title: Orientation of the N- and C-terminal lobes of the myosin regulatory light chain in cardiac muscle.
abstract: The orientations of the N- and C-terminal lobes of the cardiac isoform of the myosin regulatory light chain (cRLC) in the fully dephosphorylated state in ventricular trabeculae from rat heart were determined using polarized fluorescence from bifunctional sulforhodamine probes. cRLC mutants with one of eight pairs of surface-accessible cysteines were expressed, labeled with bifunctional sulforhodamine, and exchanged into demembranated trabeculae to replace some of the native cRLC. Polarized fluorescence data from the probes in each lobe were combined with RLC crystal structures to calculate the lobe orientation distribution with respect to the filament axis. The orientation distribution of the N-lobe had three distinct peaks (N1-N3) at similar angles in relaxation, isometric contraction, and rigor. The orientation distribution of the C-lobe had four peaks (C1-C4) in relaxation and isometric contraction, but only two of these (C2 and C4) remained in rigor. The N3 and C4 orientations are close to those of the corresponding RLC lobes in myosin head fragments bound to isolated actin filaments in the absence of ATP (in rigor), but also close to those of the pair of heads folded back against the filament surface in isolated thick filaments in the so-called J-motif conformation. The N1 and C1 orientations are close to those expected for actin-bound myosin heads with their light chain domains in a pre-powerstroke conformation. The N2 and C3 orientations have not been observed previously. The results show that the average change in orientation of the RLC region of the myosin heads on activation of cardiac muscle is small; the RLC regions of most heads remain in the same conformation as in relaxation. This suggests that the orientation of the dephosphorylated RLC region of myosin heads in cardiac muscle is primarily determined by an interaction with the thick filament surface.
authors.affiliation: Randall Division of Cell and Molecular Biophysics and British Heart Foundation Centre of Research Excellence, King's College London, London, United Kingdom.
authors.forename: Thomas
authors.initials: T
authors.lastname: Kampourakis
authors.affiliation: Randall Division of Cell and Molecular Biophysics and British Heart Foundation Centre of Research Excellence, King's College London, London, United Kingdom.
authors.forename: Yin-Biao
authors.initials: YB
authors.lastname: Sun
authors.affiliation: Randall Division of Cell and Molecular Biophysics and British Heart Foundation Centre of Research Excellence, King's College London, London, United Kingdom. Electronic address: malcolm.irving@kcl.ac.uk.
authors.forename: Malcolm
authors.initials: M
authors.lastname: Irving
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D008297:Male; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D032383:Myocytes, Cardiac / Q000378:metabolism*; D018994:Myosin Light Chains / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D017208:Rats, Wistar
chemical_list: D018994:Myosin Light Chains