title: Amplitude of the actomyosin power stroke depends strongly on the isoform of the myosin essential light chain.
abstract: We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to determine the role of myosin essential light chains (ELCs) in structural transitions within the actomyosin complex. Skeletal muscle myosins have two ELC isoforms, A1 and A2, which differ by an additional 40-45 residues at the N terminus of A1, and subfragment 1 (S1) containing A1 (S1A1) has higher catalytic efficiency and higher affinity for actin than S1A2. ELC's location at the junction between the catalytic and light-chain domains gives it the potential to play a central role in the force-generating power stroke. Therefore, we measured site-directed TR-FRET between a donor on actin and an acceptor near the C terminus of ELC, detecting directly the rotation of the light-chain domain (lever arm) relative to actin (power stroke), induced by the interaction of ATP-bound myosin with actin. TR-FRET resolved the weakly bound (W) and strongly bound (S) states of actomyosin during the W-to-S transition (power stroke). We found that the W states are essentially the same for the two isoenzymes, but the S states are quite different, indicating a much larger movement of S1A1. FRET from actin to a probe on the N-terminal extension of A1 showed close proximity to actin. We conclude that the N-terminal extension of A1-ELC modulates the W-to-S structural transition of acto-S1, so that the light-chain domain undergoes a much larger power stroke in S1A1 than in S1A2. These results have profound implications for understanding the contractile function of actomyosin, as needed in therapeutic design for muscle disorders.
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455.
authors.forename: Piyali
authors.initials: P
authors.lastname: Guhathakurta
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455.
authors.forename: Ewa
authors.initials: E
authors.lastname: Prochniewicz
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455 ddt@umn.edu.
authors.forename: David D
authors.initials: DD
authors.lastname: Thomas
mesh_terms: D000199:Actins / Q000737:chemistry / Q000378:metabolism; D000205:Actomyosin / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000465:Algorithms; D000818:Animals; D024722:Cardiac Myosins / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D007700:Kinetics; D008958:Models, Molecular; D009119:Muscle Contraction; D018482:Muscle, Skeletal / Q000378:metabolism*; D018994:Myosin Light Chains / Q000737:chemistry / Q000378:metabolism*; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D012399:Rotation; D013997:Time Factors
chemical_list: D000199:Actins; D018994:Myosin Light Chains; D015879:Myosin Subfragments; C107184:myosin light chain 2; C076659:myosin light chain I; D000255:Adenosine Triphosphate; D000205:Actomyosin; D024722:Cardiac Myosins
keywords: actomyosin; fluorescence resonance energy transfer; light chains; muscle
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