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ABSTRACT: Thermal stability and internal dynamics of myosin head in psoas muscle fibres of rabbit in the intermediate state AM.ADP.Pi - mimicked by AM.ADP.Vi - of the ATP hydrolysis cycle was studied by differential scanning calorimetry and spin label electron paramagnetic resonance
spectroscopy. Three overlapping endotherms were detected in rigor, in strongly binding ADP and weakly binding AM.ADP.Vi state of myosin to actin. The transition at 54.0°C can be assigned to the 50 k actin-binding domain. The transition at highest
temperature (67.3°C) represents the unfolding of actin and the contributions arising from the nucleotide-myosin head interaction.
The transition at 58.4°C reflects the melting of the large rod part of myosin. Nucleotide binding (ADP, ATP plus orthovanadate)
induced shifts of the melting temperatures and produced changes in the calorimetric enthalpies. The changes of the EPR parameters
indicated local rearrangements of the internal structure in myosin heads in agreement with DSC findings.
conformation of myosin–nucleotide-myosin interaction–DSC–EPR–orthovanadate–ATP hydrolysis
Journal of Thermal Analysis and Calorimetry 04/2012; 72(2):573-580. · 1.60 Impact Factor
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ABSTRACT: Force generation in muscle during contraction arises from direct interaction of the two main protein components of the muscle,
myosin and actin. The process is driven by the energy liberated from the hydrolysis of ATP. In the presence of CaATP the energy
released from hydrolysis produces conformational changes in myosin and actin, which can be manifested as an internal motion
of myosin head while bound to actin. It is suggested that myosin heads attached to actin produce conformational changes during
the hydrolysis process of ATP, which results in a strain in the head portion of myosin in an ATP-dependent manner. These structural
changes lead to a large rotation of myosin neck region relieving the strain.
Paramagnetic probes and EPR spectroscopy provide direct method in which the rotation and orientation of specifically labelled
proteins can be followed during muscle activity. In order to find correlation between local and global structural changes
in the intermediate states of the ATPase cycle, the spectroscopic measurements were combined with DSC measurements that report
domain stability and interactions.
In the review a detailed description of the application of EPR and DSC techniques in muscle protein research will be given.
The measurements show that the small local structural changes detected by EPR after nucleotide binding influence the global
structure of protein system responsible for muscle contraction.
Journal of Thermal Analysis and Calorimetry 04/2012; 90(2):611-621. · 1.60 Impact Factor
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ABSTRACT: A carotenoid-less Phaffia rhodozyma mutant (MCP 325) exhibited significantly higher resistance to oxidative stressors such as menadione, H2O2 and K2Cr2O7 than its astaxanthin-producing parental strain (MCP 324). The absence of carotenoids in the mutant did not explain this phenomenon. The cause of the decreased superoxide, hydroxyl radical and glutathione contents, the increased peroxide concentration and the elevated specific activity of catalase under uninduced conditions may be a second mutation. Peroxide treatment induced specific catalase activity in the mutant but not in the parental strain. Regulation of these processes led to the result that, in spite of the mutations, the two strains exhibited the same multiplication rate and generation time.
Acta Biologica Hungarica 06/2011; 62(2):204-10. · 0.59 Impact Factor
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ABSTRACT: A chromate-tolerant mutant chr1-663T bearing a stable one-gene mutation and its parental strain 6chr(+) were used to investigate the background of Cr(VI) tolerance in the fission yeast Schizosaccharomyces pombe. The mutant chr1-663T displayed a significantly decreased specific glutathione reductase (GR) activity coded by the pgr1 (+) gene compared with its parental strain. Transformants of the mutant chr1-663T with a nonintegrative pUR18N vector expressing the pgr1 (+) gene exhibited the same Cr(VI) sensitivity and specific GR activity as their parental strain, demonstrating the importance of the GR-NADPH system in Cr(VI) tolerance. Transformants, nevertheless, exhibited an increased intracellular peroxide concentration, a decreased Cr(VI)-reducing and HO*-producing ability, which suggested an unbalanced oxidoreduction state of cells and partial complementation of the GR function. No mutation was found in the sequences of the pgr1 (+) and the pap1 (+) (transcriptional regulatory gene of GR) genes of the Cr(VI)-tolerant mutant by sequence analysis.
