Lipid-protein interactions in frog rod outer segment disc membranes. Characterization by spin labels (original) (raw)
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The Journal of Physical Chemistry B, 2004
The dynamics of spin-labeled lipid chains in the low-temperature phases of dipalmitoyl phosphatidylcholine (DPPC) membranes, with and without equimolar cholesterol, have been investigated by pulsed electron paramagnetic resonance (EPR) spectroscopy. Echo-detected spectra from the two-pulse, primary spin-echo (pulse sequence: π/2-τ-π-τ-echo) are used to detect rapid angular motions, on the time scale of the phase memory time (T 2M) that is in the nanosecond regime. Echo-detected spectra from the three-pulse, stimulated spin-echo (pulse sequence: π/2-τ-π/2-T-π/2-τ-echo) are used to detect slow angular motions, on the time scale of the spin-lattice relaxation time (T 1) that is in the microsecond regime. Spectra recorded at very low temperature (77 K) are used to correct the two-pulse echo spectra for instantaneous diffusion that arises from dipolar spin-spin interactions between different spin labels. Echo-detected inversion recovery spectra are used to correct the three-pulse echo spectra for intrinsic spin-lattice relaxation and large-scale spectral diffusion induced by nitrogen nuclear spin flips. The dependence of the echo-detected spectral line shapes on the two time delays, τ and T, can be simulated adequately by using a simple two-state model to represent the small-amplitude librational motions in the low-temperature membrane phases. The fast librational motion has isotropic character, no singly defined direction of the librational axis, and is segmental in nature, depending on chain position and also on the presence of cholesterol. The slow librational motion is of a more global, cooperative nature, being independent of chain position and cholesterol content.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1981
Freely diffusable lipid spin labels in bovine rod outer segment disc membranes display an apparent two-component ESR spectrum. One component is markedly more immobilized than that found in fluid lipid bilayers, and is attributed to lipid interacting directly with rhodopsin. For the 14-doxyl stearic acid spin label this more immobilized component has an outer splitting of 59 G at 0°C, with a considerable temperature dependence, the effective outer splitting decreasing to 54 G at 24°C. Spin label lipid chains covalently attached to rhodopsin can also display a two-component spectrum in rod outer segment membranes. In unbleached, non-delipidated membranes the 16-doxyl stearoyl maleimide label shows an immobilized component which has an outer splitting of 59 G at 0°C and a considerable temperature dependence. This component which is not resolved at high temperatures (24--35°C), is attributed to the lipid chains interacting directly with the monomeric protein, as with the diffusable labels. In contrast, in rod outer segment membranes which have been either delipidated or extensively bleached, a strongly immobilized component is observed with the 16-doxyl maleimide label at all temperatures. This immobilized component has an outer splitting of 62--64 G at 0°C, with very little temperature dependence (61--62 G at 35°C), and is attributed to protein aggregation.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1987
We have used ESR and NMR linewidth broadening by spin-labels to determine the overall orientation of spin-labeled analogues of cholesterol and androstanol in egg lecithin bilayers. While the cholesterol analogues were found to have a single orientation in each monolayer, with the acyl chain pointing towards the center of the bilayer, the androstanol analogue appeared, at least in sonicated vesicles, to experience two opposite orientations in the same monolayer, very likely with a rapid reorientation. The possibility of rapid vertical fluctuations of the steroi molecules within the phospholipid bilayer is also discussed.
Biochemical and Biophysical Research Communications, 1980
Summary Saturation transfer electron spin resonance has been used to study the molecular motions of a C5-labelled phospholipid spin-probe in gel-phase bilayers of phosphatidylcholines, phosphatidylethanolamines and phosphatidylglycerols, and of mixtures with cholesterol. The spectra are qualitatively similar to the standard calibration spectra for isotropic motion but show differential quantitative effects in the diagnostic lineheight ratios which are indicative of anisotropic motion. A rather similar motional state ...
Time-resolved electron spin resonance studies of spin-labelled lipids in membranes
Chemistry and Physics of Lipids, 2006
Recently, developments in time-resolved spin-label electron spin resonance (ESR) spectroscopy have contributed considerably to the study of biomembranes. Two different applications of electron spin echo spectroscopy of spin-labelled phospholipids are reviewed here: (1) the use of partially relaxed echo-detected ESR spectra to study the librational lipid-chain motions in the lowtemperature phases of phospholipid bilayers; (2) the use of electron spin echo envelope modulation spectroscopy to determine the penetration of water into phospholipid membranes. Results are described for phosphatidylcholine bilayer membranes, with and without equimolar cholesterol, that are obtained with phosphatidylcholine spin probes site-specifically labelled throughout the sn-2 chain.
