Water concentrations across lipid bilayers by HYSCORE spectroscopy of nitroxides

Structural organization of cellular membranes and membrane proteins is governed by the hydration water. Distribution of water molecules across lipid bilayers is highly heterogeneous due to the amphiphilic nature of this barrier, consisting of a 3- to 4-nm-thick hydrophobic core and a hydrophilic region formed by lipid polar head groups. Hyperfine sublevel correlation (HYSCORE) spectroscopy is capable of selective detection of hyperfine interactions originating from water molecules H-bonded directly to the N‒O^⋅ group of the nitroxide. Here, we describe a high-spatial-resolution method to measure local water concentration in biological systems based on detecting the reversible formation of hydrogen-bonded (H-bonded) complex between water and nitroxides by HYSCORE spectroscopy. Via selective detection of H-bonded deuterons, HYSCORE allows for accurate measurement of the fraction of H-bonded nitroxide, while a series of calibrations in bulk mixed solvents relates the fraction to local water concentration. The applicability of the method was demonstrated by measuring local water concentration in lipid bilayers doped with 5- or 16-doxyl stearic acids. Then the water concentration across bilayers formed by 1,2-dioleoyl-sn-glycero-3-phosphocholine lipids was determined by employing a series of membrane-spanning α-helical WALP peptides spin-labeled with (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate at 17 different sites. The data were analyzed by developing a model and an iterative numerical procedure to account for a distribution of the nitroxide locations across the bilayer due to the tether flexibility and the differences in H-bonding equilibria in the polar and apolar bilayer regions. Such analysis significantly improves transmembrane water profiles obtained by spin labeling electron paramagnetic resonance, enhancing both spatial resolution and the accuracy of local water concentration. The results also indicate the presence of water molecules trapped between bilayer leaflets. The method is expected to be broadly applicable to probing local hydration in complex heterogeneous biological and chemical systems.