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Spatiotemporally Controlled Nuclear Translocation of Guests in Living Cells Using Caged Molecular Glues as Photoactivatable Tags

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Published on: January 17, 2019

Second-generation covalent TMP-tag for live cell imaging.

Zhixing Chen¹, Chaoran Jing, Sarah S Gallagher

¹Department of Chemistry, Columbia University, New York, New York 10027, USA.

Journal of the American Chemical Society

|August 10, 2012

Summary

This summary is machine-generated.

A new, improved TMP-tag enables faster covalent labeling of intracellular proteins in living cells using advanced organic fluorophores. This breakthrough enhances protein labeling techniques for biological research and synthetic biology applications.

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Area of Science:

Biochemistry
Chemical Biology
Synthetic Biology

Background:

Organic fluorophores offer advantages over fluorescent proteins for cellular labeling.
Previous TMP-tag designs had limitations in labeling speed and efficiency in live cells.

Purpose of the Study:

To develop a second-generation covalent TMP-tag with enhanced labeling kinetics.
To enable efficient labeling of diverse intracellular proteins in living cells.

Main Methods:

Engineered an acrylamide-trimethoprim-fluorophore (A-TMP-fluorophore v2.0) with an optimized linker.
Utilized a cysteine (Cys) nucleophile engineered near the Escherichia coli dihydrofolate reductase (eDHFR) binding pocket.
Screened eDHFR:Cys variants to identify optimal tag-nucleophile pairs for rapid covalent labeling.

Main Results:

Identified eDHFR:L28C variant with an 8-minute labeling half-life in vitro.
Demonstrated successful live-cell imaging of various protein targets using A-TMP-fluorophore v2.0 probes.
Developed an efficient chemical synthesis for producing A-TMP-probe v2.0 labels.

Conclusions:

The second-generation covalent TMP-tag provides a robust and efficient method for intracellular protein labeling in living cells.
Proximity-induced reactivity and organic chemistry are powerful tools for engineering chemical tags and advancing synthetic biology.