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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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Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Published on: May 29, 2011

Deciphering protein microenvironment by using a cysteine specific switch-ON fluorescent probe.

Jessy Mariam¹, Anila Hoskere Ashoka², Vandana Gaded¹

¹Department of Chemistry, IIT Bombay, Mumbai-400076, India. ruchi@chem.iitb.ac.in.

Organic & Biomolecular Chemistry

|May 26, 2021

Summary

A new coumarin-based fluorescent probe,

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Area of Science:

Biochemistry
Chemical Biology
Biophysical Chemistry

Background:

Fluorescent probes are essential for visualizing dynamic biological events.
Understanding protein unfolding is crucial for comprehending protein function and dysfunction.

Purpose of the Study:

To develop and validate a novel switch-ON fluorescent probe ('L') for tracking protein unfolding and cysteine accessibility.
To investigate the stage-wise unfolding mechanism of TadA using the developed probe.

Main Methods:

Synthesis and characterization of a coumarin-based, cysteine-specific, switch-ON fluorescent probe ('L').
Application of probe 'L' to monitor protein unfolding and cysteine residue accessibility.
X-ray crystallography of TadA and temperature-dependent fluorescence spectroscopy of native TadA and its cysteine mutants.

Main Results:

Probe 'L' demonstrated high selectivity and no artifacts with bystander species.
Probe 'L' effectively detected subtle changes in protein microenvironment and early unfolding events.
X-ray structure and fluorescence data revealed that TadA unfolds in a stage-wise manner, with functionally important regions unfolding later.

Conclusions:

Probe 'L' is a sensitive and specific tool for monitoring protein unfolding and microenvironment changes.
The study elucidated a stage-wise unfolding mechanism for TadA, highlighting the stability of functionally important sub-domains.
Probe 'L' can serve as a generic dye for studying protein unfolding and related structural dynamics.