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1.
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.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
2.
D. Lőrinczy Zsuzsanna Vértes Franciska Könczöl J. Belágyi 《Journal of Thermal Analysis and Calorimetry》2009,95(3):713-719
Actin is one of the main components in the eukaryote cells which plays significant role in many cellular processes, like force-generation,
maintenance of the shape of cells, cell-division cycle and transport processes.
In this study the thermal transitions of monomer and polymerized actins were studied to get information about the changes
induced by polymerization and binding of myosin to actin using DSC and EPR techniques. The main thermal transition of F-actin
was at 67.5°C by EPR using spin-labeled actin (the relative viscosity change was around 62°C), while the DSC denaturation
T
ms were at 60.3d°C for G-actin and at 70.5°C for F-actin.
Applying the Lumry-Eyring model to obtain the parameters of the kinetic process and calculate the activation energy, a ‘break’
was found for F-actin in the function of first-order kinetic constant vs. 1/T. This indicates that an altered interdomain interaction is present in F-actin. The addition of myosin or heavy meromyosin
(HMM) in different molar ratio of myosin to actin has changed significantly the EPR spectrum of spin-labeled F-actin, indicating
the presence of the supramolecular complex. Analyzing the DSC traces of the actomyosin complex it was possible to identify
the different structural domains of myosin and actin. 相似文献
3.
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.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
4.
Lőrinczy D. Gaszner B. Könczöl F. Belágyi J. 《Journal of Thermal Analysis and Calorimetry》2000,61(2):597-605
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.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
5.
Lőrinczy D. Könczöl F. Farkas L. Belagyi J. Schick C. 《Journal of Thermal Analysis and Calorimetry》2001,66(2):633-644
Electron paramagnetic resonance (EPR, ST-EPR) and differential scanning calorimetry(DSC) were used in conventional and temperature
modulated mode to study internal motions and energetics of myosin in skeletal muscle fibres in different states of the actomyosin
ATPase cycle. Psoas muscle fibres from rabbit were spin-labelled with an isothiocyanate-based probe molecule at the reactive
sulfhydryl site (Cys-707) of the catalytic domain of myosin. In the presence of nucleotides (ATP, ADP, AMP⋅PNP) and ATP or
ADP plus orthovanadate, the conventional EPR spectra showed changes in the ordering of the probe molecules in fibres. In MgADP
state a new distribution appeared; ATP plus orthovanadate increased the orientational disorder of myosin heads, a random population
of spin labels was superimposed on the ADP-like spectrum.
In the complex DSC pattern, higher transition referred to the head region of myosin. The enthalpy of the thermal unfolding
depended on the nucleotides, the conversion from a strongly attached state of myosin to actin to a weakly binding state was
accompanied with an increase of the transition temperature which was due to the change of the affinity of nucleotide binding
to myosin. This was more pronounced in TMDSC mode, indicating that the strong-binding state and rigor state differ energetically
from each other. The different transition temperatures indicated alterations in the internal microstructure of myosin head
region The monoton decreasing TMDSC heat capacities show that C
p of biological samples should not be temperature independent.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
6.
Lőrinczy D. Hartvig N. Farkas N. Belagyi J. 《Journal of Thermal Analysis and Calorimetry》2001,65(2):351-358
Differential scanning calorimetry and electron paramagnetic resonance experiments were performed on glycerinated muscle fibres
to study the effect of the binding of nucleotides (ADP and AMP⋅PNP) to myosin. The thermal unfolding of muscle fibres showed
three discrete domain regions with thermal stabilities of 52.2, 58.8 and 67.8°C. AMP⋅PNP markedly affected the transitions,
implying the strong interaction between AMP⋅PNP and catalytic domain, and partial dissociation of heads from actin. ADP produced
only small changes in transition temperatures.
Spectrum deconvolution performed on isothiocyanate-labelled fibres in AMP⋅PNP-state resulted in two populations; 50% of labels
was highly ordered with respect to fibre axis, whereas the other 50% of labels was randomly oriented. The conformation of
the myosin heads which showed high degree of order were in the strongly binding ADP-state, the heads being attached to actin
differ from those of heads in rigor. The results support the suggestion that the attached heads in strongly binding state
to actin might have different local conformations.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
7.
Intermediate states of myosin head during atp hydrolysis cycle in psoas muscle fibres by EPR and DSC
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. 相似文献
8.
