Fluorescently labeled proteins can improve the detection sensitivity and have been widely used in a variety of biological measurements. In single-molecule assays, site-specific labeling of proteins enables the visualization of molecular interactions, conformational changes in proteins, and enzymatic activity. In this study, based on a flexible linker in the Escherichia coli RecQ helicase, we established a scheme involving a combination of fluorophore labeling and sortase A ligation to allow site-specific labeling of the HRDC domain of RecQ with a single Cy5 fluorophore, without inletting extra fluorescent domain or peptide fragment. Using single-molecule fluorescence resonance energy transfer, we visualized that Cy5-labeled HRDC could directly interact with RecA domains and could bind to both the 3′ and 5′ ends of the overhang DNA dynamically in vitro for the first time. The present work not only reveals the functional mechanism of the HRDC domain, but also provides a feasible method for site-specific labeling of a domain with a single fluorophore used in single-molecule assays.

荧光标记的蛋白质可以提高检测灵敏度，并已广泛用于各种生物学测量中。在单分子测定中，蛋白质的位点特异性标记使分子相互作用，蛋白质构象变化和酶活性可视化。在本研究中，基于大肠杆菌RecQ解旋酶，我们建立了一个计划，涉及荧光团标记和sortase A连接的结合，以允许用单个Cy5荧光团对RecQ的HRDC域进行位点特异性标记，而无需引入额外的荧光域或肽片段。使用单分子荧光共振能量转移，我们观察到Cy5标记的HRDC可以直接与RecA结构域相互作用，并且可以动态地与突出DNA的3'和5'末端结合体外首次。目前的工作不仅揭示了HRDC域的功能机制，而且还提供了一种可行的方法，用于在单分子测定中使用单个荧光团对域进行位点特异性标记。