Movement of Smooth Muscle Tropomyosin by Myosin Heads

Abstract

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.

Phosphorylation of Smooth Muscle Myosin Heads Regulates the Head-induced Movement of Tropomyosin

“…Previously, I have prepared G-actin for labeling with the acceptor DAB by homogenizing an actin pellet, prepared from acetone powder according to Spudich and Watt (28), with G buffer plus a 1 mM concentration of the reducing agent DTT and then dialyzing extensively versus G-buffer to remove the DTT. Labeling of this preparation always resulted in a DAB/actin ratio of about 0.7, significantly less than the maximum of 1 (23). In this work, the actin pellet was homogenized with G buffer plus 10 mM DTT and incubated 30 -60 min before dialysis, which ultimately resulted in a labeling ratio very close to the maximum of 1.…”

“…Unless stated otherwise, the procedures used in this study are those described in the previous work (23). In those cases where the Lowry assay was used for protein determination in the previous work, I have substituted the BCA assay (27) in this study.…”

“…FRET was determined by the change in steady-state fluorescence of the AED donor attached to Cys 36 of smooth muscle tropomyosin (AED36smTm) in the presence of the DAB acceptor attached to actin Cys 374 (DAB-A) as a function of smHMM, as described (23). Measurements were made at 20°C in 40 mM NaCl, 5 mM MgCl 2 , 5 mM DTT, pH 7.5, with AED36smTm at 0.6 M and DAB-A at 4.8 M. Fluorescence changes were first corrected for changes that were not due to energy transfer.…”

“…Any change in the absorption spectrum would change the overlap integral and thus change FRET (see Ref. 23 and references therein). We found that both smHMM-SP and skS1 had no effect on the absorption spectrum of DAB-actin/Tm, thus ruling out such a change as responsible for the changes in FRET that we observe.…”

“…Since it is fairly well established that the movement of skeletal muscle tropomyosin plays a key role in the thin filament regulation of skeletal muscle contraction, the question arises as to whether such a movement of smooth muscle tropomyosin also exists. In our previous work, using fluorescence resonance energy transfer (FRET), we found that, indeed, smooth muscle tropomyosin moved upon the binding to actin of single-headed (skS1) and double-headed (skHMM) fragments of skeletal muscle myosin and the single-headed (smS1) fragment of smooth muscle myosin (23). Since smS1 is constitutively active (reviewed in Ref.…”