Determination of membrane immersion depth with O₂: A high-pressure ¹⁹F NMR study

Oxygen is known to partition with an increasing concentration gradient toward the hydrophobic membrane interior. At partial pressures (P_O2) of 100 Atm or more, this concentration gradient is sufficient to induce paramagnetic effects that depend sensitively on membrane immersion depth. This effect is demonstrated for the fluorine nucleus by depth-dependent paramagnetic shifts and spin-lattice relaxation rates, using a fluorinated detergent, CF3(CF₂)₅C₂H₄-O-maltose (TFOM), reconstituted into a lipid bilayer model membrane system. To interpret the spin-lattice relaxation rates (R₁^P) in terms of a precise immersion depth, two specifically fluorinated cholesterol species (6-fluorocholesterol and 25-fluorocholesterol), whose membrane immersion depths were independently estimated, were studied by 195 NMR. The paramagnetic relaxation rates, R₁/^P, of the cholesterol species were then used to parameterize a Gaussian profile that directly relates R₁^P to immersion depth z. This same Gaussian curve could then be used to determine the membrane immersion depth of all six fluorinated chain positions of TFOM and of two adjacent residues of specifically fluorinated analogs of the antibacterial peptide indolicidin. The potential of this method for determination of immersion depth and topology of membrane proteins is discussed.