Phase Diagram of Ternary Cholesterol/Palmitoylsphingomyelin/Palmitoyloleoyl-Phosphatidylcholine Mixtures: Spin-Label EPR Study of Lipid-Raft Formation

Abstract

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%<PSM<65%, 5%<chol<30-35%, 65%>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.

Communication: Orientational self-ordering of spin-labeled cholesterol analogs in lipid bilayers in diluted conditions

“…1(c) presents spectra simulated using Eqs. (1) and (2). The initial EPR spectrum E(0, B 0 ) at the zero time delay τ = 0 (not shown) was calculated using g-tensor and hyperfine interaction parameters determined in a standard way from the CW EPR spectra.…”

“…This division allows for extraction of the pure contribution of instantaneous diffusion, as all other contributions to the echo decay may be assumed to be field-independent (the influence of molecular motions is field-dependent too 22 but the temperature of measurements is too low for motions to be active); the rate of this contribution according to Eq. (1) is proportional to the difference sin 2 …”

“…Also, we note that formation of these "orientational domains" can serve as precursors for the raft formation. [1][2][3][4][5] The most important point in further studies could be the elucidation of the size of these domains of similarly oriented cholesterol molecules.…”

“…[1][2][3][4][5] These nanodomains may serve as platforms for functioning membrane proteins. To get insight into the mechanisms of raft formation, it is necessary to study properties of lipid-cholesterol interactions.…”

Live Cell Plasma Membranes Do Not Exhibit a Miscibility Phase Transition over a Wide Range of Temperatures

“…[1][2][3][4][5][6] These membrane systems are widely considered as model systems that mimic the composition of the general mammalian cell plasma membrane. Observations of the same macroscopic miscibility transitions in cell membrane blebs provide compelling confirmation that this is an inherent property of cell membrane lipids.…”