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Abstract

Profiles of polarity across biological membranes are essential determinants of the cellular permeability barrier and of the stability of transmembrane proteins. High-field electron paramagnetic resonance of systematically spin-labeled lipid chains is used here to determine the polarity profiles of cholesterol-containing phospholipid membranes. The polarity dependence of the g_xx-tensor element is opposite to the dependence on chain dynamics, and additionally has enhanced sensitivity to hydrogen bonding. Both features make high-field measurements superior to conventional determinations of local polarity from spin-label hyperfine couplings. The profile of g_xx in dimyristoyl phosphatidylcholine membranes with 5 or 40 mol% cholesterol is established with eleven positional isomers of phosphatidylcholine, spin labeled at positions n = 4-14 in the sn-2 chain. A sigmoidal barrier, centered about chain position n₀ ≈ 8, mirrors the corresponding sigmoidal trough obtained from the spin-label hyperfine coupling, A_zz. For the different positions, n, it is found that partial derivativeg_xx/partial derivativeA_zz = -2.4 T^-1, a high value that is characteristic of hydrogen-bonded spin labels. This demonstrates that the transmembrane polarity pro. le registered by spin labels corresponds to water penetration into the membrane. Inhomogeneous broadening of the g_xx-spectral feature demonstrates heterogeneities of the water distribution in the regions of higher intramembrane polarity defined by n < 8. In the transition region between high- and low-polarity regions (n ≈ 8), the g_xx-feature consists of two components characteristic of coexisting hydrated and nonhydrated states.