Protein-induced vertical lipid dislocation in a model membrane system: Spin-label relaxation studies on avidin-biotinylphosphatidylethanolamine interactions

The change in vertical location of spin-labeled N-biotinyl phosphatidyletha nolamine in fluid-phase dimyristoyl phosphatidylcholine bilayer membranes, on binding avidin to the biotinyl headgroup, has been investigated by progr essive saturation electron spin resonance measurements. Spin-labeled phosph olipids were present at a concentration of 1 mol%, relative to total membra ne lipids. For avidin-bound N-biotinyl phosphatidylethanolamine spin-labele d on the 8 C atom of the sn-2 chain, the relaxation enhancement induced by 30 mM Ni2+ ions confined to the aqueous phase was 2.5 times that induced by saturating molecular oxygen, which is preferentially concentrated in the h ydrophobic core of the membrane. For phosphatidylcholine also spin-labeled at the 8 position of the sn-2 chain, this ratio was reversed: the relaxatio n enhancement by Ni2+ ions was half that induced by molecular oxygen. In th e absence of avidin, the enhancement by either relaxant was the same for bo th spin-labeled phospholipids. For a double-labeled system, in which both N -biotinyl phosphatidylethanolamine and phosphatidylcholine were spin-labele d on the 12 C atom of the sn-2 chain, the relaxation rate in the absence of avidin was greater than that predicted from linear additivity of the corre sponding singly labeled systems, because of mutual spin-spin interactions b etween the two labeled lipid species. On binding of avidin to the N-biotiny l phosphatidylethanolamine, this relaxation enhancement by mutual spin-spin interaction was very much decreased. These results indicate that, on bindi ng of avidin to the lipid headgroup, N-biotinyl phosphatidylethanolamine is lifted vertically within the membrane, relative to the phosphatidylcholine host lipids. The specific binding of avidin to N-biotinyl phosphatidyletha nolamine parallels the liftase activity proposed for activator proteins ass ociated with the action of certain gangliosidases.