Graceffa P
Boston Biomedical Research Institute, Massachusetts 02114, USA.
Biochemistry. 1999 Sep 14;38(37):11984-92. doi: 10.1021/bi9825495.
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.
有人提出，在肌肉收缩激活过程中，肌球蛋白头部与肌动蛋白细肌丝的初始结合有助于开启细肌丝，这可能涉及与肌动蛋白结合的原肌球蛋白的移动。在这项研究中，通过测量原肌球蛋白和肌动蛋白上特定残基之间的距离变化（采用荧光共振能量转移（FRET）技术），以此作为肌球蛋白头部与肌动蛋白结合的函数，来研究平滑肌原肌球蛋白在肌动蛋白上的移动情况。将能量转移受体连接到肌动蛋白的Cys374上，并将供体连接到原肌球蛋白异二聚体的β链Cys36或α链Cys190上。添加骨骼肌或平滑肌肌球蛋白头部后，原肌球蛋白两个位置上的供体的FRET均发生变化，表明整个原肌球蛋白分子发生了移动。FRET的变化呈双曲线形，在每七个肌动蛋白亚基约结合一个头部时达到饱和，这表明每个头部协同影响几个原肌球蛋白分子，推测是通过原肌球蛋白的端对端相互作用实现的。能使肌球蛋白与肌动蛋白解离的ATP完全逆转了由头部诱导的FRET变化，而在存在ADP的情况下，头部的作用与不存在时相同。结果表明，具有和不具有ADP的肌球蛋白（肌球蛋白ATP酶水解途径中的中间体）是原肌球蛋白位置的有效调节因子，这可能在平滑肌收缩调节中发挥作用。
