Vsevolod A. Livshits

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

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.

Oxygen permeation profile in lipid membranes: comparison with transmembrane polarity profile.

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.

High-field electron spin resonance of spin labels in membranes

High-field electron spin resonance (ESR) spectroscopy is currently undergoing rapid development. This considerably increases the versatility of spin labelling which, at conventional field strengths, is already well established as a powerful physical technique in membrane biology. Among the unique advantages offered by high-field spectroscopy, particularly for spin-labelled lipids, are sensitivity to non-axial rotation and lateral ordering, a better orientational selection, an extended application to rotational dynamics, and an enhanced sensitivity to environmental polarity. These areas are treated in some depth, along with a detailed consideration of recent developments in the investigation of transmembrane polarity profiles.

Anisotropic motion effects in CW non-linear EPR spectra: relaxation enhancement of lipid spin labels

Continuous-wave (CW) EPR measurements of enhancements in spin-lattice (T(1)-) relaxation rate find wide application for determining spin-label locations in biological systems. Often, especially in membranes, the spin-label rotational motion is anisotropic and subject to an orientational potential. We investigate here the effects of anisotropic diffusion and ordering on non-linear CW-EPR methods for determining T(1) of nitroxyl spin labels. Spectral simulations are performed for progressive saturation of the conventional in-phase, first-harmonic EPR signal, and for the first-harmonic absorption EPR signals detected 90 degrees -out-of-phase with respect to the Zeeman field modulation. Motional models used are either rapid rotational diffusion, or strong-jump diffusion of unrestricted frequency, within a cone of fixed maximum amplitude. Calculations of the T(1)-sensitive parameters are made for both classes of CW-experiment by using motional parameters (i.e., order parameters and correlation times), intrinsic homogeneous and inhomogeneous linewidth parameters, and spin-Hamiltonian hyperfine- and g-tensors, that are established from simulation of the linear CW-EPR spectra. Experimental examples are given for spin-labelled lipids in membranes.

Saturation Transfer Spectroscopy of Biological Membranes

Various aspects of the branch of non-linear spectroscopy that is known as saturation transfer (ST) EPR are reviewed, ranging from its inception to the present day. Initial methodological development was by Hyde and Dalton, followed by the introduction into biology by Hyde and Thomas. ST-EPR is a continuous wave spectroscopy, which extends the sensitivity of conventional nitroxide EPR to the microsecond (or submillisecond) correlation time regime of rotational motion, for spin-labelled membranes and biopolymers. Equally, slow exchange processes are accessible to ST-EPR, as are the paramagnetic relaxation enhancements that are essential to site-directed spin-labelling strategies. Central to the latter are the principles of spin-label oximetry, as developed by Hyde and coworkers at the Milwaukee EPR Centre.

Non-linear, continuous-wave EPR spectroscopy and spin–lattice relaxation: spin-label EPR methods for structure and dynamics†

The sensitivity of continuous-wave, non-linear EPR signals to spin–lattice (T1) relaxation has been investigated. The aim was to identify those spectral displays that are most appropriate to obtain structural and dynamic information from spin-label EPR experiments that involve detection of T1-relaxation enhancements. This has been achieved by solving the Bloch equations for the various harmonics of the absorption and dispersion components of the spin magnetisation. Of interest are the magnetisation components that are out-of-phase with respect to the static magnetic field modulation, under conditions of partial saturation of the microwave absorption. It is found that both the first- and second-harmonic out-of-phase absorption EPR signals are particularly sensitive to T1-relaxation. The first-harmonic absorption quadrature-phase signal is favoured for determining T1-relaxation enhancements because of its superior intensity and relative insensitivity to transverse (T2) relaxation. The second-harmonic absorption quadrature-phase EPR signal has lower relative intensity and is more sensitive to T2-relaxation, but has a unique sensitivity to rotational diffusion that is exploited in saturation transfer EPR spectroscopy. The non-linear dispersion signals are less appropriate because of their principal sensitivity to T2-relaxation and because they saturate less readily. These novel non-linear EPR spectroscopies can be contrasted with the conventional progressive saturation behaviour of the in-phase absorption signals that are determined by the T1T2 relaxation time product.

Recent developments in biological spin-label spectroscopy

Spin-label spectroscopy deals with the electron spin resonance (ESR) of stable nitroxide free radicals. Its use in biology is based on the versatile chemistry of nitroxides that yields spin-labelled derivatives of biological ligands, lipids and macromolecules, and on the unique sensitivity of the nitroxide ESR spectra to rotational mobility and spin-spin interactions. Two new aspects of spin-label methodology that will greatly enhance its application in structural and membrane biology are non-linear ESR and high-field ESR, which are described here.