Folia Microbiologica 02/2008; 53(4):308-14. · 0.68 Impact Factor
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ABSTRACT: The heat capacity of contractile proteins actin and myosin was studied in psoas muscle of rabbit in strongly and weakly binding state of myosin to actin as a function of temperature by DSC. Deconvolution of the unfolding scans makes possible to characterize the structural domains of the macromolecules. We tried to approach the unfolding process in different intermediate state of ATP hydrolysis. The thermal transitions were calorimetrically irreversible, therefore the two-state irreversible model that describes fairly well the denaturation of different proteins was used for evaluation of the denaturation processes in muscle fibers in strongly (rigor, ADP) and weakly binding states (ATP·Vi, ADP·AlF4) of myosin to actin. Deconvolution resulted in four transitions, the first three transition temperatures were almost independent of the intermediate states of muscle, the last transition temperature was shifted to higher temperature, when the buffer solution was manipulated. The mean values in strongly binding states were Tm1=52.9±0.7°C, Tm2=57.9±0.7°C, Tm3=63.7±1.0°C and Tm4=67.8±0.7°C, but the last transition increased to higher temperature depending on the Pi analogue.
Journal of Thermal Analysis and Calorimetry 01/2005; 80(2):445-449. · 1.60 Impact Factor
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ABSTRACT: The effect of AMP.PNP on the thermal stability and dynamics of myosin head were investigated by using DSC and different spin
label technique for chemically skinned muscle fibres prepared from rabbit. The thermal unfolding of the fibres in rigor, strong
as well as weak-binding state showed a complex process characterizing at least three discrete domain regions with different
stability (T
m =54, 58.4 and 62.3°C). The unfolding at 54°C refers to the catalytic domain of myosin, whereas transition at T
m =58.4°C represents the rod-like region. In the presence of AMP.PNP only the parameters of the last transition changed significantly
(T
m =70.4°C) showing an increased interaction between actin and myosin heads being attached to actin. Measurements on MSL-fibres
(labelled at Cys-707 of myosin) in the presence of AMP.PNP showed that about half of the cross-bridges dissociated from actin.
This fraction had a dynamic disorder, the other population had the same spectral feature as in rigor. In contrast, on TCSL-fibres
AMP.PNP increased the orientational disorder of myosin heads, a random population of spin labels was superimposed on the ADP-like
spectrum showing conformational and motional changes in the internal structure of myosin heads. ST EPR measurements reported
increased rotational mobility of spin labels in the presence of AMP.PNP. The DSC and EPR results suggest that in the presence
of AMP.PNP the attached heads have the same global orientation as in rigor, but the internal structure undergoes a local conformational
change.
Journal of Thermal Analysis and Calorimetry 04/2001; 64(2):651-658. · 1.60 Impact Factor
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ABSTRACT: Conformational and dynamic properties of actin filaments polymerized from ATP- or ADP-actin monomers were compared by using fluorescence spectroscopic methods. The fluorescence intensity of IAEDANS attached to the Cys(374) residue of actin was smaller in filaments from ADP-actin than in filaments from ATP-actin monomers, which reflected a nucleotide-induced conformational difference in subdomain 1 of the monomer. Radial coordinate calculations revealed that this conformational difference did not modify the distance of Cys(374) from the longitudinal filament axis. Temperature-dependent fluorescence resonance energy transfer measurements between donor and acceptor molecules on Cys(374) of neighboring actin protomers revealed that the inter-monomer flexibility of filaments assembled from ADP-actin monomers were substantially greater than the one of filaments from ATP-actin monomers. Flexibility was reduced by phalloidin in both types of filaments.
