Collisions between nitrogen-14 and nitrogen-15 spin-labels. 2. Investigations on the specificity of the lipid environment of rhodopsin.

The method of spin-spin interactions between 15N and 14N spin-labels was used to investigate lipid-protein collision rates in reconstituted vesicles containing rhodopsin from bovine disk membranes and an equimolar mixture of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. In each sample, a fraction of one of the three phospholipids was labeled with 14N spin-label while a 15N spin-labeled fatty acid was covalently linked to rhodopsin. The extent of spin-spin interaction between 15N and 14N labels was either calculated by complete spectral simulation or evaluated from the line broadening as deducted from the intensity decrease of the low-field 15N line. It was found that all three spin-labeled phospholipids utilized for these experiments can interact magnetically with the spin-labeled rhodopsin. Above 35 degrees C little difference between the three species can be detected. Calculation of the diffusion constant of the phospholipids at the boundary of rhodopsin proves that the lifetime of the phospholipids at the protein boundary is short and that no long-lived annular lipids are segregated. At temperatures below approximately 30 degrees C the spectra of the samples containing spin-labeled phosphatidylserine depend upon the presence or absence of calcium. The extent of 15N line broadening was found weaker in the presence of Ca2+ than in the presence of ethylenediaminetetraacetate. Thus Ca2+ tends to exclude phosphatidylserine from the lipid environment of rhodopsin. This observation can be attributed to the formation of specific lipid domains within the membrane, induced by Ca2+.