Paramagnetic probes attached to a light chain on the myosin head are highly disordered in active muscle fibers.
We have measured the orientation of a region of the myosin head, close to the junction with the rod, during active force generation. Paramagnetic probes were attached specifically to a reactive cysteine (Cys 125) of purified myosin light chain 2 (LC2) and exchanged into myosin heads in glycerinated rabbit psoas muscle. Electron paramagnetic resonance spectroscopy was used to monitor the orientation of the probes. Previous work has shown that the LC2 bound spin probes are significantly ordered in rigor and muscle in the presence of adenosine diphosphate (ADP). In contrast, there is a nearly random angular distribution in relaxed muscle. We show here that during the generation of isometric tension, all of the LC2 bound spin probes (98 +/- 1.6%) show an angular distribution similar to that of relaxed muscle. These findings contrast with results obtained from probes attached to Cys 707 on the cross-bridge, located close to the actin binding site, where, during active force generation, a proportion of the spin probes were ordered as in rigor, whereas the remaining probes were disordered as in relaxation. To test the hypothesis that this ordered component is due to modification of Cys 707, we measured the spectra obtained from probes attached to LC2 in fibers modified at Cys 707. The modification of Cys 707 did not produce an ordered component in these spectra. The absence of an ordered component at the LC2 site limits the populations of some states in active fibers. An actin/myosin/ADP state is thought to be the major force-producing state. Our present results show that the populations of states with ordered probes on LC2 are < 2% in active fibers; thus, the major force-producing state is different from the one obtained by addition of ADP to rigor fibers.
Hambly
B
B
Department of Pathology, University of Sydney, New South Wales, Australia.
Franks
K
K
Cooke
R
R
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D003545:Cysteine / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D066298:In Vitro Techniques; D015394:Molecular Structure; D009119:Muscle Contraction / Q000502:physiology; D009126:Muscle Relaxation / Q000502:physiology; D009132:Muscles / Q000737:chemistry* / Q000502:physiology; D009218:Myosins / Q000737:chemistry* / Q000502:physiology; D011817:Rabbits; D013113:Spin Labels
D013113:Spin Labels; D000255:Adenosine Triphosphate; D009218:Myosins; D003545:Cysteine