Binding of Nucleotides at the Active Site Modulates the Local and Global Conformation of Myosin in Muscle Fibres (original) (raw)
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Nucleotide binding induces global and local structural changes of myosin head in muscle fibres
European journal of biochemistry / FEBS, 2001
Thermal stability and internal dynamics of myosin heads in fiber bundles from rabbit psoas muscle has been studied by electron paramagnetic resonance (EPR) spectroscopy and differential scanning calorimetry (DSC). Using ADP, ATP and orthovanadate (V(i)), three intermediate states of the ATP hydrolysis cycle were simulated in glycerinated muscle fibers. DSC transitions contained three overlapping endotherms in each state. Deconvolution showed that the transition temperature of 58.4 degrees C was almost independent of the intermediate state of myosin, while nucleotide binding shifted the melting temperatures of 54.0 and 62.3 degrees C, and changed the enthalpies. These changes suggest global rearrangements of the internal structure in myosin head. In the presence of ADP and ADP plus V(i), the conventional EPR spectra showed changes in the ordering of the probe molecules, suggesting local conformational and motional changes in the internal structure of myosin heads. Saturation transfer...
European Journal of Biochemistry, 1987
Tryptic digestion patterns reveal a close similarity of the substructure of frog subfragment-1 (Sl) to that established for rabbit S1. The 97-kDa heavy chain of chymotryptic S1 of frog myosin is preferentially cleaved into three fragments with apparent molecular masses of 29 kDa, 49 kDa and 20 kDa. These fragments correspond to the 27-kDa, 50-kDa and 20-kDa fragments of rabbit S1, respectively; this is indicated by the sequence of their appearance during digestion, by the suppression by actin of the generation of the 49-kDa and 20-kDa peptides, and by a nucleotide-promoted cleavage of the 29-kDa peptide to a 24-kDa fragment and the 49-kDa peptide to a 44-kDa fragment, analogous to the nucleotide-promoted cleavage of the 27-kDa and 50-kDa fragments of rabbit S1 to the 22-kDa and 45-kDa peptides.
Nucleotides induced changes in skeletal muscle myosin by DSC, TMDSC and EPR
2001
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.
European Journal of Biochemistry, 1990
It was previously shown that tryptic digestion of subfragment 1 (Sl) of skeletal muscle myosins at 0°C results in cleavage of the heavy chain at a specific site located 5 kDa from the NH,-terminus. This cleavage is enhanced by nucleotides and suppressed by actin and does not occur at 25"C, except in the presence of nucleotide. Here we show a similar temperature sensitivity and protection by actin of an analogous chymotryptic cleavage site in the heavy chain of gizzard S1. The results support the view that the myosin head, in general, can exist in two different conformational states even in the absence of nucleotides and actin, and indicate that the heavy chain region 5 kDa from the NH,-terminus is involved in the communication between the sites of nucleotide and actin binding.
Spin-label study of actin-myosin-nucleotide interactions in contracting glycerinated muscle fibers
Journal of Molecular Biology, 1981
This paper presents the results of simultaneous measurements of the electron paramagnetic resonance signal of spin-label bound to myosin cross-bridges and the mechanical response of glycerol-treated rabbit psoas fibers under isometric contraction. No observable change has been detected in, vitro in the local motion of spin-label bound to myosinATP with conventional electron paramagnetic resonance techniques when F-actin is added, even under conditions where more than 3074, of the myosin is expected to be in an attached state. In contrast, a clear change in the spin-label mobility is observed when cross-bridges are attached to thin filaments. Similar spectra are also observed when cross-bridges are in the rigor state or in an attached state in the presence of 5'-adenylyl imidodiphosphate in place of ATP. A good proportionality is found between the change in the electron paramagnetic resonance signal and the tension when substrate concentration is varied under conditions where no appreciable amount of rigor complex is present'. Thus, by assuming 0 and 1000/b attachment in the relaxed and rigor states, respectively, the extent of cross-bridge attachment can be estimated; it is about 8Oyb at a relatively low ATP concentration where the maximum tension is observed, while it is about 35% in the millimolar range of ATP concentration. A consistent explanation can be given for the spectra obtained both in solution and in the fiber, provided that two distinct states, the preective and active states, exist in cross-bridges attached to thin filaments. The contribution of intermediate complexes to the force generation is discussed. The effect of Ca2+ control on crossbridge attachment is also studied at various concentrations of substrate.
