Muscle myosin performance measured with a synthetic nanomachine reveals a class‐specific Ca2+‐sensitivity of the frog myosin II isoform
Muscle myosin performance measured with a synthetic nanomachine reveals a class‐specific Ca2+‐sensitivity of the frog myosin II isoform
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使用合成纳米机器测量的肌肉肌球蛋白性能揭示了青蛙肌球蛋白 II 亚型的类特异性 Ca2+ 敏感性
DOI：
10.1113/jp280976
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Bianco
中科院分区：
文献类型：
--
作者：
Pertici;Bianchi;Bongini;Manstein;Lombardi;Bianco
关键点由七个重肌球蛋白（HMM）片段的肌肉肌球蛋白与肌动蛋白丝相互作用的集合体制成的纳米机器能够模拟半肌节产生稳定的力和恒定速度缩短。为了保留Ca 2+作为自由参数，的ca 2 +不敏感的凝溶胶蛋白片段TL 40用于将正确定向的肌动蛋白丝附着到作为力传感器的激光捕获珠上。新方法表明，由来自青蛙后肢肌肉的肌球蛋白提供动力的纳米机器的性能取决于[Ca 2 +]，这是由HMM的调节轻链中的Ca 2+结合位点介导的效应。由于由哺乳动物骨骼肌肌球蛋白驱动的纳米机器的性能不依赖于Ca 2+，因此Ca 2+敏感性具有类别特异性。模型模拟能够将纳米机器的性能与肌肉的性能相结合，并提供了具有不同肌球蛋白同源异构体的肌肉功能多样性的分子解释。从青蛙（Rana esculenta）后肢肌肉中纯化的肌球蛋白-II片段用于驱动合成纳米机器，该机器在没有其他肌节蛋白质的混淆效应的情况下拉动肌动蛋白丝。在目前版本的纳米机器中，肌动蛋白丝的+端通过Ca 2+不敏感的凝溶胶蛋白片段TL 40连接到激光捕获的珠，使[Ca 2 +]成为自由参数。青蛙肌球蛋白在2 mmATP中的性能受Ca 2+的影响：在0.1 mm Ca 2+中，等长稳定力（F0，15.25 pN）增加了50%（P= 0.004）相对于无Ca 2+溶液，（V0，4.6 μm s-1）降低了27%（P= 0.46）和最大功率（Pmax，7.6aW）提高了21%（P= 0.17）。由于在低力下缺乏数据，因此V0降低不显著，尽管其通过类似的降低得到巩固（33%，P < 0.0001）。溶液中ATP水解的速率（φ）表现出类似的钙依赖性。Ca ~（2+）对Vf和φ的滴定曲线给出Kd值为~ 30 μm。当使用来自兔腰肌肌球蛋白的HMM时，所有上述机械和动力学参数均与Ca 2+无关，表明Ca 2+敏感性是肌球蛋白的类特异性。一个独特的多尺度模型允许纳米机器性能与肌肉起源的接口，并确定了负责青蛙肌球蛋白Ca 2+敏感性的动力学步骤。
Key pointsA nanomachine made of an ensemble of seven heavy‐meromyosin (HMM) fragments of muscle myosin interacting with an actin filament is able to mimic the half‐sarcomere generating steady force and constant‐velocity shortening.To preserve Ca2+as a free parameter, the Ca2+‐insensitive gelsolin fragment TL40 is used to attach the correctly oriented actin filament to the laser‐trapped bead acting as a force transducer.The new method reveals that the performance of the nanomachine powered by myosin from frog hind‐limb muscles depends on [Ca2+], an effect mediated by a Ca2+‐binding site in the regulatory light chain of HMM. The Ca2+‐sensitivity is class‐specific because the performance of the nanomachine powered by mammalian skeletal muscle myosin is Ca2+independent.A model simulation is able to interface the nanomachine performance with that of the muscle of origin and provides a molecular explanation of the functional diversity of muscles with different orthologue isoforms of myosin.AbstractAn ensemble of seven heavy‐meromyosin (HMM) fragments of myosin‐II purified from the hindlimb muscles of the frog (Rana esculenta) is used to drive a synthetic nanomachine that pulls an actin filament in the absence of confounding effects of other sarcomeric proteins. In the present version of the nanomachine the +end of the actin filament is attached to the laser trapped bead via the Ca2+‐insensitive gelsolin fragment TL40, making [Ca2+] a free parameter. Frog myosin performance in 2 mmATP is affected by Ca2+: in 0.1 mm Ca2+, the isometric steady force (F0, 15.25 pN) is increased by 50% (P= 0.004) with respect to that in Ca2+‐free solution, the maximum shortening velocity (V0, 4.6 μm s–1) is reduced by 27% (P= 0.46) and the maximum power (Pmax, 7.6 aW) is increased by 21% (P= 0.17).V0reduction is not significant for the paucity of data at low force, although it is solidified by a similar decrease (33%,P< 0.0001) in the velocity of actin sliding as indicated by anin vitromotility assay (Vf). The rate of ATP‐hydrolysis in solution (φ) exhibits a similar calcium dependence. Ca2+titration curves forVfand φ giveKdvalues of ∼30 μm. All the above mechanical and kinetic parameters are independent of Ca2+when HMM from rabbit psoas myosin is used, indicating that the Ca2+‐sensitivity is a class‐specific property of muscle myosin. A unique multiscale model allows interfacing of the nanomachine performance to that of the muscle of origin and identifies the kinetic steps responsible for the Ca2+‐sensitivity of frog myosin.
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影响因子：
5.6
作者：
CRAIG, R;OFFER, G
通讯作者：
OFFER, G
通过兔腰肌纤维中笼内 ATP 的光解研究 MgADP 存在下的跨桥动力学。
DOI：
--
发表时间：
1991
期刊：
Journal of Physiology
影响因子：
--
作者：
J. Dantzig;M. Hibberd;D. Trentham;Y. Goldman
通讯作者：
Y. Goldman
影响因子：
5.8
作者：
Barclay, C. J.
通讯作者：
Barclay, C. J.
影响因子：
5.5
作者：
Elangovan, R.;Capitanio, M.;Piazzesi, G.
通讯作者：
Piazzesi, G.
影响因子：
3.3
作者：
Szczesna, D;Zhao, JJ;Potter, JD
通讯作者：
Potter, JD