Journal of Biological Chemistry 01/2001; 275(52):41143-9. · 4.77 Impact Factor
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ABSTRACT: Differential scanning calorimetry (DSC) and electron paramagnetic resonance spectroscopy (EPR, both conventional and saturation
transfer EPR) were used to study the motional dynamics and segmental flexibility of myosin in muscle fibres in the presence
of free radical generating system.
Muscle fibre bundles isolated from psoas muscle of rabbit were spin-labelled with maleimide- and isothiocyanate-based probe
molecules at the reactive sulfhydryl sites (Cys-707) of the motor domain. In the presence of hydroxyl free radicals the spectral
intensity of the maleimide probe molecules decreased with time following a single exponential curve. MgADP and MgATP plus
orthovanadate that produce flexibility changes in the multisubunit structure of myosin enhanced the reduction of the attached
nitroxide molecules in free radical generating system. The analysis of the EPR spectra of spin-labelled and oriented fibres
showed that the narrow distribution of spin labels changed in the presence of hydroxyl free radicals. Spectrum analysis by
computer subtraction showed that short irradiation by UV light resulted in the enhancement of the ordered population at the
expense of the disordered population. This suggests a transition of myosin heads from weak- binding state into strong-binding
state.
DSC measurements performed on calf cardiac myosin resulted in two main transitions at 49.4 and 54.1°C, respectively. Addition
of MgADP produced a decrease of the 49.4°C transition, whereas a shift towards higher temperature was detected at the 54.1°C
transition. It shows that there is an inter-site communication between the domains of the myosin. Hydroxyl free radicals induced
further shifts of the transition temperatures and affected the width of the heat absorption curves.
Journal of Thermal Analysis and Calorimetry 07/2000; 61(2):597-605. · 1.60 Impact Factor
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ABSTRACT: The in vivo effects of CrCl(3) on an ergosterol-producing 33 erg(+) strain of the eukaryotic yeast Candida albicans, and on its ergosterol-deficient erg(-)2 mutant, were studied by using electron paramagnetic resonance spectroscopy. A 5-doxylstearic acid spin probe was applied to label the membranes. The absence of ergosterol, an increased accumulation of Delta(8) sterols, a decreased fatty acid chainlength and a lower proportion of unsaturated fatty acids of the erg(-)2 mutant resulted in a higher membrane rigidity and an increased sensitivity to Cr(III) than those of the parental 33 erg(+) strain. Cr(III) significantly increased the fluidity of the spin labelled membranes, this being more pronounced for the erg(-)2 mutant. The break in the slopes measured for the erg(-)2 mutant was decreased (DeltaAT approximately 4 degrees C) from 17 to 13 degrees C. Cr(III) treatment for 10 h caused a loss of metabolites adsorbing at 260 nm: this loss was 40% for 33 erg(+) and 60% for erg(-)2. This decriptification process might be the main cause of growth inhibition and cell killing by the impermeable Cr(III) ions.
FEMS Microbiology Letters 02/2000; 182(2):375-80. · 2.04 Impact Factor
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ABSTRACT: The internal dynamics and the thermal stability of myosin in rabbit psoas muscle fibres in different intermediate states of
the ATP hydrolysis cycle were studied by differential scanning calorimetry (DSC) and electron paramagnetic resonance (EPR)
spectroscopy. Three overlapping endotherms were detected in rigor, in strongly binding and weakly binding state of myosin
to actin. The transition at 58.4°C can be assigned to the nucleotide-binding domain. The transition at highest temperature
represents the unfolding of the actin and the contributions arising from the actin-myosin interaction. The transition of 54°C
reflects the interaction between the subunits of myosin. Nucleotide binding induced shifts of the melting temperatures and
produced variations in the calorimetric enthalpy changes. The changes of the EPR parameters indicated local rearrangements
of the internal structure in myosin heads.
