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Orientational distribution of spin-labeled actin oriented by flow
E M Ostap1, T Yanagida, D D Thomas
1Department of Biochemistry, University of Minnesota Medical School, Minneapolis 55455.
Abstract:
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.
Insights
Spin-labeled actin filaments exhibit distinct orientational distributions, with ordered and disordered populations observed. Protein binding does not alter this axial orientation, revealing insights into actin
Area of Science:
- Biophysics
- Structural Biology
- Biochemistry
Background:
- Previous studies using saturation transfer electron paramagnetic resonance (ST-EPR) on randomly oriented spin-labeled F-actin (MSL-actin) showed submillisecond rotational flexibility.
- These studies indicated that myosin binding affects actin flexibility but are limited in detecting slower orientational changes.
- Randomly oriented samples are insensitive to millisecond or slower orientational changes, necessitating new experimental approaches.
Purpose of the Study:
- To clarify previous findings by investigating the orientational distribution of spin-labeled actin using oriented samples.
- To detect changes in actin orientation upon binding of phalloidin and myosin subfragment 1 (S1).
- To compare the alignment of flow-oriented actin filaments with actin in muscle fibers.
Main Methods:
- Conventional electron paramagnetic resonance (EPR) experiments were performed on spin-labeled F-actin (MSL-actin) oriented by flow.
- Experimental EPR spectra were compared to simulated spectra to determine the orientational distribution of spin labels relative to the magnetic field.
- Spectra were analyzed to identify distinct populations of probes and quantify their orientational parameters (theta and phi).
Main Results:
- Flow-oriented MSL-actin exhibited two populations: a highly ordered (28%) and a disordered population.
- The ordered population showed a specific Gaussian distribution for the angle between the actin filament axis and the nitroxide z axis (theta) and x axis (phi).
- Binding of phalloidin or myosin subfragment 1 (S1) did not significantly alter the orientational distribution of MSL-actin, indicating no change in axial orientation.
- Spin-labeled S1 (MSL-S1) bound to flow-oriented actin showed similar orientational distributions to MSL-S1 in oriented muscle fibers.
Conclusions:
- Myosin and phalloidin binding does not affect the axial orientation of actin subunits.
- Flow-oriented actin filaments achieve a high degree of alignment comparable to that found in muscle fibers.
- This study provides a more detailed understanding of actin filament orientation and its stability upon protein binding.