Abstract
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.
Highlights
Labeled proteins can help dissect the detailed molecular mechanisms and have become crucial experimental tools in various research fields, especially in flow cytometry, fluorescence microscopy, and enzymatic activity measurements (Li et al, 2015)
Maleimide-activated fluorophores are monoreactive dyes that can selectively couple with cysteines in peptides or proteins to generate labeled conjugates and have been widely used in enzymology experiments
As full-length RecQ contains 11 cysteine residues, it is difficult to label the helicase and RNaseD C-terminal (HRDC) domain of RecQ with a single fluorophore directly
Summary
Labeled proteins can help dissect the detailed molecular mechanisms and have become crucial experimental tools in various research fields, especially in flow cytometry, fluorescence microscopy, and enzymatic activity measurements (Li et al, 2015). There are a variety of fluorescent labeling methods and strategies that depend on particular applications, such as genetically encoded tags (Schlichthaerle et al, 2019), quantum dots (Abu-Thabit and Ratemi, 2020), and small organic fluorophores (Rosen and Francis, 2017). It can be performed in solution and in real time, with high time resolution and high sensitivity even at the single-molecule level (Toseland, 2013). The specific strategy to label a small fluorophore to the indicated site plays a decisive role and becomes a key limiting factor in many smFRET detections
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