Journal of Thermal Analysis and Calorimetry 10/1999; 56(3):1203-1209. · 1.60 Impact Factor
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ABSTRACT: The effect of the replacement of ATP with ADP on the conformational and dynamic properties of the actin monomer was investigated, by means of electron paramagnetic resonance (EPR) and fluorescence spectroscopic methods. The measurement of the ATP concentration during these experiments provided the opportunity to estimate the time dependence of ADP-Mg-G-actin concentration in the samples. According to the results of the fluorescence resonance energy transfer experiments, the Gln-41 and Cys-374 residues are closer to each other in the ADP-Mg-G-actin than in the ATP-Mg-G-actin. The fluorescence resonance energy transfer efficiency increased simultaneously with the ADP-G-actin concentration and reached its maximum value within 30 min at 20 degrees C. The EPR data indicate the presence of an ADP-Mg-G-actin population that can be characterized by an increased rotational correlation time, which is similar to the one observed in actin filaments, and exists only transiently. We suggest that the conformational transitions, which were reflected by our EPR data, were coupled with the transient appearance of short actin oligomers during the nucleotide exchange. Besides these relatively fast conformational changes, there is a slower conformational transition that could be detected several hours after the initiation of the nucleotide exchange.
Biochemistry 10/1999; 38(39):12885-92. · 3.42 Impact Factor
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ABSTRACT: The temperature profile of the fluorescence resonance energy transfer efficiency normalized by the fluorescence quantum yield of the donor in the presence of acceptor, f', was measured in a way allowing the independent investigation of (i) the strength of interaction between the adjacent protomers (intermonomer flexibility) and (ii) the flexibility of the protein matrix within actin protomers (intramonomer flexibility). In both cases the relative increase as a function of temperature in f' is larger in calcium-F-actin than in magnesium-F-actin in the range of 5-40 degrees C, which indicates that both the intramonomer and the intermonomer flexibility of the actin filaments are larger in calcium-F-actin than those in magnesium-F-actin. The intermonomer flexibility was proved to be larger than the intramonomer one in both the calcium-F-actin and the magnesium-F-actin. The distance between Gln41 and Cys374 residues was found to be cation-independent and did not change during polymerization at 21 degrees C. The steady-state fluorescence anisotropy data of fluorophores attached to the Gln41 or Cys374 residues suggest that the microenvironments around these regions are more rigid in the magnesium-loaded actin filament than in the calcium-loaded form.
Journal of Biological Chemistry 06/1999; 274(19):12996-3001. · 4.77 Impact Factor
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ABSTRACT: The principal aim of this investigation was to study the change of the protein flexibility and/or conformational properties of actin filaments upon the replacement of Ca2+ by Mg2+. The temperature dependence of the fluorescence lifetime and the anisotropy decay of N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) attached covalently to the Cys374 residue of actin were measured. Saturation transfer electron paramagnetic resonance (ST-EPR) experiments were also carried out using N-(1-oxyl-2,2,6, 6-tetramethyl-4-piperidinyl)-maleimide (MSL) attached to the same residue (Cys374). The Arrhenius analysis of the temperature dependence of the fluorescence lifetimes shows that for Mg-F-actin, both the activation energy (E*) and the frequency factor (A) are smaller than they are for Ca-F-actin. The longer rotational correlation times resolved in the fluorescence experiments are larger in the Mg2+-loaded form of the actin filament between 6 degreesC and 28 degreesC, but this difference becomes negligible above 28 degreesC. The results of saturation transfer electron paramagnetic resonance measurements on maleimide spin-labeled actin filaments indicate that the replacement of Ca2+ by Mg2+ induced a decrease of the mobility of the label on the sub-millisecond time scale. Based upon these results, we concluded that the filaments polymerized from Ca-actin are more flexible than the filaments of Mg-actin.
