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 Label Position DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine)

6. WALP in DOPM LUVs Label Position DOPM (1,2-dioleoyl-sn-glycero-3-phosphomethanol)

7. Typical WALP/DOPC Saturation Recovery EPR CW With Oxygen Without Oxygen

8. Estimated from the CW line width From SR Walp23 in DOPC: Oxygen Transport Parameter

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.