We have examined by DSC the complexes of myosin with actin in fibre system in the absence of nucleotides and the intermediate state of ATP hydrolysis by mimicking stable complex with myosin and ADP and beryllium fluoride in muscle fibres. Comparing the DSC results with other structural analogues of phosphate Pi leads the conclusion that the AM.ADP.BeFx complex favours the AM.ADP.Pi complex in fibre system. The deconvolution of DSC scans resulted in four transitions, the first three transition temperatures were almost independent of the intermediate state of the muscle, the last transition temperature was shifted to higher temperature, depending on the actual intermediate states of ATP hydrolysis. In AM.ADP.Vi state the transition temperature at the second and third transitions (actin binding domain and myosin rod) varied only slightly, whereas the last one (the fourth transition) shifted markedly to higher temperature depending on the ternary complex, e.g. in case of ADP plus BeFx it was 77.7 °C, the highest value in weakly binding state of myosin to actin. The sum of calorimetric enthalpies of the first and last curves was practically constant, but their fractions depended on the state of the muscle. In strongly binding state of myosin to actin (rigor, ADP state) the fraction of the first transition was much larger, than the last one, whereas in weakly binding state of myosin to actin, the fraction of the first transition decreased at the expense of the last one. It supports also the view that these transitions are parts of the same portion of the myosin molecule. 相似文献
9.
Differential scanning calorimetry and electron paramagnetic resonance experiments were performed on glycerinated skeletal
muscle fibres to study the effect of the binding of nucleotides and nucleotide analogues to myosin. The thermal unfolding
of muscle fibres in rigor showed three discrete domain regions with thermal stability of 52.2, 58.8 and 67.8°C. AMP.PNP and
ATP plus AlF3 or BeF2 affected markedly the transitions, which implies the strong interaction between AMP.PNP or nucleotide analogues and catalytic
domain of myosin, and a partial dissociation of heads from actin. ADP.BeFx and states model the transition states of the ATP
hydrolysis cycle which precede the powerstroke of the muscle fibres. Spectrum deconvolution on isothiocyanate-labelled fibres
in AMP.PNP-state resulted in two populations; 50% of labels was highly ordered with respect to fibre axis, whereas the other
50% of labels was randomly oriented. The myosin heads which showed high degree of order were in the strongly binding ADP-state.
The spectra in - and ADP.BeFx state reflected random orientation of labels with increased rotational mobility in comparison
with rigor. The results suggest that myosin in muscle fibres in ADP.BeFx state exists in two forms.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
10.
T. Dergez F. Könczöl N. Farkas J. Belőgyi D. Lõrinczy 《Journal of Thermal Analysis and Calorimetry》2005,80(2):445-449
Summary 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. 相似文献
11.
K. Türmer F. Könczöl D. Lőrinczy J. Belágyi 《Journal of Thermal Analysis and Calorimetry》2012,108(1):95-100
Actin is the component of several biological systems and it plays important role in different biological processes, especially
in cell motility. The actin-based motility is accompanied with ATP-consume, and the irreversible ATP hydrolysis is coupled
with the polymerization of monomer actin into filamentous form. When an actin monomer is incorporated into a filament, the
ATPase is activated, and thereby the polymer formation is promoted. The polymer formation and the ATP hydrolysis is associated
with internal motions and significant changes of the conformation in reaction partners. In this article, the ATP nucleotide
in monomer actin was exchanged by its non-hydrolyzable analogue adenylyl-imidodiphosphate (AMP.PNP), and using two biophysical
methods, electron paramagnetic resonance spectroscopy (EPR) and differential scanning calorimetry (DSC), we studied the local
and global changes in globular and fibrous actin following the nucleotide exchange. The paramagnetic probe molecule—a maleimide
spin label—was attached to Cys-374 site of monomer actin, and its rotational mobility was derived at different temperature.
In DSC measurements the transition temperatures of samples with different bound nucleotides were compared. From the measurements
we could conclude, that the nucleotide exchange induces changes in the internal rigidity of the actin systems, AMP.PNP-actins
showed longer rotational correlation time and increased thermal transition temperature. 相似文献
12.