Effects of nucleotide binding on thermal transitions and domain structure of myosin subfragment 1
European Journal of Biochemistry, 1992
The thermal unfolding and domain structure of myosin subfragment 1 (SI) from rabbit skeletal muscles and their changes induced by nucleotide binding were studied by differential scanning calorimetry. The binding of ADP to S1 practically does not influence the position of the thermal transition (maximum at 47.2 "C), while the binding of the nun-hydrolysable analogue of ATP, adenosine 5'-[/Y~-imido]triphosphate (AdoPP[NH]P) to S1, or trapping of ADP in S1 by orthovanadate (V,), shift the maximum of the heat adsorption curve for S1 up to 53.2 and 56.loC, respectively. Such an increase of S1 thermostability in the complexes S1-AdoPP[NH]P and S1-ADP-Vi is confirmed by results of turbidity and tryptophan fluorescence measurements. The total heat adsorption curves for S1 and its complexes with nucleotides were decomposed into elementary peaks corresponding to the melting of structural domains in the S1 molecule. Quantitative analysis of the data shows that the domain structure of S1 in the complexes Sl-AdoPP[NH]P and S1-ADP-V, is similar and differs radically from that of nucleotide-free S1 and S1 in the S1-ADP complex. These data are the first direct evidence that the S1 molecule can be in two main conformations which may correspond to different states during the ATP hydrolysis : one of them corresponds to nucleotide-free S1 and to the complex S1-ADP, and the other corresponds to the intermediate complexes S1-ATP and S1-ADP-P,. Surprisingly it turned out that the domain structure of S1 with ADP trapped by p-phenylene-N, N'-dimaleimide @PDM) thiol cross-linking almost does not differ from that of the nucleotide-free S1. This means that pPDM-cross-linked S1 in contrast to S1-AdoPP[NH]P and S1-ADP-V, can not be considered a structural analogue of the intermediate complexes S1-ATP and S1-ADP-P,.
DSC study on the motor protein myosin in fibre system
Thermochimica Acta, 2006
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⋅V i , ADP⋅AlF 4 ) 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 T m1 =52.9±0.7°C, T m2 =57.9±0.7°C, T m3 =63.7±1.0°C and T m4 =67.8±0.7°C, but the last transition increased to higher temperature depending on the P i analogue.
FEBS Letters, 2001
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,NP P-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 5³C 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.
Biophysical journal, 1995
The structures of the MgADP-beryllium fluoride and MgADP-aluminum fluoride complexes of the truncated myosin head from Dictyostelium myosin II are reported. These reveal the location of the nucleotide complex and define the amino acid residues that form the active site. The tertiary structure of the beryllium fluoride complex is essentially identical to that seen previously in the three-dimensional structure of chicken skeletal muscle myosin. By contrast, significant domain movements are observed in the aluminum fluoride complex. These structural findings form the basis of a revised model for the structural basis of the contractile cycle. It is now suggested that the narrow cleft that splits the central 50-kDa segment of the heavy chain provides not only the communication route between the nucleotide-binding pocket and actin but also transmits the conformational change necessary for movement.
European Journal of Biochemistry, 1988
The effects of temperature, Mg", ATP, and actin on the conformation of the neck region of the myosin head were studied by limited proteolysis of heavy meromyosin (HMM) and subfragment 1 (Sl) preparations obtained by papain digestion of myosin in the presence of Mg2+ (Mg-S1) or EDTA (EDTA-Sl). The preparations were fluorescently labelled at the SH1 thiol group to enable identification of the COOH-terminal fragments of the head portion of the heavy chain where this group is located.