Abstract：
Tropomyosin (Tpm) is an actin-binding protein central to muscle contraction regulation. The Tpm sequence consists of periodic repeats corresponding to seven actin-binding sites, further divided in two functionally distinct halves. To clarify the importance of the first and second halves of the actin-binding periods in regulating the interaction of myosin with actin, we introduced hypercontractile mutations D20H, E181K located in the N-terminal halves of periods 1 and 5 and hypocontractile mutations E41K, N202K located in the C-terminal halves of periods 1 and 5 of the skeletal muscle Tpm isoform Tpm2.2. Wild-type and mutant Tpms displayed similar actin-binding properties, however, as revealed by FRET experiments, the hypercontractile mutations affected the binding geometry and orientation of Tpm2.2 on actin, causing a stimulation of myosin motor performance. Contrary, the hypocontractile mutations led to an inhibition of both, actin activation of the myosin ATPase and motor activity, that was more pronounced than with wild-type Tpm2.2. Single ATP turnover kinetic experiments indicate that the introduced mutations have opposite effects on product release kinetics. While the hypercontractile Tpm2.2 mutants accelerated product release, the hypocontractile mutants decelerated product release from myosin, thus having either an activating or inhibitory influence on myosin motor performance, which agrees with the muscle disease phenotypes caused by these mutations.
摘要：
原肌球蛋白（Tpm）是一种肌动蛋白结合蛋白，对肌肉收缩调节至关重要。Tpm序列由对应于七个肌动蛋白结合位点的周期性重复组成，进一步分为两个功能不同的一半。为了阐明肌动蛋白结合期的第一和第二半部在调节肌球蛋白与肌动蛋白的相互作用中的重要性，我们引入了过度收缩突变D20H，E181K位于第1期和第5期的N末端一半和收缩不足突变E41K，N202K位于骨骼肌Tpm同工型Tpm2.2的第1期和第5期的C末端一半。野生型和突变型Tpms表现出相似的肌动蛋白结合特性，然而,正如FRET实验所揭示的，过度收缩突变影响Tpm2.2在肌动蛋白上的结合几何和方向，刺激肌球蛋白运动性能。相反，收缩不足的突变导致两者的抑制，肌动蛋白激活肌球蛋白ATP酶和运动活动，比野生型Tpm2.2更明显。单ATP周转动力学实验表明，引入的突变对产物释放动力学具有相反的影响。而过度收缩的Tpm2.2突变体加速了产品的释放，收缩不足的突变体减缓了肌球蛋白的产品释放，因此对肌球蛋白运动性能有激活或抑制影响，这与这些突变引起的肌肉疾病表型一致。