Recent reports in the literature have indicated a physical association of creatine-phosphokinase (CPK) with the tail portion of the myosin molecule. The present paper describes further studies on the interaction of CPK with myosin and myosin fragments, using the techniques of electron paramagnetic resonance (EPR) and nanosecond fluorescence depolarization. From EPR work, spin-labeled CPK appears to interact with myosin, tail-less myosin (heavy meromyosin [HMM]), and myosin heads (subfragment-1 [S1]), the extent of interaction being proportional to the S1 content of myosin or its fragments. Spin-labeled CPK did not evidence interaction with the headless myosin "rods," with myosin tails (light meromyosin [LMM]), with S2 necks (which connect S1 to the rest of the myosin molecule), or with actin. When a fluorescent dye is directed to the essential epsilon-amino group of CPK, nanosecond fluorescence depolarization studies indicate a substantial interaction with myosin, HMM, and S1, but very little with F-actin. When the "fast-reacting" thiol of the S1 moiety or the "essential thiol" of CPK was labeled with either a fluorescent dye or a spin label, no interaction between CPK and myosin (or S1) was detected. 相似文献
13.
An optical biosensor with a stirred cuvette has been used to monitor the interaction between immobilized human serum albumin (HSA) and three water-soluble cationic porphyrins. The binding constants at 25℃ obtained from biosensor analysis were compared with those from fluorescence spectroscopy. The interactions were further investigated at temperatures from 15℃ to 30℃. The thermodynamics parameters, changes of free energy (△G), enthalpy (△H) and entropy (△S), were evaluated from equilibrium data. It appeared that the binding process was governed primarily by electrostatic forces. 相似文献
14.
A high molecular weight copolymer of N-isopropylacrylamide (NiPAAm) and vinyl imidazole (VI) was synthesized and its phase transition behavior in aqueous solutions (5 wt%) by simultaneous changes of ionic strength and temperature was investigated. At low ionic strengths, the copolymer solution showed two phases (clear and opaque solutions), which were freely mobile, as increasing temperatures up to 65°C due to repulsion interaction of positive charges developed by basic imidazole group on the polymer aggregates. However, at the physiological condition (I=0.15, T=37°C), four distinctive phases (clear solution, opaque solution, gel and shrunken gel) were observed because of charge shielding effect by added salts. The gel state was stable and maintained from 32°C to 55°C. In particular, the phase transition from opaque solution to gel rapidly occurred by the change in ionic strength (from ∼ 0 to 0.15) at 37°C. This characteristic can be utilized as a liquid embolic agent. 相似文献
15.
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 segmental flexibility of cardiac myosins.Cardiac myosins isolated from bovine and human heart muscle were spin-labelled with isothiocyanate- or maleimide-based probe molecules at the reactive sulfhydryl sites (Cys-697 and Cys-707) of the motor domain. The maleimide probe molecules attached to human cardiac myosin rotated with an effective rotational correlation time of 33 ns which was at least eight times shorter than the rotational correlation time of the same label on skeletal myosin (260 ns). In the presence of MgADP and MgADP plus orthovanadate, 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. Significant difference in the internal flexibility was detected on myosin samples isolated from ischemic tissue, the rotational correlation time decreased to 25 ns.DSC measurements supported the view that addition of nucleotides produced additional loosening in the multisubunit structure of cardiac myosin. It is postulated that there is an intersite communication between the nucleotide binding domain and the 20 kDa subunit where the reactive thiol sites are located.This work was supported by grants from the National Research Foundation (OTKA T 017099) and Ministry of Social Welfare (ETT 737/1993). The Bruker ESP 300 E spectrometer and the SETARAM Micro DSC-II used in the experiments were purchased with funds provided by the National Research Foundation Grants CO-123 and CO-272. The computer (PC-386) was supported by POPEX Ltd., Pécs, Hungary. The authors thank to Prof. Dr. K. Hideg (Central Research Laboratory) for providing the iodoacetamide spin label. 相似文献
16.
Yu. M. Shul’ga A. V. Kulikov V. M. Martynenko V. V. Open’ko V. G. Karatevskii Yu. G. Morozov 《Russian Journal of Physical Chemistry A, Focus on Chemistry》2008,82(8):1314-1317
For fullerite C60 with intercalated oxygen, a sharp (by three orders of magnitude) increase in the intensity of the EPR signal with a g-factor of 2.0024 was observed at ~200°C. Studies of gases formed in heating of the sample in a vacuum showed that molecular oxygen was largely released at temperatures below 100°C, whereas the gas phase formed as the temperature increased to 200°C contained carbon oxides CO and CO2 in addition to oxygen. The conclusion was drawn that the intensity of the EPR signal was determined by the products of oxygen interaction with fullerene rather than the concentration of oxygen in the sample. 相似文献
17.
