(4)(-), i.e., the established structural analogs of the Myosin weakly bound states. To distinguish between the conformational effects of SH1-SH2 cross-linking and those due to their monofunctional modification, we used S1 with the SH1 and SH2 groups labeled with N-phenylmaleimide (NPM-S1) as a control in our experiments. The state of the nucleotide pocket was probed using a hydrophobic fluorescent dye, 3-[4-(3-phenyl-2-pyrazolin-1-yl)benzene-1-sulfonylamido]phen ylboronic acid (PPBA). Differential scanning calorimetry (DSC) was used to study the thermal stability of S1. By both methods the conformational state of pPDM-S1 was different from that of unmodified S1 in the S1.ADP.BeF(x) and S1.ADP.AlF(4)(-) complexes and closer to that of nucleotide-free S1. Moreover, BeF(x) and AlF(4)(-) binding failed to induce conformational changes in pPDM-S1 similar to those observed in unmodified S1. Surprisingly, when pPDM cross-linking was performed on S1.ADP.BeF(x) complex, ADP.BeF(x) protected to some extent the nucleotide pocket of S1 from the effects of pPDM modification. NPM-S1 behaved similarly to pPDM-S1 in our experiments. Overall, this work presents new evidence that the conformational state of pPDM-S1 is different from that of the weakly bound state analogs, S1.ADP.BeF(x) and S1.ADP.AlF(4)(-). The similar structural effects of pPDM cross-linking of SH1 and SH2 groups and their monofunctional labeling with NPM are ascribed to the inhibitory effects of these modifications on the flexibility/mobility of the SH1-SH2 helix.
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actin and temperature effects on the cross linking of the sh1 sh2 helix in Myosin Subfragment 1
Biophysical Journal, 2000Co-Authors: Lisa K Nitao, Emil ReislerAbstract:Past biochemical work on Myosin Subfragment 1 (S1) has shown that the bent alpha-helix containing the reactive thiols SH1 (Cys(707)) and SH2 (Cys(697)) changes upon nucleotide and actin binding. In this study, we investigated the conformational dynamics of the SH1-SH2 helix in two actin-bound states of Myosin and examined the effect of temperature on this helix, using five cross-linking reagents that are 5-15 A in length. Actin inhibited the cross-linking of SH1 to SH2 on both S1 and S1.MgADP for all of the reagents. Because the rate of SH2 modification was not altered by actin, the inhibition of cross-linking must result from a strong stabilization of the SH1-SH2 helix in the actin-bound states of S1. The dynamics of the helix is also influenced by temperature. At 25 degrees C, the rate constants for cross-linking in S1 alone are low, with values of approximately 0.010 min(-1) for all of the reagents. At 4 degrees C, the rate constants, except for the shortest reagent, range between 0.030 and 0.070 min(-1). The rate constants for SH2 modification in SH1-modified S1 show the opposite trend; they increase with the increases in temperature. The greater cross-linking at the lower temperature indicates destabilization of the SH1-SH2 helix at 4 degrees C. These results are discussed in terms of conformational dynamics of the SH1-SH2 helix.
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activation of regulated actin by sh1 modified Myosin Subfragment 1
Biochemistry, 1997Co-Authors: Andrey A Bobkov, Elena Bobkova, Earl Homsher, Emil ReislerAbstract:The reactive SH1 (Cys-707) group of the Myosin Subfragment 1 (S1) has been used frequently as an attachment site for fluorescent and spin probes in solution and muscle fiber experiments. In this study we examined (i) the motor function of SH1 spin-labeled heavy meroMyosin (HMM) in the in vitro motility assays and (ii) the effect of SH1-modified S1 on the motility of regulated actin, i.e., actin complexed with tropoMyosin and troponin. N-ethylmaleimide (NEM), N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-iodacetamide (IASL), N-[[(iodoacetyl)amino]ethyl]1-sulfo-5-naphthylamine (IAEDANS), and iodoacetamide (IAA) were used to selectively modify the SH1 group on S1; the SH1 group on HMM was labeled with IASL. In the in vitro motility assays, 10-20% of unregulated actin filaments moved at a speed of approximately 1 microm/s over a surface coated with 90-95% modified IASL-HMM. Actin sliding was not observed with 95-98% modified IASL-HMM. The sliding of regulated actin over unmodified HMM was activated by the addition of S1 modified with any of the SH1 reagents to the in vitro motility assay solutions; both the speeds and the percentage of the moving filaments increased at pCa 5, 7, and 8. To shed light on the activation of regulated actin sliding by SH1-modifed S1, acto-S1 ATPase and the binding to actin were determined for IASL-S1. While the binding affinities to actin were similar for IASL-S1 and unmodified S1 in the presence and absence of ADP and ATP, the Km and Vmax values were approximately 10-fold lower for the modified protein. It is concluded that the activation of regulated actin by SH1-modifed S1 facilitates the interaction of unmodified HMM heads with actin and thus can increase the sliding speeds and the percentage of regulated actin filaments that move in the in vitro motility assays.
