High-Resolution Detection of muscle Crossbridge Orientation by Electron Paramagnetic Resonance

Barnett, Vincent A.; Fajer, Piotr G.; Polnaszek, Carl F.; Thomas, David D.

doi:10.1016/s0006-3495(86)83628-1

Cited by 32 publications

(39 citation statements)

References 6 publications

(10 reference statements)

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“…The spectrum of the MSL-S 1 bound to the oriented actin was fitted assuming two orientational distributions, and the spectrum of MSL-S1 bound to actin in the oriented fibers was fitted assuming a single orientational distribution. The fit of the MSL-Sl decorated fibers resulted in an orientational distribution very similar to that previously reported (Barnett et al, 1986;Fajer et al, 1990b). As expected, the fit of the spectrum of MSL-S1 bound to actin oriented by flow resulted in a highly ordered component and a disordered component resembling a powder spectrum.…”

Section: Orientational Distribution Of Msl-actin Oriented By Flowsupporting

confidence: 83%

“…4, Table 2) was good, but the residual spectrum showed significant disagreement, especially in the high-field part of the experimental spectrum. The sharper high-field peaks of the experimental spectrum clearly indicate a narrower Gaussian distribution (AO) about the average angle 00 than found by the computer fit (Barnett et al, 1986).…”

Section: Orientational Distribution Of Msl-actin Oriented By Flowmentioning

confidence: 58%

“…2, bottom) are the intense and narrow low-field peak and the positive high-field peak. These features are indicative ofhigh orientation (Barnett et al, 1986). If the MSL-actin were not oriented, or if there were no preferred angular distribution of spin labels on the oriented actin, the spectra of the samples flowing parallel and perpendicular to the magnetic field would both resemble Fig.…”

Section: Conventional Epr Of Msl-actinmentioning

confidence: 99%

“…In the fitting ofthe two components, the values of 00 and AO of the first component (component A) were restricted between the angles 0 and 450. This restriction was justified by the measurement of the spectral splitting (2T') and the splitting of the highfield peaks (AHPP) (Barnett et al, 1986;Fajer et al, 1990a). All other angles were allowed to vary between 0 and 900.…”

Section: Orientational Distribution Of Msl-actin Oriented By Flowmentioning

confidence: 99%

“…Spectra of MSL-Sl decorated fibers and spectra of oriented solutions of 100 ,uM actin and 100 ,uM MSL-S1 were acquired and computer fitted ( Table 2). The tensor and line-width parameters used in the fits were determined previously (Barnett et al, 1986; al., 1 990b). The spectrum of the MSL-S 1 bound to the oriented actin was fitted assuming two orientational distributions, and the spectrum of MSL-S1 bound to actin in the oriented fibers was fitted assuming a single orientational distribution.…”

Section: Orientational Distribution Of Msl-actin Oriented By Flowmentioning

confidence: 99%

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Orientational distribution of spin-labeled actin oriented by flow

Ostap¹,

Yanagida²,

Thomas³

1992

Biophysical Journal

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Previous studies on spin-labeled F-actin (MSL-actin), using saturation transfer electron paramagnetic resonance (ST-EPR), have demonstrated that actin has submillisecond rotational flexibility and that this flexibility is affected by the binding of myosin and its subfragments. This rotational flexibility does not change during the active interaction of myosin heads, actin, and adenosine triphosphate. However, these ST-EPR studies, performed on randomly oriented actin, would not be sensitive to orientational changes on the millisecond time scale or slower. In the present study, we have clarified these results by performing conventional EPR experiments on MSL-actin oriented by flow to detect changes in the orientational distribution. We have determined the orientational distribution of the spin labels relative to the magnetic field (flow direction) by comparing experimental EPR spectra to simulated EPR spectra corresponding to known orientational distributions. Spectra acquired during flow indicate two populations of probes: a highly ordered population and a disordered population. For the ordered population (28% of the total spin concentration), the angle between the actin filament axis and the nitroxide z axis (theta) fits a Gaussian distribution centered at 32.0 +/- 0.9 degrees, with a full width at half maximum of 20.7 +/- 3.9 degrees. The angle between the nitroxide x axis and the projection of the field in the xy plane (phi) is centered at 37.5 +/- 9.2 degrees with a full width of 24.9 +/- 10.7 degrees. This orientational distribution is not significantly changed upon the binding of phalloidin or myosin subfragment 1 (S1), indicating that these proteins do not affect the axial orientation of actin subunits. Spectra of spin-labeled S1 (MSL-S1) bound to actin oriented by flow have about the same orientational distribution as MSL-S1 bound to actin in oriented fibers. Thus, the oriented fraction of flow-oriented actin filaments has nearly the same high degree of alignment as the actin filaments in muscle fibers.

