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Related Concept Videos

Protein Dynamics in Living Cells01:19

Protein Dynamics in Living Cells

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Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
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Probing Protein-DNA Interactions with Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy Utilizing

Sandeep Yadav1, Kunihiko Ishii1,2, Tahei Tahara1,2

  • 1Molecular Spectroscopy Laboratory, RIKEN, 2-1 Hirosawa, Wako 351-0198, Japan.

The Journal of Physical Chemistry. B
|October 31, 2025
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Summary
This summary is machine-generated.

This study introduces a novel method combining protein-induced fluorescence enhancement (PIFE) with two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS) to analyze single-molecule protein-DNA interactions. The technique successfully quantifies binding events and determines molecular properties in complex biological systems.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biophysics

Background:

  • Protein-DNA interactions are fundamental to cellular functions.
  • Single-molecule protein-induced fluorescence enhancement (smPIFE) offers sensitive detection of these interactions.
  • Quantifying smPIFE for freely diffusing molecules is challenging due to intensity measurement limitations.

Purpose of the Study:

  • To develop a quantitative method for assessing protein-induced fluorescence enhancement (PIFE) in freely diffusing molecules.
  • To overcome the limitations of traditional smPIFE by utilizing fluorescence lifetime information.
  • To apply the novel technique to study specific protein-DNA interactions at the single-molecule level.

Main Methods:

  • Integration of protein-induced fluorescence enhancement (PIFE) with two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS).
  • Utilizing a Cy3-labeled DNA hairpin and the restriction enzyme BamHI as a model system.
  • Analysis of fluorescence lifetime data to distinguish different molecular states.

Main Results:

  • Successfully identified three distinct fluorescence components: unbound DNA, DNA bound to the enzyme, and a non-binding population.
  • Quantitatively assessed PIFE through fluorescence lifetime measurements.
  • Determined the dissociation constant and diffusion coefficients for both bound and unbound DNA states.

Conclusions:

  • The combined PIFE-2D FLCS technique provides a robust method for studying protein-DNA interactions at the single-molecule level.
  • This approach enables quantitative analysis of binding events and molecular dynamics in complex environments.
  • Highlights the potential of PIFE-2D FLCS for advancing research in molecular recognition and cellular processes.