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

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.

Conformationally Trapping the Actin-binding Cleft of Myosin with a Bifunctional Spin Label

“…6C, BSL), comparable to that induced in the absence of crosslinking by ATP (31, 54 -57) and in sharp contrast to the high degree of orientational order observed in the absence of ATP for uncrosslinked myosin bound to actin (Fig. 6C, MSL) (29,31,54,58). In contrast, the rotational motion of the crosslinked catalytic domain is very slow, undetectable on the submillisecond time scale (Fig.…”

Electron paramagnetic resonance reveals age-related myosin structural changes in rat skeletal muscle fibers

“…Most previous EPR studies of spin-labeled myosin have been conducted on purified proteins or filaments isolated from rabbit skeletal muscle (5,29) or on fibers from rabbit psoas muscle (2,6,8,12,24,31,37). The rabbit psoas muscle has been preferred for EPR fiber studies because it is a large fusiform muscle that is easily exposed in vivo from end to end.…”

Pulsed EPR Distance Measurements in Soluble Proteins by Site‐Directed Spin Labeling (SDSL)

“…These experiments used spin labels with maleimide (MSL) and iodoacetamide (IASL) reactivities that target thiol groups on myosin heads (Thomas and Cooke, 1980; Thomas et al, 1980; Fajer et al, 1988; Fajer, 1994; Roopnarine and Thomas, 1994). Another study on rabbit skeletal muscles utilized the spin label 3-(5-fluoro-2,4-dinitroanilino)proxyl (FDNA) that is specific to the ε-amino group on a lysine residue in G-actin (Waring and Cooke, 1987).…”