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Using O 2 to probe membrane immersion depth by 19 F NMR

A ﬂuorinated detergent, CF 3 (CF 2 ) 5 C 2 H 4 -O-maltose, was reconsti- tuted into a lipid bilayer model membrane system to demonstrate the feasibility of determining solvent accessibility and membrane immersion depth of each ﬂuorinated group by 19 F NMR. Apolar oxygen, which is known to partition with an increasing concentration gradient toward the hydrophobic membrane interior, ex-hibits a range of paramagnetic relaxation effects on 19 F nuclei, depending on its depth in the membrane. This effect, which is predominately associated with spin-lattice relaxation rates ( R 1 ) and chemical shifts, can be ampliﬁed greatly with minimal line broadening by increasing the partial pressure of O 2 at least 100-fold (i.e., P O 2 greater than 20 bar). The differences of longitudinal relaxation rates at 20 bar of oxygen pressure to those under ambient pressure ( R 120bar 2 R 10 ) are largest for those ﬂuorine groups expected to be most deeply buried in the membrane bilayer. This result contrasts with the reverse trend, which is observed on addition of a membrane surface-associated paramagnetic species, 4-( N , N -dimethyl- N -hexadecyl) ammonium-2,2,6,6-tetramethylpip-eridine-1-oxyl iodide (CAT-16) at ambient pressures. Thus, differential relaxation rates may be observed in 19 F-labeled membrane-associated molecules resulting from the addition of apolar oxygen under high pressure. The results demonstrate that the degree of solvent accessibility and membrane immersion depth of speciﬁc ﬂuorinated species in membrane-associated macromolecules can be probed by 19 F NMR.