Dominique Pantaloni - One of the best experts on this subject based on the ideXlab platform.
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mechanism of Myosin Subfragment 1 induced assembly of cag actin and mgg actin into f actin s1 decorated filaments
Biochemistry, 1997Co-Authors: Stéphane Fievez, Marie-france Carlier, Dominique PantaloniAbstract:The kinetics and mechanism of Myosin Subfragment-1-induced polymerization of G-actin into F-actin-S1-decorated filaments have been investigated in low ionic strength buffer and in the absence of free ATP. The mechanism of assembly of F-actin-S1 differs from salt-induced assembly of F-actin. Initial condensation of G-actin and S1 into oligomers in reversible equilibrium is a prerequisite step in the formation of F-actin-S1 . Oligomers have a relatively low stability (10(6) M-1) and contain S1 in a molar ratio to actin close to 0.5. Increased binding of S1 up to a 1:1 molar ratio to actin is associated with further irreversible condensation of oligomers into large F-actin-S1 structures of very high stability. In contrast to salt-induced assembly of F-actin, no monomer-polymer equilibrium, characterized by a critical concentration, can be defined for F-actin-S1 assembly, and end-to-end annealing of oligomers is predominant over growth from nuclei in the kinetics. Simultaneous recordings of the changes in light scattering, pyrenyl- and NBD-actin fluorescence, ATP hydrolysis, and release of Pi during the polymerization process have been analyzed to propose a minimum kinetic scheme for assembly, within which several elementary steps, following oligomer formation, are required for assembly of F-actin-S1. ATP hydrolysis occurs before polymerization of MgATP-G-actin but not of CaATP-G-actin. The release of inorganic phosphate occurs on F-actin-S1 at the same rate as on F-actin.
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kinetics of association of Myosin Subfragment 1 to unlabeled and pyrenyl labeled actin
Journal of Biological Chemistry, 1996Co-Authors: Laurent Blanchoin, Marie-france Carlier, Dominique Didry, Dominique PantaloniAbstract:Abstract The kinetics of reaction of Myosin Subfragment-1 (S) with F-actin have been monitored by the changes in light scattering and in pyrenyl-actin fluorescence at 20°C, pH 7.5, and physiological ionic strength. The association rate constant of S to F-actin decreases about 10-fold as the molar ratio of bound S increases from 0 to 1. This decrease in k is most likely due to the steric hindrance of available binding sites by initially bound S. The apparent rate constant for association of S to bare filaments is 9 μM s, a value 1 order of magnitude higher than the one previously estimated from experiments in which S was in excess over F-actin. The anticooperative binding kinetics of S to F-actin are consistent with the negative cooperativity displayed in the equilibrium binding curves of S to pyrenyl-F-actin. Fluorescence titration curves of partially labeled pyrenyl-F-actin by S are sigmoidal, consistent with a 4-fold higher affinity of S for unlabeled than for labeled actin. This conclusion is strengthened by kinetic data of S binding to partially labeled F-actin, which exhibit a biphasic behavior due to the slower dissociation of S from unlabeled than from labeled actin.
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Kinetics of the Interaction of Myosin Subfragment-1 with G-Actin EFFECT OF NUCLEOTIDES AND DNaseI
Journal of Biological Chemistry, 1995Co-Authors: Laurent Blanchoin, Stéphane Fievez, Franck Travers, Marie-france Carlier, Dominique PantaloniAbstract:Abstract The kinetics of interaction of monomeric pyrenyllabeled G-actin with Myosin Subfragment-1 (S1 (A1) and S1(A2) isomers) has been examined in the stopped-flow at low ionic strength. The data confirm the previously reported existence of binary GS and ternary G2S complexes. The increase in pyrenyl-actin fluorescence which monitors the G-actin-S1 interactions is linked to the isomerization of these complexes following rapid equilibrium binding steps. The rates of isomerization are ∼200 s−1 for GS and ∼50 s−1 for G2S at 4°C and in the absence of ATP. DNaseI and S1 bind G-actin essentially in a mutually exclusive fashion. Both GS and G2S are dissociated by MgATP and MgADP. The kinetics and mechanism of ATP-induced dissociation of G2S are quantitatively close to the ATP-induced dissociation of F-actin-S1, which indicates the G2S is a good model for the F-actin-S1 interface. GS and G2S display different kinetic behaviors in response to nucleotides, GS being less efficiently dissociated than G2S by MgATP. This result suggests that different mechanical properties of the cross-bridge might correlate with different orientations of the Myosin head and different actin/Myosin binding ratios.
