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Protein Dynamics in Living Cells

Protein Dynamics in Living Cells

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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Tracking protein transitions through fluorescence spectral phasor analysis with ACDAN.

Leandro Cruz Rodríguez¹, Nahuel Naum Foressi¹, María Soledad Celej¹

¹Departamento de Química Biológica Ranwel Caputto, Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC, CONICET), Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Haya de la Torre y Medina Allende, Ciudad Universitaria, Córdoba, Argentina.

Biophysical Reports

|April 19, 2025

Summary

This summary is machine-generated.

This study uses spectral phasor analysis and ACDAN fluorescence to reveal early protein changes during unfolding, aggregation, and phase separation, offering a new tool for biophysical research.

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Area of Science:

Biophysics
Biochemistry
Molecular Biology

Background:

Protein conformational changes are crucial for biological function and disease.
Understanding protein transitions like unfolding, aggregation, and phase separation is vital.
Existing methods have limitations in detecting early-stage events and microenvironmental details.

Purpose of the Study:

To investigate the utility of spectral phasor analysis and ACDAN fluorescence for studying protein transitions.
To detect subtle conformational changes and microenvironmental shifts during protein unfolding.
To gain insights into early amyloid aggregation events and the properties of protein biocondensates.

Main Methods:

Utilizing spectral phasor analysis combined with hyperspectral imaging.
Employing 6-acetyl-2-dimethylaminonaphthalene (ACDAN) as a fluorescent probe.
Comparing ACDAN fluorescence with conventional thioflavin T in amyloid formation studies.

Main Results:

ACDAN fluorescence sensitively detects early protein microenvironmental shifts preceding complete unfolding.
ACDAN identifies solvent dipolar relaxation events during amyloid formation, missed by thioflavin T.
Physicochemical properties and distinct microenvironments within protein biocondensates are mapped.

Conclusions:

Spectral phasor analysis and ACDAN fluorescence offer a sensitive and versatile toolkit for studying protein transitions.
The approach provides critical insights into early events in protein unfolding and aggregation.
This method enhances the understanding of liquid-liquid phase separation and biocondensate properties.