Biophysical Journal 01/1999; 75(6):3015-22. · 3.65 Impact Factor
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ABSTRACT: Temperature dependence of the fluorescence intensity and anisotropy decay of N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine attached to Cys374 of actin monomer was investigated to characterize conformational differences between Ca- and Mg-G-actin. The fluorescence lifetime is longer in Mg-G-actin than that in Ca-G-actin in the temperature range of 5-34 degrees C. The width of the lifetime distribution is smaller by 30% in Mg-saturated actin monomer at 5 degrees C, and the difference becomes negligible above 30 degrees C. The semiangle of the cone within which the fluorophore can rotate is larger in Ca-G-actin at all temperatures. Electron paramagnetic resonance measurements on maleimide spin-labeled (on Cys374) monomer actin gave evidence that exchange of Ca2+ for Mg2+ induced a rapid decrease in the mobility of the label immediately after the addition of Mg2+. These results suggest that the C-terminal region of the monomer becomes more rigid as a result of the replacement of Ca2+ by Mg2+. The change can be related to the difference between the polymerization abilities of the two forms of G-actin.
Biophysical Journal 11/1997; 73(4):2023-32. · 3.65 Impact Factor
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ABSTRACT: The parameters characterizing the quenching of fluorescence emitted by the four tryptophans of actin were measured by time resolved techniques in the monomeric (G) and the polymerized (F) forms of the protein. Acrylamide as a neutral and cesium ions as positively charged quenchers were used to characterize the subdomain 1, which contains all the four tryptophan residues. Use of acrylamide did not reveal any difference between the G- and F-forms, cesium, however, did so. The value of the quenching rate constant, k+, is significantly higher for the F-form than that for the G-form. This difference is present independently of the model (discrete or continuous lifetime distribution) used to process the data. These results are compatible with the conclusion that the charge distribution of the microenvironment around at least one of the four tryptophans is changed. This means that this region becomes more attracted to the cesium ion as a result of transition from the G- to the F-form.
Journal of Photochemistry and Photobiology B Biology 10/1996; 35(3):175-9. · 2.81 Impact Factor
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ABSTRACT: Conventional and saturation transfer electron paramagnetic resonance spectroscopy (EPR and ST EPR) and differential scanning calorimetry (DSC) were used to study the motional dynamics and structural stability of cardiac myosins. Cardiac myosins isolated from bovine and human heart muscle were spin-labelled with a maleimide- or an iodoacetamide-based probe molecule at the reactive sulhydryl sites (Cys-697 and Cys-707). The probe molecules rotated with an effective rotational correlation time of 0.04 microsecond which was at least six times shorter than the rotational correlation time of the same label on skeletal myosin. In the presence of MgADP, flexibility changes in the multisubunit structure of myosins were detected, but this did not lead to changes of the overall rotational property of the myosin heads. Temperature dependence of the EPR spectra of maleimide spin-labelled myosin showed continuous decrease of the spectral parameters (amplitude ratio of the diagnostic peak heights in the ST EPR time domain and hyperfine splitting constant) at increasing temperature. In contrast, marked changes were obtained at about 17 degrees C in light chain-2 deficient myosin. DSC measurements supported the view that the removal of the light chain-2 produced additional loosening in the multisubunit structure of cardiac myosin. It is postulated that the light chain-2 is an integral part of myosin, and there is an intersite communication between light chain-2 and the 20 kDa subunit.
Acta Physiologica Hungarica 02/1995; 83(4):373-87. · 0.82 Impact Factor
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ABSTRACT: Rotational dynamics and ordering of myosin heads in glycerinated skeletal muscle fibres were studied using an isothiocyanate-based spin label attached to the fast-reacting thiol sites of myosin and were compared with data obtained for maleimide and iodoacetamide spin labels attached to the same sites. The ordering of probe molecules on the millisecond time scale in the rigor state, at sarcomere length 2.2-2.3 +/- 0.1 microns, was static. Isothiocyanate probe molecules showed greater mobility; the segment holding the label rotated in the microsecond time range. In the saturation transfer EPR time domain, MgADP did not produce a significant change in the mobility of spin labels. The spectra of isothiocyanate spin-labelled fibres were analyzed in terms of two narrow distributions with mean angles of 75 degrees and 56 degrees. In the rigor state, the fractions represented approximately 76% and 24% of the total EPR absorbance. In the presence of MgADP, the conventional EPR spectra showed large changes in the ordering of isothiocyanate probe molecules towards a new distribution, the population with a theta value of 56% increased from 24% to 71% at the expense of the 75% population with no change in the mean angles of the distributions. In the case of maleimide and iodoacetamide spin-labelled fibres, however, the effect of MgADP on the probe angular distribution was small.