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Myosin Subfragment-1-induced polymerization of G-actin. Formation of partially decorated filaments at high actin-S1 ratios.
The Journal of biological chemistry, 1994Co-Authors: Marie-france Carlier, Dominique Didry, Inge Erk, Jean Lepault, Dominique PantaloniAbstract:Myosin Subfragment-1-induced polymerization of G-actin into arrowhead-decorated F-actin-Myosin Subfragment-1 (S1) filaments has been studied at low ionic strength and in the absence of ATP, using a combination of light scattering, fluorescence of 4-nitrobenz-2-oxa-1,3-diazol-7-yl- or pyrenyl-labeled actin, sedimentation, and electron microscopy techniques. When G-actin is in excess over Myosin Subfragment-1, the initial formation of fully decorated F-actin-S1 filaments, in which the actin:S1 molar ratio is 1:1, is followed by further incorporation of G-actin subunits in the polymer concomitant with the redistribution of the Myosin heads along the polymer, leading to partially decorated filaments containing less than one S1/actin, in equilibrium with G-actin. This process leads to an overshoot in the light-scattering polymerization curves at high actin:S1 ratios. The concentration of G-actin at equilibrium with partially decorated filaments is a nonlinear function of the molar fraction of S1 in the polymer, indicating that actin-actin-S1 interactions are energetically more favorable than actin-actin or actin-S1-actin-S1 interactions.
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Myosin Subfragment-1 interacts with two G-actin molecules in the absence of ATP.
The Journal of biological chemistry, 1991Co-Authors: C Valentin-ranc, C Combeau, Dominique PantaloniAbstract:The interaction between G-actin and Myosin Subfragment-1 (S1) has been monitored by pyrenyl-actin fluorescence and light scattering. In low ionic strength buffer and in the absence of ATP the polymerization of G-actin induced by Myosin Subfragment-1 is preceded by the formation of binary GS and ternary G2S complexes in which S1 interacts tightly in rapid equilibrium (K greater than 10(7) M-1) with one and two G-actin molecules, respectively. Pyrenyl fluorescence of G-actin is enhanced 4-fold in GS and 3-fold in G2S. At concentrations of G-actin and S1 in the micromolar range and above, G2S is the predominant species at G-actin/S1 ratios equal to or greater than 1. The isomer of Myosin Subfragment-1 carrying the A1 light chain, S1(A1), forms a tighter ternary complex than the isomer S1(A2). Actin-bound ATP is not hydrolyzed upon formation of GS and G2S. In the presence of one molar equivalent or more of Myosin Subfragment-1/mol of G-actin, in low ionic strength buffer containing no nucleotides, G-actin polymerizes faster in the presence of S1(A1) than in the presence of S1(A2). The interaction of S1 with G-actin is inhibited by the binding of ATP or ADP to S1, ATP having a higher affinity for S1 than ADP. The possible structural similarity of the G2S complex to the F-acto-S1 complex in the rigor state and the potential significance of a ternary (actin)2-Myosin interaction for actoMyosin-based motility are discussed.
Andrey A Bobkov - One of the best experts on this subject based on the ideXlab platform.
