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Regulatory light chain mutants linked to heart disease modify the cardiac myosin lever arm.

ABSTRACT: Myosin is the chemomechanical energy transducer in striated heart muscle. The myosin cross-bridge applies impulsive force to actin while consuming ATP chemical energy to propel myosin thick filaments relative to actin thin filaments in the fiber. Transduction begins with ATP hydrolysis in the cross-bridge driving rotary movement of a lever arm converting torque into linear displacement. Myosin regulatory light chain (RLC) binds to the lever arm and modifies its ability to translate actin. Gene sequencing implicated several RLC mutations in heart disease, and three of them are investigated here using photoactivatable GFP-tagged RLC (RLC-PAGFP) exchanged into permeabilized papillary muscle fibers. A single-lever arm probe orientation is detected in the crowded environment of the muscle fiber by using RLC-PAGFP with dipole orientation deduced from the three-spatial dimension fluorescence emission pattern of the single molecule. Symmetry and selection rules locate dipoles in their half-sarcomere, identify those at the minimal free energy, and specify active dipole contraction intermediates. Experiments were performed in a microfluidic chamber designed for isometric contraction, total internal reflection fluorescence detection, and two-photon excitation second harmonic generation to evaluate sarcomere length. The RLC-PAGFP reports apparently discretized lever arm orientation intermediates in active isometric fibers that on average produce the stall force. Disease-linked mutants introduced into RLC move intermediate occupancy further down the free energy gradient, implying lever arms rotate more to reach stall force because mutant RLC increases lever arm shear strain. A lower free energy intermediate occupancy involves a lower energy conversion efficiency in the fiber relating a specific myosin function modification to the disease-implicated mutant.

SUBMITTER: Burghardt TP

PROVIDER: S-EPMC3587134 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Regulatory light chain mutants linked to heart disease modify the cardiac myosin lever arm.

Burghardt Thomas P TP Sikkink Laura A LA

Biochemistry 20130206 7

Myosin is the chemomechanical energy transducer in striated heart muscle. The myosin cross-bridge applies impulsive force to actin while consuming ATP chemical energy to propel myosin thick filaments relative to actin thin filaments in the fiber. Transduction begins with ATP hydrolysis in the cross-bridge driving rotary movement of a lever arm converting torque into linear displacement. Myosin regulatory light chain (RLC) binds to the lever arm and modifies its ability to translate actin. Gene s ...[more]

PMID: 23343568

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Project description:Myosin is the molecular motor in muscle-binding actin and executing a power stroke by rotating its lever arm through an angle of approximately 70 degrees to translate actin against resistive force. A green fluorescent protein (GFP)-tagged human cardiac myosin regulatory light chain (HCRLC) was constructed to study in situ lever arm orientation one molecule at a time by polarized fluorescence emitted from the GFP probe. The recombinant protein physically and functionally replaced the native RLC on myosin lever arms in the thick filaments of permeabilized skeletal muscle fibers. Detecting single molecules in fibers where myosin concentration reaches 300 microM is accomplished using total internal reflection fluorescence microscopy. With total internal reflection fluorescence, evanescent field excitation, supercritical angle fluorescence detection, and CCD detector pixel size limits detection volume to just a few attoliters. Data analysis manages both the perturbing effect of the TIR interface on probe emission and the effect of high numerical aperture collection of light. The natural myosin concentration gradient in a muscle fiber allows observation of fluorescence polarization from C-term GFP-tagged HCRLC exchanged myosin from regions in the thick filament containing low and high myosin concentrations. In rigor, cross-bridges at low concentration at the end of the thick filament maintain GFP dipole moments at two distinct polar angles relative to the fiber symmetry axis. The lower angle, where the dipole is nearly parallel to fiber axis, is more highly populated than the alternative, larger angle. Cross-bridges at higher concentration in the center of the thick filament are oriented in a homogeneous band at approximately 45 degrees to the fiber axis. The data suggests molecular crowding impacts myosin conformation, implying mutual interactions between cross-bridges alter how the muscle generates force. The GFP-tagged RLC is a novel probe to assess single-lever-arm orientation characteristics in situ.

Dataset Information

Regulatory light chain mutants linked to heart disease modify the cardiac myosin lever arm.

Publications

Regulatory light chain mutants linked to heart disease modify the cardiac myosin lever arm.

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