European Journal of Biochemistry 09/1994; 224(1):215-22. · 3.58 Impact Factor
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ABSTRACT: The radical scavenging capacity of garlic (allium sativum) has been investigated in vitro in radical generating systems by electron paramagnetic resonance (EPR) and low-level chemiluminescence measurements. Garlic (both the homogenate of 10% in physiological saline solution and its supernatant) was able to reduce the radicals generated by the Fenton reaction and trapped by phenyl-butyl-nitron for EPR measurements. Also radicals present in cigarette smoke could be reduced by garlic as judged from chemiluminescence in the presence of tert.-butyl-hydroperoxide. According to the in vitro measurements garlic contains substances which have significant radical scavenging capacity.
Arzneimittel-Forschung 06/1994; 44(5):608-11. · 0.72 Impact Factor
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ABSTRACT: The effect of 6,6'-methylene-bis-2,2,4-trimethyl-1,2-dihydroquinoline (MTDQ) and its water soluble species (MTDQ-DA) was studied in biological membrane and model systems using EPR spectroscopy for detecting molecular motion and radical formation. Both compounds influenced the rotational mobility of maleimide spin labels attached to proteins of the nerve membrane: the addition of MTDQ or MTDQ-DA induced an increase of the rigidity of the membrane in the environment of the attaching sites. The reaction of MTDQ-DA with hydroxyl and superoxide free radicals showed that this compound was also a competitive OH and superoxide free radical scavenger. The reaction rate constant of the formation of MTDQ-DA free radical was k = (4.0 +/- 0.5) x 10(9) M-1 s-1 in the hydroxyl free radical generating system. Simulation of EPR spectra supported that MTDQ-DA free radical was very likely a stable nitroxide free radical.
Biochimica et Biophysica Acta 03/1994; 1190(1):123-8. · 4.66 Impact Factor
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ABSTRACT: Thermal behavior of intact and LC-2 deficient myosin obtained from bovine heart was studied using EPR and DSC techniques. The reactive thiol sites (Cys 704) of myosin was labelled with 4-maleimidopiperidine-nitroxyl, and the measurements were taken in X-band in the conventional and saturation transfer EPR time domains. DSC scans were made from 5 degrees up to 60 degrees C with 0.25 degree C/min scan rate. Bovine heart myosin was isolated by standard methods. The LC-2 deficient myosin was prepared by cleaving myosin with alpha-chymotrypsin (400:1 molar ratio) for 1.5 min at 25 degrees. Our basic finding was a conformational change in LC-2 deficient myosin detected at 18 degrees C. It was not observed in intact myosin suggesting that the dissociation of the regulatory light chain resulted in a local structural change in the neighbourhood of the attached label in the 20 kD domain. The rotational correlation time of the label and the microwave saturation behavior of myosin at 25 degrees C exhibited no significant differences after removal of the LC-2 light chain. However, the mobility of the same label was significantly diminished in skeletal muscle. Studying the melting behavior of myosin, six endothermic peaks were detected at 19; 41.3; 43.3; 45.5; 48.5; and 54.3 degrees C (enthalpies: 708.4; 399; 773.8; 1089; 1612.8; and 3304.8 kJ/mol). They were assigned to the segment containing the essential thiols: HMM S-2, HMM S-1 (50kD and 20kD plus 27kD) and LMM. Removal of the LC-2 light chain was associated with the disappearance of the 18 degrees transition showing again a structural change in LC-2 deficient myosin which extended to a larger region.
General Physiology and Biophysics 01/1991; 9(6):589-603. · 1.19 Impact Factor