Biochemistry, 1979
In order to fix spin-labeled acids at the boundary layer of membrane-bound proteins, spin-labeled long-chain derivatives (m,n)MSL (general formula, CH,(CH2),R-(CH2),COO(CH2)2-M, where R is an oxazolidine ring containing a nitroxide and M is a maleimide residue) were synthesized. The spin-labeled molecules bind covalently to at least two different classes of sulfhydryl groups on rhodopsin in disc membrane fragments from bovine retina. One class of sites is hydrophilic and corresponds to the two SH groups labeled readily by N-ethylmaleimide; the second class of sites is only reached by hydrophobic probes. (10,3)MSL binds equally well to the two classes of sites on rhodopsin, whereas (1 , I 4)MSL, more hydrophobic, binds preferentially to the hydrophobic sites. Apparently a third class of SH groups can be labeled if a very large excess of (m,n)MSL is employed, but proteins may be denatured in this latter case. Labels not covalently bound are removed from the membranes by incubation with fatty acid free bovine serum albumin. However, it is found that the probes do not bind only to rhodopsin in the disc membranes. (m,n)MSL also binds covalently to phosphatidylethanolamine in the rod outer segments or in liposomes. This covalent binding to phospholipids is demonstrated by lipid extraction and thin-layer chromatographic analysis. In order to obtain the pure EPR spectra of the spin-labeled fatty acids bound to the protein, the spectra corresponding to phospholipid-bound spin labels have been I t is often admitted that intrinsic membrane proteins are surrounded by a boundary layer or "annulus" of rigidly bound lipid. The immobilization of this shell of lipid has been deduced essentially from EPR experiments involving spin-labeled fatty acids incorporated into reconstituted systems containing variable lipid to protein ratios. were the first to propose from spin-label experiments the model of a boundary layer of lipid surrounding an intrinsic membrane protein, namely cytochrome oxidase. Later, Hesketh et al. (1976) reported similar experiments with Ca2+-ATPase, while Chapman et al. (1977) showed that gramcidin A can lead to the same EPR results, if this polypeptide is dissolved in a small amount of lipid.
Applied Magnetic Resonance, 2013
Electron spin echo (ESE) spectroscopy, a pulsed version of electron paramagnetic resonance (EPR), was applied to spin-labeled stearic acids in phospholipid bilayers hydrated in the presence of sucrose and sorbitol, which are known for their cryoprotective action on biological membranes. The phospholipids were 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine (POPC). Stearic acids were labeled by nitroxide 4,4-dimethyl-oxazolidine-1-oxyl (DOXYL) attached rigidly at either the 5th or 16th specific carbon positions. ESE detects fast stochastic small-angle restricted molecular rotations (stochastic molecular librations) with correlation times on the nanosecond timescale. These motions are believed to have the same nature as the anharmonic motions of hydrogen atoms in biological substances detected by neutron scattering and Mössbauer spectroscopy, which become active above 200 K. To ensure that the echo decays indeed originate from fast stochastic molecular librations, a three-pulse stimulated spin echo was employed. It was found that the presence of sucrose or sorbitol suppresses the observed molecular motions. The observed effect was nearly the same for both label positions, indicating that the motions are similarly suppressed near the bilayer surface and in the bilayer interior. This finding suggests non-specific interactions of sugars with bilayer surface, which are likely to influence only the bulk physical properties of hydrated membranes. The results obtained show the usefulness of spin-echo EPR of spin labels when applied to investigate the molecular mechanisms of action of cryoprotective agents on biological systems.
Biophysical Journal, 2006
The importance of membrane-based compartmentalization in eukaryotic cell function has become broadly appreciated, and a number of studies indicate that these eukaryotic cell membranes contain coexisting liquid-ordered (L o ) and liquid-disordered (L d ) lipid domains. However, the current evidence for such phase separation is indirect, and so far there has been no direct demonstration of differences in the ordering and dynamics for the lipids in these two types of regions or their relative amounts in the plasma membranes of live cells. In this study, we provide direct evidence for the presence of two different types of lipid populations in the plasma membranes of live cells from four different cell lines by electron spin resonance. Analysis of the electron spin resonance spectra recorded over a range of temperatures, from 5 to 37°C, shows that the spinlabeled phospholipids incorporated experience two types of environments, L o and L d , with distinct order parameters and rotational diffusion coefficients but with some differences among the four cell lines. These results suggest that coexistence of lipid domains that differ significantly in their dynamic order in the plasma membrane is a general phenomenon. The L o region is found to be a major component in contrast to a model in which small liquid-ordered lipid rafts exist in a 'sea' of disordered lipids. The results on ordering and dynamics for the live cells are also compared with those from model membranes exhibiting coexisting L o and L d phases.