SPIN-LABEL STUDIES OF PROTEIN-PROTEIN INTERACTIONS IN RETINAL ROD OUTER SEGMENT MEMBRANES - SATURATION TRANSFER ELECTRON-PARAMAGNETIC RESONANCE SPECTROSCOPY

Rhodopsin in disc membrane fragments from bovine retina was labeled with a maleimide spin label on hydrophilic sites of the protein, and saturation transfer electron paramagnetic resonance spectra were recorded. This technique allows the measurement of the rotational diffusion of membrane-bound proteins and consequently the detection of any restriction in the mobility. Protein-protein interactions in the disc membranes induced by various treatments of the membranes were systematically analyzed by this technique. In large-size membrane fragments, the fast rotation of rhodopsin, first observed by R. A. Cone ((1972) Nature (London), New Biol. 236, 39-43) (τ ≃ 20 μs), is found also by saturation transfer spectroscopy. A brief sonication of the membranes decreases the size of the vesicles but leaves the saturation transfer spectra unchanged, thus confirming that the motion observed is due to the rotation of the proteins and not to tumbling of the vesicles. The addition of sucrose reduces the apparent motion of the protein, while glutaraldehyde (5%) stops the rotation completely (τ > 10-3 s). Illumination for a few seconds has no effect on the saturation transfer spectra. However, prolonged illumination at 37 °C results in a progressive immobilization of the proteins. This immobilization is completed within 30 min at 37 °C but requires several hours at 20 °C. Very likely the observed phenomena reflect artificial protein aggregation. The protein-protein interactions seen in such experiments do not appear to be physiologically relevant. Progressive removal of the lipids by treatment with phos-pholipase A2, followed by several washes with bovine serum albumin, slows down the motion of the proteins. When approximately 70% of the lipids has been removed, no more motion can be detected. Relipidation by fusion with egg lecithin vesicles does not restore the initial motion. The relationship between the mobility of the proteins and the amount of lipid might reflect an average viscosity depending upon the lipid to protein ratio. However, the nonreversibility of this phenomenon suggests an increasing protein aggregation induced by delipidation. The general conclusion is that a minimum amount of phospholipid is required (a) to provide a low viscosity allowing for protein mobility and (b) to protect against protein-protein aggregation. There is no need for tight binding of phospholipids to the proteins to achieve such goals. © 1979, American Chemical Society. All rights reserved.