The equilibrium concentration of oxygen in membranes can be 3 to 4 times that in water. This concentration enhancement has been used to assess the depth of penetration of protein residues into membranes combined with site direct spin labeling and

1
Introduction

• The equilibrium concentration of oxygen in
  membranes can be 3 to 4 times that in water. This
  concentration enhancement has been used to assess
  the depth of penetration of protein residues into
  membranes combined with site direct spin labeling
  and EPR-based collisional relaxation
  measurements.
• These studies have been very useful in
  characterizing the nature of protein-membrane
  interactions and in determining the orientation
  of a protein with respect to the membrane.
  However, the profile and absolute concentration
  of oxygen is not well characterized and, thus
  far, any method to position an amino acid residue
  using oxygen alone has only been qualitative.
  Therefore, we have prepared a series of
  polypeptides consisting of around 23 amino acids
  that form a single alpha-helix that spans the
  membrane. The polypeptide, a variation on the
  WALP-23 of Demmers, et al., JBC, 276,
  34501-34508, consists of Leu-Ala repeats that
  form an alpha helix within the membrane and
  terminate in a tryptophan and uncharged groups at
  either end. The terminal groups aid in
  registering the polypeptide with respect to the
  membrane.

2

• A series of polypeptides was made in which the
  position of a single cysteine was systematically
  varied from one end of WALP23 to the other. A
  spin label was covalently attached to the
  cysteine. We recorded the spin lattice relaxation
  rate for the spin-label on the polypeptide as a
  function of residue position with and without
  oxygen using pulsed saturation recovery. We
  determined the oxygen transport parameter, which
  is the product of the relaxivity and the local
  concentration of oxygen, as a function of
  position on the alpha helix. The profile is
  symmetric about the middle of the membrane, and
  the effect is about three fold larger in the
  middle of the membrane than in bulk water. The
  effect of the spin relaxant Ni(EDDA) at 50mM was
  also measured.

3
The WALP23 Polypeptide

• WALP23 is a single alpha helical
  membrane-spanning polypeptide.
• The sequence is 23 residues long
• HCO-NH-G-WW-L-(AL)8-WW-A-CO-NH2
• L and A are both hydrophobic. In a membrane this
  forms a single turn alpha helix.
• The membrane using di-oleic (DO) is about 28-30
  Ang thick. The two outer Tryptophans (W) are
  about 30 Angs apart. The membrane will stretch
  (or shrink) to accommodate the protein.
• Demmers et al J. Biol. Chem., 276, 34501-34508,
  2001

4
Oxygen Transport Parameter

• The Oxygen transport parameter is the change
  in the spin-lattice relaxation rate due to oxygen
  collisional relaxation
• ,
• where

Depends on transport properties (e.g. Diffusion)
of Oxygen in the local environment of the spin
label
5
WALP in DOPC LUVs
DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine)
Label Position
6
WALP in DOPM LUVs
DOPM (1,2-dioleoyl-sn-glycero-3-phosphomethanol)
Label Position
7
Typical WALP/DOPC Saturation Recovery EPR
With Oxygen
CW
Without Oxygen
8
Walp23 in DOPC Oxygen Transport Parameter
From SR
Estimated from the CW line width
9
Walp23 in DOPM Oxygen Transport Parameter
10
Ratio Parameter
Altenbach, C. et al. PNAS (1994) V 91 n5 pgs.
1667-71
11
Results

• The Oxygen transport parameter achieves a maximum
  in the center of the bi-layer for both DOPC and
  DOPM membranes (filled squares)
• The oxygen transport parameter approaches a
  common solution value at each end of the
  polypeptide, indicating the ends are exposed to
  solvent
• The spin-lattice (filled diamonds) and spin-spin
  (open squares) relaxation rates have a maximum
  and a minimum ,respectively, in the center of the
  bi-layer and reflect local mobility of the spin
  label. The bi-layer is more fluid in the middle
  and near the ends.
• Ni(EDDA) data (filled circles, DOPC) shows
  partial solubility of the Ni relaxant in the
  bi-layer. A gradient of the Ni(EDDA) transport
  parameter is observed, and diminishes toward the
  center of the bi-layer.
• The ratio of oxygen and nickel transport
  parameters introduced by Hubbell and co-workers
  is measured here by time domain EPR for first
  time

12
Conclusions

• The gradient of the oxygen transport parameter
  measured on WALP 23 is ideal as a ruler for
  determination of spin label position in membranes
  because WALP is a uniform helical polypeptide
  that has known registration in various membrane
  bi-layers
• The spin-lattice and spin-spin relaxation rates
  show dependence on local mobility of the spin
  label in the bi-layer. Therefore, the oxygen
  transport parameter cannot be separated into its
  two components the oxygen concentration and
  transport-dependent coefficient.
• The ratio parameter, which is designed to
  cancel out transport effects, and can be a
  measure of relaxant concentration seems not to
  scale simply with nitroxide positions in the
  membrane.