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Is SH1-SH2-cross-linked Myosin Subfragment 1 a structural analog of the weakly-bound state of Myosin
2016Co-Authors: Andrey A Bobkov, Emil ReislerAbstract:ABSTRACT Myosin Subfragment 1 (S1) with SH1 (Cys707) and SH2 (Cys697) groups cross-linked by p-phenylenedimaleimide (pPDM-S1) is thought to be an analog of the weakly bound states of Myosin bound to actin. The structural properties of pPDM-S1 were compared in this study to those of S1zADPzBeFx and S1zADPzAlF4 2, i.e., the established structural analogs of the Myosin weakly bound states. To distinguish between the conformational effects of SH1-SH2 cross-linking and those due to their monofunctional modification, we used S1 with the SH1 and SH2 groups labeled with N-phenylmaleimide (NPM-S1) as a control in our experiments. The state of the nucleotide pocket was probed using a hydrophobic fluorescent dye, 3-[4-(3-phenyl-2-pyrazolin-1-yl)benzene-1-sulfonylamido]phenylboronic acid (PPBA). Differential scanning calorimetry (DSC) was used to study the thermal stability of S1. By both methods the conformational state of pPDM-S1 was different from that of unmodified S1 in the S1zADPzBeFx and S1zADPzAlF4 2 complexes and closer to that of nucleotide-free S1. Moreover, BeFx and AlF4 2 binding failed to induce conformational changes in pPDM-S1 similar to those observed in unmodified S1. Surprisingly, when pPDM cross-linking was performed on S1zADPzBeFx complex, ADPzBeFx protected to some extent the nucleotide pocket of S1 from the effects of pPDM modification. NPM-S1 behaved similarly to pPDM-S1 in our experiments
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is sh1 sh2 cross linked Myosin Subfragment 1 a structural analog of the weakly bound state of Myosin
Biophysical Journal, 2000Co-Authors: Andrey A Bobkov, Emil ReislerAbstract:Myosin Subfragment 1 (S1) with SH1 (Cys(707)) and SH2 (Cys(697)) groups cross-linked by p-phenylenedimaleimide (pPDM-S1) is thought to be an analog of the weakly bound states of Myosin bound to actin. The structural properties of pPDM-S1 were compared in this study to those of S1.ADP.BeF(x) and S1.ADP.AlF(4)(-), i.e., the established structural analogs of the Myosin weakly bound states. To distinguish between the conformational effects of SH1-SH2 cross-linking and those due to their monofunctional modification, we used S1 with the SH1 and SH2 groups labeled with N-phenylmaleimide (NPM-S1) as a control in our experiments. The state of the nucleotide pocket was probed using a hydrophobic fluorescent dye, 3-[4-(3-phenyl-2-pyrazolin-1-yl)benzene-1-sulfonylamido]phen ylboronic acid (PPBA). Differential scanning calorimetry (DSC) was used to study the thermal stability of S1. By both methods the conformational state of pPDM-S1 was different from that of unmodified S1 in the S1.ADP.BeF(x) and S1.ADP.AlF(4)(-) complexes and closer to that of nucleotide-free S1. Moreover, BeF(x) and AlF(4)(-) binding failed to induce conformational changes in pPDM-S1 similar to those observed in unmodified S1. Surprisingly, when pPDM cross-linking was performed on S1.ADP.BeF(x) complex, ADP.BeF(x) protected to some extent the nucleotide pocket of S1 from the effects of pPDM modification. NPM-S1 behaved similarly to pPDM-S1 in our experiments. Overall, this work presents new evidence that the conformational state of pPDM-S1 is different from that of the weakly bound state analogs, S1.ADP.BeF(x) and S1.ADP.AlF(4)(-). The similar structural effects of pPDM cross-linking of SH1 and SH2 groups and their monofunctional labeling with NPM are ascribed to the inhibitory effects of these modifications on the flexibility/mobility of the SH1-SH2 helix.
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activation of regulated actin by sh1 modified Myosin Subfragment 1
Biochemistry, 1997Co-Authors: Andrey A Bobkov, Elena Bobkova, Earl Homsher, Emil ReislerAbstract:The reactive SH1 (Cys-707) group of the Myosin Subfragment 1 (S1) has been used frequently as an attachment site for fluorescent and spin probes in solution and muscle fiber experiments. In this study we examined (i) the motor function of SH1 spin-labeled heavy meroMyosin (HMM) in the in vitro motility assays and (ii) the effect of SH1-modified S1 on the motility of regulated actin, i.e., actin complexed with tropoMyosin and troponin. N-ethylmaleimide (NEM), N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-iodacetamide (IASL), N-[[(iodoacetyl)amino]ethyl]1-sulfo-5-naphthylamine (IAEDANS), and iodoacetamide (IAA) were used to selectively modify the SH1 group on S1; the SH1 group on HMM was labeled with IASL. In the in vitro motility assays, 10-20% of unregulated actin filaments moved at a speed of approximately 1 microm/s over a surface coated with 90-95% modified IASL-HMM. Actin sliding was not observed with 95-98% modified IASL-HMM. The sliding of regulated actin over unmodified HMM was activated by the addition of S1 modified with any of the SH1 reagents to the in vitro motility assay solutions; both the speeds and the percentage of the moving filaments increased at pCa 5, 7, and 8. To shed light on the activation of regulated actin sliding by SH1-modifed S1, acto-S1 ATPase and the binding to actin were determined for IASL-S1. While the binding affinities to actin were similar for IASL-S1 and unmodified S1 in the presence and absence of ADP and ATP, the Km and Vmax values were approximately 10-fold lower for the modified protein. It is concluded that the activation of regulated actin by SH1-modifed S1 facilitates the interaction of unmodified HMM heads with actin and thus can increase the sliding speeds and the percentage of regulated actin filaments that move in the in vitro motility assays.