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Section: Orientational Distribution Of Msl-actin Oriented By Flowsupporting

confidence: 83%

Section: Orientational Distribution Of Msl-actin Oriented By Flowmentioning

confidence: 58%

Section: Conventional Epr Of Msl-actinmentioning

confidence: 99%

Section: Orientational Distribution Of Msl-actin Oriented By Flowmentioning

confidence: 99%

Section: Orientational Distribution Of Msl-actin Oriented By Flowmentioning

confidence: 99%

See 3 more Smart Citations

Orientational distribution of spin-labeled actin oriented by flow

Ostap¹,

Yanagida²,

Thomas³

1992

Biophysical Journal

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Orientation and rotational dynamics of spin‐labeled phalloidin bound to actin in muscle fibers

Naber

et al. 1993

Proteins

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We have used electron paramagnetic resonance spectroscopy (EPR) to investigate the orientational distribution of actin in thin filaments of glycerinated muscle fibers in rigor, relaxation, and contraction. A spin-labeled derivative of a mushroom toxin, phalloidin (PHSL), was bound to actin in the muscle fibers (PHSL-fibers). The EPR spectrum of unoriented PHSL-labeled myofibrils consisted of three sharp lines with a splitting between the outer extrema (2T parallel') of 42.8 +/- 0.1 G, indicating that the spin labels undergo restricted nanosecond rotational motion within an estimated half-cone angle of 76 degrees. When the PHSL-fiber bundle was oriented parallel to the magnetic field, the splitting between the zero-crossing points (2T') was 42.7 +/- 0.1 G. When the fiber bundle was perpendicular to the magnetic field, 2T' decreased to 34.5 +/- 0.2 G. This anisotropy shows that the motion of the probe is restricted in orientation by its binding site on actin, so that the EPR spectrum of PHSL-fiber bundles would be sensitive to small changes in the mean axial orientation of the PHSL-actin interface. No differences in the EPR spectra were observed in fibers during rigor, relaxation, or contraction, indicating that the mean axial orientation of the PHSL binding site changes by less than 5 degrees, and that the amplitude of nanosecond probe rotational motion, which should be quite sensitive to the local environment of the phalloidin, changes by no more than 1 degree.(ABSTRACT TRUNCATED AT 250 WORDS)

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Structural Determination of Spin Label Immobilization and Orientation: A Monte Carlo Minimization Approach

Sale

Sár

Sharp

et al. 2002

Journal of Magnetic Resonance

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Electron paramagnetic resonance (EPR) is often used in the study of the orientation and dynamics of proteins. However, there are two major obstacles in the interpretation of EPR signals: (a) most spin labels are not fully immobilized by the protein, hence it is difficult to distinguish the mobility of the label with respect to the protein from the reorientation of the protein itself; (b) even in cases where the label is fully immobilized its orientation with respect to the protein is not known, which prevents interpretation of probe reorientation in terms of protein reorientation. We have developed a computational strategy for determining whether or not a spin label is immobilized and, if immobilized, predicting its conformation within the protein. The method uses a Monte Carlo minimization algorithm to search the conformational space of labels within known atomic level structures of proteins. To validate the method a series of spin labels of varying size and geometry were docked to sites on the myosin head catalytic and regulatory domains. The predicted immobilization and conformation compared well with

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High-Resolution Detection of muscle Crossbridge Orientation by Electron Paramagnetic Resonance

Cited by 32 publications

References 6 publications

Orientational distribution of spin-labeled actin oriented by flow

Orientational distribution of spin-labeled actin oriented by flow

Orientation and rotational dynamics of spin‐labeled phalloidin bound to actin in muscle fibers

Structural Determination of Spin Label Immobilization and Orientation: A Monte Carlo Minimization Approach

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