Membrane lipids spin labeling

It is clear from a variety of spectroscopic techniques that biomembranes are dynamic not static structures. It is also known that certain membrane functions depend critically on the fluidity of the membrane lipids. Spin-labelled and fluorescent-labelled lipid probes are found to perform rotational motions in the nanosecond timescale in fluid lipid bilayers and membranes. For diffusive rotation the characteristic correlation times are given by the Debye equation (t = n V/kT) and correspond to effective viscosities in the range q 0.1-1 poise. A spin-labelled steroid analogue of cholesterol for instance rotates rapidly about its long... 

The membranes used in EPR measurements are usually multilamellar dispersions of lipids (mul-tilamellar liposomes) containing an investigated carotenoid and 0.5-1.0mol% of an appropriate lipid spin label (Figure 10.1). The total amount of lipids usually is 5-10 pmol per sample. 

The cell membrane is one of the most significant constituents for the life. In general, the cell can absorb required substances and exclude wrong ones through the membrane. In order to understand the mechanism of the peptide transport for example, we have to clarify the structure of the membrane, the fluidity of the lipid chain and the interaction between the chain and the peptide in the membrane. The spin label method is very effective for the characterization of the cell membrane because we can allow for selective spin labeling with nitroxide radicals at particular sites in the lipid chain. The labels can also be selectively bonded at particular molecules such as lipid chains and at peptides. The fluidity of membrane by the spin-labeUng method is reviewed by Toyoshima [17]. 

Griffith, O. H. Jost, P. C. Lipid spin labels in biological membranes. In Spin Labeling Theory and Application Berliner, L. J., Ed. New Yoik-San Francisco-London, Acad. Press, 1979,489-569. 

The involvement of free radicals in lipid peroxidation in the biological systems has been studied by ESR methods (9—11). The highly sensitive, non-invasive ESR oximetry method enables one to study early stages of lipid peroxidation by measuring oxygen uptake (12—17). ESR oximetry methods are based on bimolecular collisions of with spin labels that effectively shorten the spin-lattice relaxation time and lead to the broadening of the observed line width of the ESR signal. In addition, the lipid spin label enables the determination of membrane structure alteration with ESR techniques. 

Membranes with a relatively high protein content frequently display a second component in the ESR spectra of lipid spin labels, in addition to the fluid lipid bilayer component discussed in the previous section. This component is best resolved with labels close to the end of the chain, since a large degree of averaged spectral anisotropy is available to detect any immobilization induced by the protein. The ESR spectra of the 16-SASL stearic acid spin label in acetylcholine receptor-rich membranes and in bilayers of the extracted lipids is given in Fig. 3.3. A motionally restricted spin label component is seen in the outer wings of the spectrum from the membranes which is not present in the spectrum from the lipids alone. 

A motionally restricted lipid spin label component has been observed in several different membrane systems, including rod outer segment disc membranes, and (Na" , )-ATPase-containing membranes. Our results are summarized in Table 3.1, Only for the cytochrome... 

The ESR spectra of similar lipid spin labels, but with differing headgroups, are given in Fig. 3.5 for three membrane systems. These show a degree of specificity of the motionally restricted component for various lipids, and also a different pattern of selectivity for the different proteins. The spectra indicate a greater preference for stearic acid and for cardiolipin than for phosphatidylcholine in the first lipid shell of both cytochrome oxidase... 

Fretten, P., Morris, S.J., Watts, A., and Marsh, D., 1980, Lipid-lipid and lipid-protein interactions in chromaffin granule membranes. A spin label ESR study, Biochim. Biophys. Acta, 598 247. 

In any case, undesirable exclusion of spin-labelled lipids from the bilayer, and the presence of U-shaped lipid conformations, can be ruled out in experiments with D20-hydrated membranes for lipids spin-labelled at the terminal position - if the freezing protocol is correct, the ESEEM signal here must be zero. ... 

