Biophysical Journal 67: 1634-1645 (1994)
© 1994 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 Roopnarine, O Articles by Thomas, D D Search for Related Content PubMed PubMed Citation Articles by Roopnarine, O Articles by Thomas, D D

A spin label that binds to myosin heads in muscle fibers with its principal axis parallel to the fiber axis.

Department of Biochemistry, University of Minnesota Medical School, Minneapolis 55455.

ABSTRACT

We have used an indane-dione spin label (2-[-oxyl-2,2,5,5-tetramethyl-3-pyrrolin-3-yl)methenyl]in dane-1,3-dione), designated InVSL, to study the orientation of myosin heads in bundles of chemically skinned rabbit psoas muscle fibers, with electron paramagnetic resonance (EPR) spectroscopy. After reversible preblocking with 5,5'-dithiobis(2-nitro-benzoic acid) (DTNB), we were able to attach most of the spin label covalently and rigidly to either Cys 707 (SH1) or Cys 697 (SH2) on myosin heads. EPR spectra of labeled fibers contained substantial contributions from both oriented and disordered populations of spin labels. Similar spectra were obtained from fibers decorated with InVSL-labeled myosin heads (subfragment 1), indicating that virtually all the spin labels in labeled fibers are on the myosin head. We specifically labeled SH2 with InVSL after reversible preblocking of the SH1 sites with 1-fluoro-2,4-dinitrobenzene (FDNB), resulting in a spectrum that indicated only disordered spin labels. Therefore, the oriented and disordered populations correspond to labels on SH1 and SH2, respectively. The spectrum of SH2-bound labels was subtracted to produce a spectrum corresponding to SH1-bound labels, which was used for further analysis. For this corrected spectrum, the angle between the fiber axis and the principal axis of the spin label was fitted well by a Gaussian distribution centered at theta o = 11 +/- 1 degree, with a full width at half-maximum of delta theta = 15 +/- 2 degrees. The unique orientation of InVSL, with its principal axis almost parallel to the fiber axis, makes it complementary to spin labels previously studied in this system. This label can provide unambiguous information about axial rotations of myosin heads, since any axial rotation of the head must be reflected in the same axial rotation of the principal axis of the probe, thus changing the hyperfine splitting. Therefore, InVSL-labeled fibers have ideal properties needed for further exploration myosin head orientation and rotational motion in muscle.

This article has been cited by other articles:

				O. Roopnarine Mechanical Defects of Muscle Fibers with Myosin Light Chain Mutants that Cause Cardiomyopathy Biophys. J., April 1, 2003; 84(4): 2440 - 2449. [Abstract] [Full Text] [PDF]

				D. A. Lowe, J. T. Surek, D. D. Thomas, and L. V. Thompson Electron paramagnetic resonance reveals age-related myosin structural changes in rat skeletal muscle fibers Am J Physiol Cell Physiol, March 1, 2001; 280(3): C540 - C547. [Abstract] [Full Text] [PDF]