title: Spectroscopic Studies of the Super Relaxed State of Skeletal Muscle.
abstract: In the super-relaxed state of myosin, ATPase activity is strongly inhibited by binding of the myosin heads to the core of the thick filament in a structure known as the interacting-heads motif. In the disordered relaxed state myosin heads are not bound to the core of the thick filament and have an ATPase rate that is 10 fold greater. In the interacting-heads motif the two regulatory light chains appear to bind to each other. We have made single cysteine mutants of the regulatory light chain, placed both paramagnetic and fluorescent probes on them, and exchanged them into skinned skeletal muscle fibers. Many of the labeled light chains tended to disrupt the stability of the super-relaxed state, and showed spectral changes in the transition from the disordered relaxed state to the super-relaxed state. These data support the putative interface between the two regulatory light chains identified by cryo electron microscopy and show that both the divalent cation bound to the regulatory light chain and the N-terminus of the regulatory light chain play a role in the stability of the super-relaxed state. One probe showed a shift to shorter wavelengths in the super-relaxed state such that a ratio of intensities at 440nm to that at 520nm provided a measure of the population of the super-relaxed state amenable for high throughput screens for finding potential pharmaceuticals. The results provide a proof of concept that small molecules that bind to this region can destabilize the super-relaxed state and provide a method to search for small molecules that do so leading to a potentially effective treatment for Type 2 diabetes and obesity.
authors.affiliation: Dipartimento di Scienze Biomediche, University of Padua, Padua Italy.
authors.forename: Leonardo
authors.identifier: http://orcid.org/0000-0002-7009-8707
authors.initials: L
authors.lastname: Nogara
authors.affiliation: Department of Biochemistry/Biophysics, University of California San Francisco, San Francisco, California, United States of America.
authors.forename: Nariman
authors.initials: N
authors.lastname: Naber
authors.affiliation: Voiland School of Bioengineering, Washington State University, Pullman, Washington, United States of America.
authors.forename: Edward
authors.initials: E
authors.lastname: Pate
authors.affiliation: Dipartimento di Scienze Biomediche, University of Padua, Padua Italy.
authors.forename: Marcella
authors.initials: M
authors.lastname: Canton
authors.affiliation: Dipartimento di Scienze Biomediche, University of Padua, Padua Italy.
authors.forename: Carlo
authors.initials: C
authors.lastname: Reggiani
authors.affiliation: Department of Biochemistry/Biophysics, University of California San Francisco, San Francisco, California, United States of America.
authors.forename: Roger
authors.initials: R
authors.lastname: Cooke
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D020285:Cryoelectron Microscopy; D004578:Electron Spin Resonance Spectroscopy; D005456:Fluorescent Dyes / Q000737:chemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D018485:Muscle Fibers, Skeletal / Q000737:chemistry* / Q000378:metabolism*; D009126:Muscle Relaxation / Q000502:physiology*; D018994:Myosin Light Chains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020836:Protein Structure, Quaternary; D011817:Rabbits; D013050:Spectrometry, Fluorescence
chemical_list: D005456:Fluorescent Dyes; D018994:Myosin Light Chains; D000255:Adenosine Triphosphate