Biophysical Journal 63: 966-975 (1992)
© 1992 the Biophysical Society

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ostap, E M Articles by Thomas, D D Search for Related Content PubMed PubMed Citation Articles by Ostap, E M Articles by Thomas, D D

Orientational distribution of spin-labeled actin oriented by flow.

Department 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.

This article has been cited by other articles:

				J. N. Forkey, M. E. Quinlan, and Y. E. Goldman Measurement of Single Macromolecule Orientation by Total Internal Reflection Fluorescence Polarization Microscopy Biophys. J., August 1, 2005; 89(2): 1261 - 1271. [Abstract] [Full Text] [PDF]