In cell Gd³⁺-based site-directed spin labeling and EPR spectroscopy of eGFP

Area of Science:

• Biophysics
• Chemical Biology
• Molecular Imaging

Background:

• Functional studies of biomolecules necessitate labeling within their native cellular environment.
• Existing methods face challenges in achieving stable labeling and sensitive detection in living cells.

Purpose of the Study:

• To establish an efficient in-cell spin labeling technique for biomolecule studies.
• To enable Electron Paramagnetic Resonance (EPR) spectroscopy of labeled proteins in living cells.

Main Methods:

• Utilized alkyne-azide click chemistry for covalent labeling of biomolecules inside cells.
• Employed a Gadolinium(III)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (Gd3+-DOTAM)-azide complex as the spin label.
• Performed EPR spectroscopy on spin-labeled proteins in living cells at ambient temperatures.

Main Results:

• Achieved high stability of the spin label attached to the protein within the cellular environment.
• Obtained narrow Electron Paramagnetic Resonance (EPR) lines, indicative of a well-defined and stable spin label.
• Successfully detected spin-labeled proteins in living cells using EPR spectroscopy.

Conclusions:

• In-cell spin labeling with Gd3+-DOTAM-azide click chemistry is a viable method for studying biomolecules.
• The technique offers high spin label stability and enables sensitive EPR detection of proteins in living systems.
• This approach advances the capability for functional studies of biomolecules in their native cellular context.