SPIN-LABEL STUDIES OF LIPID-PROTEIN INTERACTIONS IN RETINAL ROD OUTER SEGMENT MEMBRANES - FLUIDITY OF THE BOUNDARY-LAYER

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, CH3(CH2)mR-(CH2)nCOO(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, 14)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 subtracted. (1, 14)MSL corresponds to the spin label with the nitroxide near the ω-2 carbon of the acyl chain. When this spin label is bound to rhodopsin in the disc membranes, it gives rise to an EPR spectrum not very different from the spectrum of the corresponding fatty acid diffusing freely in the lipid phase. This result suggests that, in native membranes, a high degree of fluidity exists in the boundary layer of phospholipids and therefore indicates that the lipid phase of the rod outer segment membranes is largely homogeneous. If membranes are illuminated at 37 °C for an hour, an immobilized component appears, superimposed on the former spectrum of (1, 14)MSL. Similarly if membranes are partially delipidated with phospholipase A2, a strongly immobilized component is always seen. The (10, 3)MSL, which has a probe closer to the maleimide residue, is more immobilized than the corresponding free fatty acid. However, saturation transfer spectroscopy demonstrates that, in this latter case, the motion of the probe still does not reflect the rotation of the protein; thus, it is not rigidly fixed to the protein. Only when membranes are highly delipidated is it possible to liken the protein motion to the remaining hydrocarbon chain motion. However, in this latter case the apparent correlation time describing the motion is increased by more than two orders of magnitude, showing that lipid-depleted membranes cannot be used to characterize the viscosity of the boundary layer of native membranes. © 1979, American Chemical Society. All rights reserved.