The regulatory light chain (RLC) of myosin is commonly tagged to monitor myosin behavior in vitro, in muscle fibers, and in cells. The goal of this study was to prepare smooth muscle myosin (SMM) filaments containing a single head labeled with a quantum dot (QD) on the RLC. We show that when the RLC is coupled to a QD at Cys‐108 and exchanged into SMM, subsequent filament assembly is severely disrupted. To address this, we used a novel approach for myosin by implementing the SpyTag002 SpyCatcher002 system to prepare SMM incorporated with RLC constructs fused to SpyTag or SpyCatcher. We show that filament assembly, actin‐activated steady‐state ATPase activities, ability to be phosphorylated, and selected enzymatic and mechanical properties were essentially unaffected if either SpyTag or SpyCatcher were fused to the C‐terminus of the RLC. Crucially for our application, we also show that a QD coupled to SpyCatcher can be covalently attached to a RLC‐Spy incorporated into a SMM filament without disrupting the filament, and that the filaments can move along actin in vitro.

中文翻译：

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使用SpyTag SpyCatcher系统在调节性轻链上的量子点标记平滑肌肌球蛋白II细丝。

肌球蛋白的调节性轻链（RLC）通常被标记为在体外，肌纤维和细胞中监测肌球蛋白的行为。这项研究的目的是制备包含在RLC上标有量子点（QD）的单个头部的平滑肌肌球蛋白（SMM）细丝。我们显示，当RLC在Cys-108处耦合到QD并交换为SMM时，随后的灯丝组件将受到严重破坏。为了解决这个问题，我们通过实施SpyTag002 SpyCatcher002系统为肌球蛋白使用了一种新颖的方法，以准备与融合到SpyTag或SpyCatcher的RLC结构结合的SMM。我们显示，如果将SpyTag或SpyCatcher融合到RLC的C末端，则丝状体的组装，肌动蛋白激活的稳态ATPase活性，被磷酸化的能力以及所选的酶和机械性能基本不受影响。