Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.6.4.1 (myosin ATPase)
 1,140 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have used saturation-transfer electron paramagnetic resonance (ST-EPR) to detect the microsecond rotational motions of spin-labeled myosin subfragment one (MSL-S1) bound to actin in the presence of the ATP analogues AMPPNP (5'-adenylylimido diphosphate) and ATP gamma S [adenosine 5'-O-(3-thiotriphosphate)], which are believed to trap myosin in strongly and weakly bound intermediate states of the actomyosin ATPase cycle, respectively. Sedimentation binding measurements were used to determine the fraction of myosin heads bound to actin under ST-EPR conditions and the fraction of heads containing bound nucleotide. ST-EPR spectra were then corrected to obtain the spectrum corresponding to the ternary complex (actin.MSL-S1.nucleotide). The ST-EPR spectrum of MSL-S1.AMPPNP bound to actin is identical to that obtained in the absence of nucleotide (rigor complex), indicating no rotational motion of MSL-S1 relative to actin on the microsecond time scale. However, MSL-S1-ATP gamma S bound to actin is rotationally mobile, with an effective rotational correlation time (tau r) of 17 +/- 2 microseconds. This motion is similar to that observed previously for actin-bound MSL-S1 during the steady-state hydrolysis of ATP [Berger et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 8753-8757]. We conclude that, in solution, the weakly bound actin-attached states of the myosin ATPase cycle undergo microsecond rotational motions, while the strongly bound intermediates do not, and that these motions are likely to be involved in the molecular mechanism of muscle contraction.
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PMID:Rotational dynamics of actin-bound intermediates in the myosin ATPase cycle. 165 57

Spin-labeling and multifrequency EPR spectroscopy were used to probe the dynamic local structure of skeletal myosin in the region of force generation. Subfragment 1 (S1) of rabbit skeletal myosin was labeled with an iodoacetamide spin label at C707 (SH1). X- and W-band EPR spectra were recorded for the apo state and in the presence of ADP and nucleotide analogs. EPR spectra were analyzed in terms of spin-label rotational motion within myosin by fitting them with simulated spectra. Two models were considered: rapid-limit oscillation (spectrum-dependent on the orientational distribution only) and slow restricted motion (spectrum-dependent on the rotational correlation time and the orientational distribution). The global analysis of spectra obtained at two microwave frequencies (9.4 GHz and 94 GHz) produced clear support for the second model and enabled detailed determination of rates and amplitudes of rotational motion and resolution of multiple conformational states. The apo biochemical state is well-described by a single structural state of myosin (M) with very restricted slow motion of the spin label. The ADP-bound biochemical state of myosin also reveals a single structural state (M*, shown previously to be the same as the post-powerstroke ATP-bound state), with less restricted slow motion of the spin label. In contrast, the extra resolution available at 94 GHz reveals that the EPR spectrum of the S1.ADP.V(i)-bound biochemical state of myosin, which presumably mimics the S1.ADP.P(i) state, is resolved clearly into three spectral components (structural states). One state is indistinguishable from that of the ADP-bound state (M*) and is characterized by moderate restriction and slow motion, with a mole fraction of 16%. The remaining 84% (M**) contains two additional components and is characterized by fast rotation about the x axis of the spin label. After analyzing EPR spectra, myosin ATPase activity, and available structural information for myosin II, we conclude that post-powerstroke and pre-powerstroke structural states (M* and M**) coexist in the S1.ADP.V(i) biochemical state. We propose that the pre-powerstroke state M** is characterized by two structural states that could reflect flexibility between the converter and N-terminal domains of myosin.
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PMID:Structure and dynamics of the force-generating domain of myosin probed by multifrequency electron paramagnetic resonance. 1833 64

Incubation of actin with decavanadate induces cysteine oxidation and oxidovanadium(IV) formation. The studies were performed combining kinetic with spectroscopic (NMR and EPR) methodologies. Although decavanadate is converted to labile oxovanadates, the rate of deoligomerization can be very slow (half-life time of 5.4 h, at 25 degrees C, with a first order kinetics), which effectively allows decavanadate to exist for some time under experimental conditions. It was observed that decavanadate inhibits F-actin-stimulated myosin ATPase activity with an IC(50) of 0.8 microM V(10) species, whereas 50 microM of vanadate or oxidovanadium(IV) only inhibits enzyme activity up to 25%. Moreover, from these three vanadium forms, only decavanadate induces the oxidation of the so called "fast" cysteines (or exposed cysteine, Cys-374) when the enzyme is in the polymerized and active form, F-actin, with an IC(50) of 1 microM V(10) species. Decavanadate exposition to F- and G-actin (monomeric form) promotes vanadate reduction since a typical EPR oxidovanadium(IV) spectrum was observed. Upon observation that V(10) reduces to oxidovanadium(IV), it is proposed that this cation interacts with G-actin (K(d) of 7.48 +/- 1.11 microM), and with F-actin (K(d) = 43.05 +/- 5.34 microM) with 1:1 and 4:1 stoichiometries, respectively, as observed by EPR upon protein titration with oxidovanadium(IV). The interaction of oxidovanadium(IV) with the protein may occur close to the ATP binding site of actin, eventually with lysine-336 and 3 water molecules.
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PMID:Decavanadate interactions with actin: cysteine oxidation and vanadyl formation. 1977 61

We have trapped the catalytic domain of Dictyostelium (Dicty) myosin II in a weak actin-binding conformation by chemically crosslinking two engineered cysteines across the actin-binding cleft, using a bifunctional spin label (BSL). By connecting the lower and upper 50 kDa domains of myosin, the crosslink restricts the conformation of the actin-binding cleft. Crosslinking has no effect on the basal ATPase activity of isolated myosin, but it impairs rigor actin binding and actin-activation of myosin ATPase. EPR spectra of BSL provide insight into actomyosin structural dynamics. BSL is highly immobilized within the actin-binding cleft and is thus exquisitely sensitive to the global orientation and rotational motions of the myosin head. Conventional EPR shows that myosin heads bound to oriented actin filaments are highly disordered with respect to the actin filament axis, in contrast to the nearly crystalline order of myosin heads in rigor. This disorder is similar to that of weakly bound heads induced by ATP, but saturation transfer EPR shows that the disorder of crosslinked myosin is at least 100 times slower. Thus this cleft-crosslinked myosin is remarkably similar, in both actin affinity and rotational dynamics, to SH1-SH2 crosslinked BSL-myosin S1. We conclude that, whether myosin is trapped at the actin-myosin interface or in the force-generating region between the active site and lever arm, the structural state of myosin is intermediate between the weak-binding state preceding phosphate release and the strong-binding state that succeeds it. We propose that it represents the threshold of force generation.
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PMID:Conformationally trapping the actin-binding cleft of myosin with a bifunctional spin label. 2325 Jul 50