Specific surface association of avidin with N-biotinylphosphatidylethanolamine membrane assemblies: effect on lipid phase behavior and acyl-chain dynamics.

The interaction of avidin with aqueous dispersions of N-biotinylphosphatidylethanolamines, of acyl chain lengths C(14:0), C(16:0), and C(18:0), was studied by using spin-label electron spin resonance (ESR) spectroscopy, (31)P nuclear magnetic resonance ((31)P NMR) spectroscopy, differential scanning calorimetry, and chemical binding assays. In neutral buffer containing 1 M NaCl, binding of avidin is due to specific interaction with the biotinyl lipid headgroup because avidin presaturated with biotin does not bind. Saturation binding of the protein corresponds to a ratio of 50 lipid molecules per tetrameric avidin. Phospholipid probes spin-labeled at various positions between C-4 and C-14 in the sn-2 chain were used to characterize the effects of avidin binding on the lipid chain dynamics. In the fluid phase, protein binding results in a decrease of chain mobility at all positions of labeling while the flexibility gradient characteristic of a liquid-crystalline lipid phase is maintained. There is no evidence from the spin-label ESR spectra for penetration of the protein into the hydrophobic interior of the membrane. At temperatures corresponding to the gel phase, the lipid chain mobility increases on binding protein. The near constancy in mobility found with chain position, however, suggests that in the gel phase the lipid chains remain interdigitated upon binding avidin. Binding of increasing amounts of avidin results in a gradual decrease of the lipid chain-melting transition enthalpy with only small change in the transition temperature. At saturation binding, the calorimetric enthalpy is reduced to zero. (31)P NMR spectroscopy indicates that protein binding increases the surface curvature of dispersions of all three biotin lipids. The C(14:0) biotin lipid yields isotropic (31)P NMR spectra in the presence of avidin at all temperatures between 10 and 70 degrees C, in contrast to dispersions of the lipid alone, which give lamellar spectra at low temperature that become isotropic at the chain-melting temperature. In the presence of avidin, the C(16:0) and C(18:0) biotin lipids yield primarily lamellar (31)P NMR spectra at low temperature with a small isotropic component; the intensity of the isotropic component increases with temperature, and the spectra narrow and become totally isotropic at high temperature, in contrast to dispersions of the lipids alone, which give lamellar spectra in the fluid phase. The binding of avidin therefore reduces the cooperativity of the biotin lipid packing, regulates the mobility of the lipid chains, and enhances the surface curvature of the lipid aggregates. These effects may be important for both lateral and transbilayer communication in the membrane.