Andrea Vig Réka Dudás Tünde Kupi J. Orbán G. Hild D. Lőrinczy M. Nyitrai 《Journal of Thermal Analysis and Calorimetry》2009,95(3):721-725
The effect of phalloidin on filaments polymerized from ADP-actin monomers of the heart muscle was investigated with differential
scanning calorimetry. Heart muscle contains α-skeletal and α-cardiac actin isoforms. In the absence of phalloidin the melting
temperature was 55°C for the α-cardiac actin isoform and 58°C for the α-skeletal one when the filaments were generated from
ADP-actin monomers. After the binding of phalloidin the melting temperature was isoform independent (85.5°C). We concluded
that phalloidin stabilized the actin filaments of α-skeletal and α-cardiac actin isoforms to the same extent when they were
polymerized from ADP-actin monomers. 相似文献
18.
G. Rezaei Behbehani A. A. Saboury E. Yahaghi 《Journal of Thermal Analysis and Calorimetry》2010,100(1):283-288
A thermodynamic study on the interaction of bovine carbonic anhydrase II (CAII) with nickel ions was performed by using isothermal
titration calorimetry (ITC) at 27 °C in Tris buffer solution at pH = 7.5. The enthalpies of Ni2+ + CAII interaction are reported and analysed in terms of the new solvation theory. It was indicated that there are three
identical and non-cooperative sites for Ni2+. The binding of a nickle ion is exothermic with dissociation equilibrium constants of 81.306 and 99.126 μM at 27°C and 37°C,
respectively. The binding of nickel ions can cause some changes in the stability of the enzyme at low and high Ni2+ concentrations. 相似文献
19.
A. A. Saboury H. Ghourchaei M. H. Sanati M. S. Atri M. Rezaei-Tawirani G. H. Hakimelahi 《Journal of Thermal Analysis and Calorimetry》2007,89(3):921-927
Binding properties and structural changes of human growth hormone (hGH) due to the interaction by cobalt ion (Co2+) were done at 27°C in NaCl solution, 50 mM, using different techniques of UV-Vis spectroscopy, circular dichroism (CD), isothermal
titration calorimetry (ITC) and differential scanning calorimetry (DSC) techniques. There is a set of three identical and
non-interacting binding sites for cobalt ions. The intrinsic association equilibrium constant and the molar enthalpy of binding
obtained by ITC are 0.80 mM−1 and −16.70 kJ mol−1, respectively. The intrinsic association equilibrium constant obtained by a standard isothermal titration UV-Vis spectrophotometry
method is also 0.79 mM−1, which is in good agreement with the value obtained from ITC. The Gibbs free energy and entropy changes due to the binding
of cobalt ion on hGH are −16.67 kJ mol−1 and −0.1 J K−1 mol−1, respectively. Energetic domains analysis by DSC shows that phase transition of hGH in the presence of cobalt occurs at one
main transition. Deconvolution of the main transition provides two sub-transitions with different values of the melting point
and enthalpy of unfolding (33°C and 164 kJ mol−1 for the first and 49°C and 177 kJ mol−1 for the second, respectively). Interaction of cobalt ions with hGH prevents aggregation by an affect on the hydrophobicity
of the protein macromolecule and provide useful information about its structure due to becoming reversible of protein thermal
denaturation. 相似文献
20.
Jan Cocquyt Ulf Olsson Gerd Olofsson Paul Van der Meeren 《Colloid and polymer science》2005,283(12):1376-1381
Two endothermic transitions, at 36°C and 44°C, were observed with differential scanning calorimetry (DSC) upon heating dioctadecyldimethylammonium
bromide vesicle dispersions that were equilibrated below 15°C while in samples kept at 25°C there was only the transition
at 44°C, which was shown to be the gel to liquid–crystalline transition by 1H-NMR measurements. The transition at 36°C was reversed in an exothermic transition around 13°C upon cooling. The slowness
of this transition at ambient temperatures suggests that the presence of the transition at 36°C in a DSC upscan depends strongly
on the sample history. 相似文献