For canonical lipid raft mixtures of cholesterol (chol), N-palmitoylsphingomyelin (PSM), and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), electron paramagnetic resonance (EPR) of spin-labeled phospholipids--which is insensitive to domain size--is used to determine the ternary phase diagram at 23°C. No phase boundaries are found for binary POPC/chol mixtures, nor for ternary mixtures with PSM content <24 mol %. EPR lineshapes indicate that conversion from the liquid-disordered (L(α)) to liquid-ordered (L(o)) phase occurs continuously in this region. Two-component EPR spectra and several tie lines attributable to coexistence of gel (L(β)) and fluid phases are found for ternary mixtures with low cholesterol or low POPC content. For PSM/POPC alone, coexistence of L(α) and L(β) phases occurs over the range 50-95.5 mol % PSM. A further tie line is found at 3 mol % chol with endpoints at 50 and ≥77 mol % PSM. For PSM/chol, L(β)-L(o) coexistence occurs over the range 10-38 mol % chol and further tie lines are found at 4.5 and 7 mol % POPC. Two-component EPR spectra indicative of fluid-fluid (L(α)-L(o)) phase separation are found for lipid compositions: 25%POPC>10%, and confirmed by nonlinear EPR. Tie lines are identified in the L(α)-L(o) coexistence region, indicating that the fluid domains are of sufficient size to obey the phase rule. The three-phase triangle is bounded approximately by the compositions 40 and 75 mol % PSM with 10 mol % chol, and 60 mol % PSM with 25 mol % chol. These studies define the compositions of raft-like L(o) phases for a minimal realistic biological lipid mixture.
Permeation of oxygen into membranes is relevant not only to physiological function, but also to depth determinations in membranes by site-directed spin labeling. Spin-lattice (T(1)) relaxation enhancements by air or molecular oxygen were determined for phosphatidylcholines spin labeled at positions (n = 4-14, 16) of the sn-2 chain in fluid membranes of dimyristoyl phosphatidylcholine, by using nonlinear continuous-wave electron paramagnetic resonance (EPR). Both progressive saturation and out-of-phase continuous-wave EPR measurements yield similar oxygen permeation profiles. With pure oxygen, the T(2)-relaxation enhancements determined from homogeneous linewidths of the linear EPR spectra are equal to the T(1)-relaxation enhancements determined by nonlinear EPR. This confirms that both relaxation enhancements occur by Heisenberg exchange, which requires direct contact between oxygen and spin label. Oxygen concentrates in the hydrophobic interior of phospholipid bilayer membranes with a sigmoidal permeation profile that is the inverse of the polarity profile established earlier for these spin-labeled lipids. The shape of the oxygen permeation profile in fluid lipid membranes is controlled partly by the penetration of water, via the transmembrane polarity profile. At the protein interface of the KcsA ion channel, the oxygen profile is more diffuse than that in fluid lipid bilayers.
Progressive saturation EPR measurements and EPR linewidth determinations have been performed on spin-labeled lipids in fluid phospholipid bilayer membranes to elucidate the mechanisms of relaxation enhancement by different paramagnetic ion salts. Such paramagnetic relaxation agents are widely used for structural EPR studies in biological systems, particularly with membranes. Metal ions of the 3d and 4f series were used as their chloride, sulfate, and perchlorate salts. For a given anion, the efficiency of relaxation enhancement is in the order Mn(2+) > or = Cu(2+) > Ni(2+) > Co(2+) approximately Dy(3+). A pronounced dependence of the paramagnetic relaxation enhancement on the anion is found in the order ClO(-)(4) > Cl(-) > SO(2-)(4). This is in the order of the octanol partition coefficients multiplied by spin exchange rate constants that were determined for the different paramagnetic salts in methanol. Detailed studies coupled with theoretical estimates reveal that, for the chlorides and perchlorates of Ni(2+) (and Co(2+)), the relaxation enhancements are dominated by Heisenberg spin exchange interactions with paramagnetic ions dissolved in fluid membranes. The dependence on membrane composition of the relaxation enhancement by intramembrane Heisenberg exchange indicates that the diffusion of the ions within the membrane takes place via water-filled defects. For the corresponding Cu(2+) salts, additional relaxation enhancements arise from dipolar interactions with ions within the membrane. For the case of Mn(2+) salts, static dipolar interactions with paramagnetic ions in the aqueous phase also make a further appreciable contribution to the spin-label relaxation enhancement. On this basis, different paramagnetic agents may be chosen to optimize sensitivity to different structurally correlated interactions. These results therefore will aid further spin-label EPR studies in structural biology.
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