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differential scanning calorimetric study of the complexes of modified Myosin Subfragment 1 with adp and vanadate or beryllium fluoride
Journal of Muscle Research and Cell Motility, 1996Co-Authors: Nina L Golitsina, Olga P. Nikolaeva, Victor N. Orlov, Irina Dedova, Andrey A Bobkov, D A Pavlov, Dmitrii I. LevitskyAbstract:The effects of various modifications of rabbit skeletal Myosin Subfragment 1 on thermal denaturation of Subfragment 1 in ternary complexes with Mg-ADP and orthovanadate (Vi) 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.
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differential scanning calorimetric study of the complexes of Myosin Subfragment 1 with nucleoside diphosphates and vanadate or beryllium fluoride
Biochemistry, 1995Co-Authors: Andrey A Bobkov, Dmitrii I. LevitskyAbstract:It has been recently shown by differential scanning calorimetry (DSC) that the formation of stable complexes of Myosin Subfragment 1 (S1) with Mg-ADP and orthovanadate (Vi) or beryllium fluoride (BeFx) causes a global conformational change in the S1 molecule which is reflected in a pronounced increase of S1 thermal stability and in a significant change of S1 domain structure [Shriver, J. W., & Kamath U. (1990) Biochemistry 29, 2556-2564; Levitsky, D. I., Shnyrov, V. L., Khvorov, N. V., Bukatina, A. E., Vedenkina, N. S., Permyakov, E. A., Nikolaeva, O. P., & Poglazov, B. F. (1992) Eur. J. Biochem. 209, 829-835; Bobkov, A. A., Khvorov, N. V., Golitsina, N. L., & Levitsky, D. I. (1993) FEBS Lett. 332, 64-66]. In this work, which continues the previous investigations, we report on a DSC study of the complexes of S1 with various nucleoside diphosphates (NDP). In the absence of Vi or BeFx the various Mg(2+)-NDP and Mg(2+)-PPi had a similar effect on the S1 conformation. All of them had practically no influence on the temperature of the thermal transition but increased its sharpness. However, in the presence of Vi or BeFx the effects of Mg(2+)-NDP complexes were quite different from each other and strongly depended on the base structure of NDP; their effectiveness in inducing conformational changes in S1 and the stability of these complexes decreased in the following order: ADP > CDP >> UDP >> IDP > GDP.(ABSTRACT TRUNCATED AT 250 WORDS)
Olga P. Nikolaeva - One of the best experts on this subject based on the ideXlab platform.
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intermolecular interactions of Myosin Subfragment 1 induced by the n terminal extension of essential light chain 1
Biochemistry, 2017Co-Authors: Olga P. Nikolaeva, Dmitrii I. Levitsky, Daria S LogvinovaAbstract: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).
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thermal denaturation and aggregation of Myosin Subfragment 1 isoforms with different essential light chains
International Journal of Molecular Sciences, 2010Co-Authors: Denis I. Markov, Olga P. Nikolaeva, Eugene O Zubov, B I Kurganov, Dmitrii I. LevitskyAbstract: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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changes in the thermal unfolding of p phenylenedimaleimide modified Myosin Subfragment 1 induced by its weak binding to f actin
FEBS Letters, 2001Co-Authors: Olga V Kaspieva, Olga P. Nikolaeva, Dmitrii I. Levitsky, Victor N. Orlov, V. A. Drachev, Michael A PonomarevAbstract: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°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.
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differential scanning calorimetric study of the complexes of modified Myosin Subfragment 1 with adp and vanadate or beryllium fluoride
Journal of Muscle Research and Cell Motility, 1996Co-Authors: Nina L Golitsina, Olga P. Nikolaeva, Victor N. Orlov, Irina Dedova, Andrey A Bobkov, D A Pavlov, Dmitrii I. LevitskyAbstract:The effects of various modifications of rabbit skeletal Myosin Subfragment 1 on thermal denaturation of Subfragment 1 in ternary complexes with Mg-ADP and orthovanadate (Vi) 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.
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Interaction of Myosin Subfragment 1 with F-actin studied by differential scanning calorimetry.
Biochemistry and molecular biology international, 1996Co-Authors: Olga P. Nikolaeva, Victor N. Orlov, Irina Dedova, V. A. Drachev, Dmitrii I. LevitskyAbstract: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.
