Dataset Information

A Toolbox for Site-Specific Labeling of RecQ Helicase With a Single Fluorophore Used in the Single-Molecule Assay.

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.

SUBMITTER: Teng FY

PROVIDER: S-EPMC7545742 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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A Toolbox for Site-Specific Labeling of RecQ Helicase With a Single Fluorophore Used in the Single-Molecule Assay.

Teng Fang-Yuan FY Jiang Zong-Zhe ZZ Huang Ling-Yun LY Guo Man M Chen Feng F Hou Xi-Miao XM Xi Xu-Guang XG Xu Yong Y

Frontiers in molecular biosciences 20200925

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 <i>Escherichia coli</i> RecQ helicase, we established a scheme involving a combination of fluorophore labeling and sortase A ligation to all ...[more]

PMID: 33102530

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Project description:The lactose operon repressor protein LacI has long served as a paradigm of the bacterial transcription factors. However, the mechanisms whereby LacI rapidly locates its cognate binding site on the bacterial chromosome are still elusive. Single-molecule fluorescence imaging approaches are well suited for the study of these mechanisms but rely on a functionally compatible fluorescence labeling of LacI. Particularly attractive for protein fluorescence labeling are synthetic fluorophores due to their small size and favorable photophysical characteristics. Synthetic fluorophores are often conjugated to natively occurring cysteine residues using maleimide chemistry. For a site-specific and functionally compatible labeling with maleimide fluorophores, the target protein often needs to be redesigned to remove unwanted native cysteines and to introduce cysteines at locations better suited for fluorophore attachment. Biochemical screens can then be employed to probe for the functional activity of the redesigned protein both before and after dye labeling. Here, we report a mutagenesis-based redesign of LacI to enable a functionally compatible labeling with maleimide fluorophores. To provide an easily accessible labeling site in LacI, we introduced a single cysteine residue at position 28 in the DNA-binding headpiece of LacI and replaced two native cysteines with alanines where derivatization with bulky substituents is known to compromise the protein's activity. We find that the redesigned LacI retains a robust activity in vitro and in vivo, provided that the third native cysteine at position 281 is retained in LacI. In a total internal reflection microscopy assay, we observed individual Cy3-labeled LacI molecules bound to immobilized DNA harboring the cognate O1 operator sequence, indicating that the dye-labeled LacI is functionally active. We have thus been able to generate a functional fluorescently labeled LacI that can be used to unravel mechanistic details of LacI target search at the single molecule level.

Dataset Information

A Toolbox for Site-Specific Labeling of RecQ Helicase With a Single Fluorophore Used in the Single-Molecule Assay.

Publications

A Toolbox for Site-Specific Labeling of RecQ Helicase With a Single Fluorophore Used in the Single-Molecule Assay.

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