The TOAC spin label is used to substitute for Aib in trichogin, at the three possible sites. The spin-labelled peptide was studied by ESEEM in D20-hydrated dipalmitoyl phosphatidylcholine (DPPC), " and egg phosphatidylcholine (ePC), model membranes. To determine the localization of the peptide, reference data on the density profile of water in phospholipid model membranes were obtained from ESEEM of spin-labelled lipids. At low peptide concentration (peptide/lipid ratio less than 1 200 mol mol ), ESEEM amplitudes were found to be similar for labels close to the N-terminus and to the C-terminus. The ESEEM amplitudes turned out to be close to those found previously for lipids spin-labelled at carbon-atom of the lipid acyl chain nearest to the membrane surface. Therefore, it can be concluded that the peptide is oriented parallel to the membrane surface. The molecular structure of the peptide in the lipid environment that is estimated from these data is shown in Fig. 7. ... 

Additional confirmation of these results was obtained in experiments with cholesterol-dy, deuterium-substituted in the allqrl residue -CH(CH3)2, in bilayers hydrated with regular water. Note that, from experiments with membranes containing deuterium-substituted cholesterol and lipids spin-labelled at different positions along the acyl chain, one can elucidate the exact position of cholesterol in the membrane. 

Schreier, S. Frezzatti, W. A., Jr. Araujo, R S. Chaimovich, H. Cuccovia, I. M., Effect of lipid membranes on the apparent pK of the local anesthetic tetracaine Spin label and titration studies, Biochim. Biophys. Acta 769, 231-237 (1984). 

In the membrane lipid alkyl chains of n-SASL and n-PC spin labels undergo rapid rotational motion about the long axis of the spin label and wobble within the confines of a cone imposed by the... 

We would like to point out that an order parameter indicates the static property of the lipid bilayer, whereas the rotational motion, the oxygen transport parameter (Section 4.1), and the chain bending (Section 4.4) characterize membrane dynamics (membrane fluidity) that report on rotational diffusion of alkyl chains, translational diffusion of oxygen molecules, and frequency of alkyl chain bending, respectively. The EPR spin-labeling approach also makes it possible to monitor another bulk property of lipid bilayer membranes, namely local membrane hydrophobicity. 

Recently, due to increased interest in membrane raft domains, extensive attention has been paid to the cholesterol-dependent liquid-ordered phase in the membrane (Subczynski and Kusumi 2003). The pulse EPR spin-labeling DOT method detected two coexisting phases in the DMPC/cholesterol membranes the liquid-ordered and the liquid-disordered domains above the phase-transition temperature (Subczynski et al. 2007b). However, using the same method for DMPC/lutein (zeaxanthin) membranes, only the liquid-ordered-like phase was detected above the phase-transition temperature (Widomska, Wisniewska, and Subczynski, unpublished data). No significant differences were found in the effects of lutein and zeaxanthin on the lateral organization of lipid bilayer membranes. We can conclude that lutein and zeaxanthin—macular xanthophylls that parallel cholesterol in its function as a regulator of both membrane fluidity and hydrophobicity—cannot parallel the ability of cholesterol to induce liquid-ordered-disordered phase separation. 

Ashikawa, I., J.-J. Yin, W. K. Subczynski, T. Kouyama, J. S. Hyde, and A. Kusumi. 1994. Molecular organization and dynamics in bacteriorhodopsin-rich reconstituted membranes Discrimination of lipid environments by the oxygen transport parameter using a pulse ESR spin-labeling technique. Biochemistry 33 4947 1952. 

Subczynski, W. K., J. Widomska, and J. B. Feix. 2009. Physical properties of lipid bilayers from EPR spin labeling and their influence on chemical reactions in a membrane environment. Free Radic. Biol. Med., 46, 707-718. 

Widomska, J., M. Raguz, J. Dillon, E. R. Gaillard, and W. K. Subczynski. 2007. Physical properties of the lipid bilayer membrane made of calf lens lipids EPR spin labeling studies. Biochim. Biophys. Acta 1768 1454-1465. 

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