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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.
Biophysical Journal
|October 1, 1992
Summary
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.