Water accessibility in a membrane-inserting peptide comparing Overhauser DNP and pulse EPR methods

Area of Science:

• Biophysics
• Structural Biology
• Spectroscopy

Background:

• Water accessibility is vital for understanding biomolecular structures, particularly membrane proteins.
• Electron Paramagnetic Resonance (EPR) spectroscopy with site-directed spin labeling offers experimental approaches to probe water accessibility.
• Comparing different EPR techniques is essential for optimizing structural studies.

Purpose of the Study:

• To compare the efficacy of relaxation time measurements and electron spin echo envelope modulation (ESEEM) with Overhauser dynamic nuclear polarization (DNP) for determining water accessibility.
• To evaluate these techniques using a model membrane protein system (WALP23 in liposomes).
• To assess the sensitivity of these methods in discriminating water accessibility at different labeled positions.

Main Methods:

• Utilized X-band EPR spectroscopy at 0.33 T.
• Employed Overhauser DNP to transfer electron spin polarization to nuclear spins via cross-relaxation.
• Measured changes in the intensity of the 1H NMR spectrum of H2O under microwave irradiation of nitroxide spin labels.
• Investigated eight spin-label positions on the WALP23 peptide within unilamellar DOPC liposomes at low radical concentrations (10-20 μM).

Main Results:

• All tested EPR techniques consistently indicated very low water accessibility, even for labels near the helix terminus.
• Overhauser DNP provided the most informative profile of water accessibility.
• Overhauser DNP was the only method capable of distinguishing between neighboring spin-label positions.
• The DNP parameter ϵ was normalized by electron spin concentration, determined via continuous-wave EPR, to account for variations in peptide concentration.

Conclusions:

• Overhauser DNP is a highly effective technique for quantifying water accessibility around spin-labeled sites in membrane proteins.
• The study highlights the limited water penetration even in hydrophobic membrane protein environments.
• The findings provide a basis for selecting optimal EPR methods for structural and accessibility studies of membrane proteins.