Familial hypertrophic cardiomyopathy (HCM) affects .2% of the world's population and is inherited in an autosomal dominant manner. Mutations in cardiac α‐actin are the cause in 1%–5% of all observed cases. Here, we describe the recombinant production, purification, and characterization of the HCM‐linked cardiac α‐actin variants p.A21V and p.D26N. Mass spectrometric analysis of the initially purified recombinant cardiac α‐actin variants and wild‐type protein revealed improper N‐terminal processing in the Spodoptera frugiperda (Sf‐9) insect cell system, compromising the labeling of the protein with fluorescent probes for biochemical studies. Therefore, we produced N‐terminal deletion mutants lacking the N‐terminal cysteine (ΔC2). The ΔC2 wild‐type construct behaved similar to porcine cardiac α‐actin purified from native Sus scrofa heart tissue and all ΔC2 constructs showed improved fluorescent labeling. Further analysis of untruncated and ΔC2 constructs showed that while neither the A21V nor the D26N mutation affects nucleotide binding, they cause a similar slowing of the rate of filament formation as well as a reduction in the thermal stability of monomeric and filamentous cardiac α‐actin. In vitro motility assays and transient‐kinetic studies probing the interaction of the actin variants with cardiac β‐myosin revealed perturbed actomyosin interactions and a reduced motile activity for the p.D26N variant. Addition of the small molecule effector EMD 57033, which targets cardiac β‐myosin, rescued the approximately 40% drop in velocity observed with the p.D26N constructs and activated the motile activity of wild‐type and p.D26N to the same level of 1100 nm s−1.

中文翻译：

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与肥厚型心肌病相关的心脏 α-肌动蛋白突变 A21V 和 D26N 的生化特征

家族性肥厚型心肌病 (HCM) 影响世界上 0.2% 的人口，并以常染色体显性遗传方式遗传。心脏 α-肌动蛋白突变是所有观察到的病例中 1%–5% 的原因。在这里，我们描述了 HCM 连接的心脏 α-肌动蛋白变体 p.A21V 和 p.D26N 的重组生产、纯化和表征。对最初纯化的重组心脏 α-肌动蛋白变体和野生型蛋白的质谱分析揭示了 N 末端加工不当草地贪夜蛾(Sf-9) 昆虫细胞系统，损害了用于生化研究的荧光探针对蛋白质的标记。因此，我们产生了缺乏 N 端半胱氨酸 (ΔC2) 的 N 端缺失突变体。ΔC2 野生型构建体的行为类似于从天然纯化的猪心脏 α-肌动蛋白野猪心脏组织和所有 ΔC2 构建体均显示出改进的荧光标记。对未截短和 ΔC2 构建体的进一步分析表明，虽然 A21V 和 D26N 突变都不影响核苷酸结合，但它们会导致类似的丝形成速率减慢以及单体和丝状心脏 α-肌动蛋白的热稳定性降低。体外运动测定和瞬态动力学研究探讨了肌动蛋白变体与心脏 β-肌球蛋白的相互作用，揭示了 p.D26N 变体的肌动球蛋白相互作用受到干扰，运动活性降低。添加针对心脏 β-肌球蛋白的小分子效应器 EMD 57033，挽救了 p.D26N 构建体观察到的约 40% 的速度下降，并将野生型和 p.D26N 的运动活性激活至相同水平 1100纳米级−1。