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Updated: Jul 13, 2025

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Published on: February 7, 2019

A framework to validate fluorescently labeled DNA-binding proteins for single-molecule experiments.

Miranda Molina¹, Lindsey E Way², Zhongqing Ren²

¹Biochemistry and Molecular Biology Program, University of Texas Rio Grande Valley, Edinburg, TX 78539, USA; Department of Physics and Astronomy, University of Texas Rio Grande Valley, Edinburg, TX 78539, USA.

Cell Reports Methods

|October 13, 2023

Summary

This summary is machine-generated.

Lysine-cysteine-lysine (KCK) tags, used for protein visualization, can significantly alter DNA-binding protein properties. Researchers must carefully validate tags, as even subtle changes impact protein function and DNA interactions.

Keywords:

Bacillus subtilis CP: Molecular biology ChIP-seq DNA flow-stretching assay KCK ParB maleimide-conjugated fluorescent dyes single-molecule imaging

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Area of Science:

Molecular Biology
Biophysics
Protein Engineering

Background:

Maleimide-based fluorescent probes utilize lysine-cysteine-lysine (KCK) tags for protein visualization.
Short peptide tags are generally assumed to minimally affect protein function.

Purpose of the Study:

To assess the impact of KCK tags on DNA-binding protein properties using an in vitro single-molecule DNA flow-stretching assay.
To evaluate the KCK tag's influence on Bacillus subtilis ParB's DNA interaction dynamics.

Main Methods:

In vitro single-molecule DNA flow-stretching assay.
Comparison with in vivo fluorescence imaging and chromatin immunoprecipitation (ChIP) assays.
Analysis of protein response to nucleotide binding and specific DNA sequences (parS).

Main Results:

The KCK tag substantially altered ParB's DNA compaction rates.
KCK tagging affected ParB's response to nucleotide binding and the presence of the parS sequence.
No significant changes were detected by in vivo fluorescence imaging and ChIP assays, highlighting the sensitivity of the single-molecule assay.

Conclusions:

Short peptide tags, including KCK, can significantly perturb DNA-binding protein function.
Researchers should rigorously validate the impact of tags on protein behavior, especially in sensitive assays.
The single-molecule DNA flow-stretching assay provides a comprehensive method for assessing tag effects on DNA-binding proteins.