A point mutation in the SH1 helix alters elasticity and thermal stability of myosin II
A point mutation in the SH1 helix alters elasticity and thermal stability of myosin II
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DOI：
10.1074/jbc.m605365200
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发表时间：
2006-10-13
影响因子：
4.8
通讯作者：
Chaen, Shigeru
中科院分区：
文献类型：
--
作者：
Iwai, Sosuke;Hanamoto, Daisuke;Chaen, Shigeru
肌凝蛋白运动产生的运动通常被认为是由肌凝蛋白分子内弹性元件的扭曲和随后产生的应变释放驱动的。然而，这种弹性元件在肌凝蛋白中的位置仍不清楚。肌凝蛋白运动结构域由四个主要的子结构域组成，这些子结构域由柔性关节连接。SH1螺旋是连接转换器子域和其他域的关节，并且被认为在转换器相对于电机的布置中起重要作用。为了研究SH1螺旋在肌球蛋白弹性扭曲中的作用，我们在Dictyostelium myosin II （R689H）的SH1螺旋中引入了一个点突变，该突变在人类非肌性肌球蛋白IIA中导致非综合征性遗传性耳聋，DFNA17。突变导致运动能力明显受损，而肌动蛋白激活的atp酶活性仅受到轻微影响。利用光阱进行的单分子力学测量表明，突变并没有缩短步长，这表明运动速度变慢是由动力学改变引起的。单分子测量表明，突变显著降低了桥间刚度。野生型和突变型肌凝蛋白混合产生的运动活性也表明突变影响了肌凝蛋白的弹性。这些结果表明SH1螺旋参与肌凝蛋白弹性的调节，可能是通过调节转化器的灵活性。与此一致的是，该突变还显示降低热稳定性并诱导蛋白质的热聚集，这可能与疾病过程有关。
Movement generated by the myosin motor is generally thought to be driven by distortion of an elastic element within the myosin molecule and subsequent release of the resulting strain. However, the location of this elastic element in myosin remains unclear. The myosin motor domain consists of four major subdomains connected by flexible joints. The SH1 helix is the joint that connects the converter subdomain to the other domains, and is thought to play an important role in arrangements of the converter relative to the motor. To investigate the involvement of the SH1 helix in elastic distortion in myosin, we have introduced a point mutation into the SH1 helix of Dictyostelium myosin II (R689H), which in human nonmuscle myosin IIA causes nonsyndromic hereditary deafness, DFNA17. The mutation resulted in a significant impairment in motile activities, whereas actin-activated ATPase activity was only slightly affected. Single molecule mechanical measurements using optical trap showed that the step size was not shortened by the mutation, suggesting that the slower motility is caused by altered kinetics. The single molecule measurements demonstrated that the mutation significantly reduced cross-bridge stiffness. Motile activities produced by mixtures of wild-type and mutant myosins also suggested that the mutation affected the elasticity of myosin. These results suggest that the SH1 helix is involved in modulation of myosin elasticity, presumably by modulating the converter flexibility. Consistent with this, the mutation was also shown to reduce thermal stability and induce thermal aggregation of the protein, which might be implicated in the disease process.