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Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy
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Coumarin-based fluorogenic probes for no-wash protein labeling.

Yingche Chen1, Christopher M Clouthier, Kelvin Tsao

  • 1Department of Chemistry, University of Ottawa, 10 Marie-Curie, Ottawa, ON K1N 6N5 (Canada).

Angewandte Chemie (International Ed. in English)
|October 15, 2014
PubMed
Summary
This summary is machine-generated.

A new method enables specific labeling of intracellular proteins using a genetically encoded dithiol tag and novel fluorogens. This approach allows for rapid and precise visualization of proteins within living cells.

Keywords:
bioorthogonal chemistryfluorescent probesglutathionemaleimideprotein labeling

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Chemical Biology

Background:

  • Visualizing proteins within living cells is crucial for understanding cellular processes.
  • Existing protein labeling methods often face challenges with specificity, reactivity, or intracellular compatibility.

Purpose of the Study:

  • To develop a novel, rapid, and specific fluorescent protein-labeling strategy for intracellular applications.
  • To design and synthesize fluorogenic reagents that selectively label a unique protein tag within the cell.

Main Methods:

  • Genetically encoding a protein of interest (POI) with a short peptide sequence containing two cysteine (Cys) residues.
  • Synthesizing novel fluorogens featuring a fluorophore and two maleimide groups that are quenched until thiol addition.
  • Characterizing the kinetics of novel fluorogens, including evaluating the impact of a methoxy substituent on maleimide reactivity.

Main Results:

  • A robust fluorescent protein-labeling system was established using a dithiol tag and custom-synthesized fluorogens.
  • The developed fluorogens demonstrated selective reactivity with the intracellular dithiol tag, minimizing off-target reactions with endogenous thiols like glutathione.
  • The methoxy substituent on the maleimide was found to be critical for suppressing reactivity with glutathione while maintaining efficient labeling of the dithiol tag.

Conclusions:

  • The novel fluorescent labeling strategy enables rapid and specific visualization of intracellular proteins of interest.
  • This method offers a valuable tool for studying protein dynamics and localization in live cells.
  • The design principles for the fluorogens can be applied to develop other targeted labeling reagents.