Gd(III)-PyMTA label is suitable for in-cell EPR

Area of Science:

• Biophysics
• Structural Biology
• Biochemistry

Background:

• Electron Paramagnetic Resonance (EPR) spectroscopy with site-directed spin labeling is crucial for studying biomacromolecules.
• In-cell applications are limited by the instability of common nitroxide spin labels in cellular environments.

Purpose of the Study:

• To evaluate the suitability of a Gadolinium(III)-based spin label (Gd-PyMTA) for in-cell EPR.
• To measure distances and analyze peptide conformation within living cells.

Main Methods:

• Site-directed spin labeling of a proline-rich peptide with Gd-PyMTA.
• Microinjection into Xenopus laevis oocytes.
• Distance measurements using double electron-electron resonance (DEER) spectroscopy.
• Conformational analysis using a rotamer library.

Main Results:

• Gd-PyMTA proved to be cell-compatible and stable in cellular extracts for over 24 hours.
• Intracellular distance measurements of a labeled peptide were successfully performed using DEER.
• The study suggests the peptide inserts into cell membranes, inducing a conformational change from PPII to PPI helix.

Conclusions:

• Gd(III)-based spin labels like Gd-PyMTA are viable for in-cell EPR studies.
• This approach enables the investigation of biomacromolecular structure and dynamics within their native cellular context.
• The findings provide insights into peptide-membrane interactions and conformational dynamics in vivo.