Movement of smooth muscle tropomyosin by myosin heads.
It has been proposed that during the activation of muscle contraction the initial binding of myosin heads to the actin thin filament contributes to switching on the thin filament and that this might involve the movement of actin-bound tropomyosin. The movement of smooth muscle tropomyosin on actin was investigated in this work by measuring the change in distance between specific residues on tropomyosin and actin by fluorescence resonance energy transfer (FRET) as a function of myosin head binding to actin. An energy transfer acceptor was attached to Cys374 of actin and a donor to the tropomyosin heterodimer at either Cys36 of the beta-chain or Cys190 of the alpha-chain. FRET changed for the donor at both positions of tropomyosin upon addition of skeletal or smooth muscle myosin heads, indicating a movement of the whole tropomyosin molecule. The changes in FRET were hyperbolic and saturated at about one head per seven actin subunits, indicating that each head cooperatively affects several tropomyosin molecules, presumably via tropomyosin's end-to-end interaction. ATP, which dissociates myosin from actin, completely reversed the changes in FRET induced by heads, whereas in the presence of ADP the effect of heads was the same as in its absence. The results indicate that myosin with and without ADP, intermediates in the myosin ATPase hydrolytic pathway, are effective regulators of tropomyosin position, which might play a role in the regulation of smooth muscle contraction.
Graceffa
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Boston Biomedical Research Institute, Massachusetts 02114, USA. graceffa@bbri.org
D000199:Actins / Q000378:metabolism; D000818:Animals; D002645:Chickens; D019281:Dimerization; D004735:Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000378:metabolism*; D009119:Muscle Contraction; D018482:Muscle, Skeletal / Q000737:chemistry / Q000378:metabolism; D009130:Muscle, Smooth / Q000737:chemistry / Q000378:metabolism*; D009218:Myosins / Q000737:chemistry / Q000378:metabolism*; D009282:Naphthalenesulfonates / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014335:Tropomyosin / Q000737:chemistry / Q000378:metabolism*
D000199:Actins; D005456:Fluorescent Dyes; D020409:Molecular Motor Proteins; D009282:Naphthalenesulfonates; D014335:Tropomyosin; C006421:1,5-I-AEDANS; D009218:Myosins