title: Water accessibility in a membrane-inserting peptide comparing Overhauser DNP and pulse EPR methods.
abstract: Water accessibility is a key parameter for the understanding of the structure of biomolecules, especially membrane proteins. Several experimental techniques based on the combination of electron paramagnetic resonance (EPR) spectroscopy with site-directed spin labeling are currently available. Among those, we compare relaxation time measurements and electron spin echo envelope modulation (ESEEM) experiments using pulse EPR with Overhauser dynamic nuclear polarization (DNP) at X-band frequency and a magnetic field of 0.33 T. Overhauser DNP transfers the electron spin polarization to nuclear spins via cross-relaxation. The change in the intensity of the (1)H NMR spectrum of H2O at a Larmor frequency of 14 MHz under a continuous-wave microwave irradiation of the nitroxide spin label contains information on the water accessibility of the labeled site. As a model system for a membrane protein, we use the hydrophobic α-helical peptide WALP23 in unilamellar liposomes of DOPC. Water accessibility measurements with all techniques are conducted for eight peptides with different spin label positions and low radical concentrations (10-20 μM). Consistently in all experiments, the water accessibility appears to be very low, even for labels positioned near the end of the helix. The best profile is obtained by Overhauser DNP, which is the only technique that succeeds in discriminating neighboring positions in WALP23. Since the concentration of the spin-labeled peptides varied, we normalized the DNP parameter ϵ, being the relative change of the NMR intensity, by the electron spin concentration, which was determined from a continuous-wave EPR spectrum.
authors.affiliation: Laboratory of Physical Chemistry, ETH Zurich, Vladimir-Prelog-Weg 2, CH-8093 Zurich, Switzerland.
authors.forename: Takuya F
authors.initials: TF
authors.lastname: Segawa
authors.affiliation: Laboratory of Physical Chemistry, ETH Zurich, Vladimir-Prelog-Weg 2, CH-8093 Zurich, Switzerland.
authors.forename: Maximilian
authors.initials: M
authors.lastname: Doppelbauer
authors.affiliation: Laboratory of Physical Chemistry, ETH Zurich, Vladimir-Prelog-Weg 2, CH-8093 Zurich, Switzerland.
authors.forename: Luca
authors.initials: L
authors.lastname: Garbuio
authors.affiliation: Laboratory of Physical Chemistry, ETH Zurich, Vladimir-Prelog-Weg 2, CH-8093 Zurich, Switzerland.
authors.forename: Andrin
authors.initials: A
authors.lastname: Doll
authors.affiliation: Laboratory of Physical Chemistry, ETH Zurich, Vladimir-Prelog-Weg 2, CH-8093 Zurich, Switzerland.
authors.forename: Yevhen O
authors.initials: YO
authors.lastname: Polyhach
authors.affiliation: Laboratory of Physical Chemistry, ETH Zurich, Vladimir-Prelog-Weg 2, CH-8093 Zurich, Switzerland.
authors.forename: Gunnar
authors.initials: G
authors.lastname: Jeschke
mesh_terms: D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D008051:Lipid Bilayers / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008872:Microwaves; D008956:Models, Chemical; D010455:Peptides / Q000737:chemistry*; D010713:Phosphatidylcholines / Q000737:chemistry; D011758:Pyrroles / Q000737:chemistry; D013113:Spin Labels; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014867:Water / Q000737:chemistry*
chemical_list: C000614674:2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate; D008051:Lipid Bilayers; D010455:Peptides; D010713:Phosphatidylcholines; D011758:Pyrroles; D013113:Spin Labels; D013438:Sulfhydryl Compounds; C571014:WALP23; D014867:Water; C017251:1,2-oleoylphosphatidylcholine