Gd^III -¹⁹ F Distance Measurements for Proteins in Cells by Electron-Nuclear Double Resonance

Area of Science:

• Biophysics
• Structural Biology
• Cellular Biophysics

Background:

• Protein structure and dynamics studies are typically done in dilute solutions, not mimicking the crowded cellular environment.
• Double electron-electron resonance (DEER) can track protein conformations in cells but has a distance limitation below 1.8 nm.

Purpose of the Study:

• To demonstrate the utility of Gadolinium (Gd^III)-fluorine (¹⁹F) Mims electron-nuclear double resonance (ENDOR) for measuring short distances within proteins inside cells.
• To assess if proteins retain their structure when delivered into human cells.

Main Methods:

• Performed low-temperature solution and in-cell ENDOR measurements.
• Utilized room-temperature solution and in-cell Gd^III-¹⁹F paramagnetic relaxation enhancement (PRE) NMR.
• Studied fluorinated GB1 and ubiquitin (Ub) spin-labeled with Gd^III tags, delivered into human cells via electroporation.

Main Results:

• Gd^III-¹⁹F distances measured by ENDOR and PRE NMR were comparable in solution and within cells.
• The determined Gd^III-¹⁹F distances ranged from 1 to 1.5 nm.
• GB1 and ubiquitin proteins maintained their overall structure in the labeled regions after cellular delivery.

Conclusions:

• Gd^III-¹⁹F Mims ENDOR is effective for measuring short distances (1-1.5 nm) in proteins within the cellular environment.
• This technique complements DEER by covering distances below its lower limit.
• Proteins GB1 and Ub retain their structural integrity in cellular environments when studied with these methods.