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Protein Dynamics in Living Cells

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A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

Published on: December 1, 2018

A fluorescence-based binding assay for proteins using the cell surface as a sensing platform.

Kazuki Hirano¹, Shinji Sueda²

¹Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, 680-4 Kawazu, Iizuka, 820-8502, Japan.

Analytical Sciences : the International Journal of the Japan Society for Analytical Chemistry

|December 13, 2023

Summary

This study introduces a novel cell-surface protein-protein interaction (PPI) analysis system. This method avoids protein immobilization, enabling more accurate PPI quantification and function elucidation in living cells.

Keywords:

Binding affinity data Binding equilibrium analysis Confocal laser scanning microscopy Fluorescence imaging Protein–protein interaction

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Area of Science:

Biochemistry
Molecular Biology
Cell Biology

Background:

Protein-protein interactions (PPIs) are crucial for cellular functions.
Current PPI analysis methods often require protein immobilization, which can alter binding characteristics.
A need exists for methods that analyze PPIs in a more biologically relevant context.

Purpose of the Study:

To develop and validate a novel cell-surface-based system for protein-protein interaction (PPI) analysis.
To overcome limitations of traditional methods that require protein immobilization.
To enable quantitative PPI analysis directly on living cell surfaces.

Main Methods:

Engineered cells to display target proteins (biotin carboxyl carrier protein, BCCP) on their surface via fusion with membrane proteins.
Utilized fluorescently labeled binding partners (biotin protein ligase, BPL) to detect interactions.
Quantified binding using fluorescence intensity ratios (fluorescein to mApple) for real-time monitoring.

Main Results:

Successfully demonstrated PPI analysis using a cell-surface sensing platform.
Achieved stable and reliable binding level evaluation over a 60-minute observation period.
Determined the binding dissociation constant (Kd) for the BPL-BCCP interaction to be 0.33 ± 0.05 μM.

Conclusions:

The proposed cell-surface system offers a viable alternative for quantitative PPI analysis.
This method preserves the native state of proteins, providing more accurate interaction data.
The system facilitates the elucidation of protein functions by studying interactions in a cellular context.