Motility Assay to Probe the Calcium Sensitivity of Myosin and Regulated Thin Filaments.
Motility Assay to Probe the Calcium Sensitivity of Myosin and Regulated Thin Filaments.
复制标题
用于探测肌球蛋白和调节细丝的钙敏感性的运动测定。
DOI：
10.1007/978-1-0716-3527-8_10
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发表时间：
2024
期刊：
影响因子：
--
通讯作者：
Spudich,JamesA
中科院分区：
文献类型：
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作者：
Liu,Chao;Ruppel,KathleenM;Spudich,JamesA
肌钙蛋白-原肌球蛋白复合物介导的细丝的钙依赖性激活是基于肌动蛋白-肌球蛋白的肌肉收缩调节的关键。对该系统的扰动，无论是生理的（例如，肌球蛋白轻链的磷酸化）或病理性（例如，导致家族性心肌病的突变），可以改变钙敏感性，因此对人类健康和疾病具有重要意义。体外运动试验为研究重组肌球蛋白-细丝运动系统的钙敏感性提供了一种定量和精确的方法。在这里，我们提出了一个简单而强大的协议，执行钙依赖性运动的β-心肌肌球蛋白和调节细丝。该实验在多通道微流体载玻片上进行，需要最少量的蛋白质。一个完整的速度与钙浓度曲线是从一个实验在1小时内产生的。
Calcium-dependent activation of the thin filament mediated by the troponin-tropomyosin complex is key in the regulation of actin-myosin based muscle contraction. Perturbations to this system, either physiological (e.g., phosphorylation of myosin light chains) or pathological (e.g., mutations that cause familial cardiomyopathies), can alter calcium sensitivity and thus have important implications in human health and disease. The in vitro motility assay provides a quantitative and precise method to study the calcium sensitivity of the reconstituted myosin-thin filament motile system. Here we present a simple and robust protocol to perform calcium-dependent motility ofβ-cardiac myosin and regulated thin filaments. The experiment is done on a multichannel microfluidic slide requiring minimal amounts of proteins. A complete velocity vs. calcium concentration curve is produced from one experiment in under 1 h.