Specific surface association of avidin with N-biotinylphosphatidylethallolamine membrane assemblies: Effect on lipid phase behavior and acyl-chain dynamics

The interaction of avidin with aqueous dispersions of N-biotinylphosphatidy lethanolamines, of acyl chain lengths C(14:0), C(16:0), and C(18:0), was st udied by using spin-label electron spin resonance (ESR) spectroscopy, P-31 nuclear magnetic resonance (P-31 NMR) spectroscopy, differential scanning c alorimetry, and chemical binding assays. In neutral buffer containing 1 M N aCl, binding of avidin is due to specific interaction with the biotinyl lip id head-roup because avidin presaturated with biotin does not bind. Saturat ion binding of the protein corresponds to a ratio of 50 lipid molecules per tetrameric avidin. Phospholipid probes spin-labeled at various positions b etween 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 labeli ng while the flexibility gradient characteristic of a liquid-crystalline li pid phase is maintained. There is no evidence from the spin-label ESR spect ra for penetration of the protein into the hydrophobic interior of the memb rane. At temperatures corresponding to the gel phase, the lipid chain mobil ity increases on binding protein. The near constancy in mobility found with chain position, however, suggests that in the gel phase the lipid chains r emain 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 saturatio n binding, the calorimetric enthalpy is reduced to zero. P-31 NMR spectrosc opy indicates that protein binding increases the surface curvature of dispe rsions of all three biotin lipids. The C(14:0) biotin lipid yields isotropi c P-31 NMR spectra in the presence of avidin at all temperatures between 10 and 70 degreesC, in contrast to dispersions of the lipid alone, which give lamellar spectra at low temperature that become isotropic at the chain-mel ting temperature. In the presence of avidin, the C(16:0) and C(18:0) biotin lipids yield primarily lamellar P-31 NMR spectra at low temperature with a small isotropic component; the intensity of the isotropic component increa ses with temperature, and the spectra narrow and become totally isotropic a t high temperature, in contrast to dispersions of the lipids alone, which g ive lamellar spectra in the fluid phase. The binding of avidin therefore re duces the cooperativity of the biotin lipid packing, regulates the mobility of the lipid chains, and enhances the surface curvature of the lipid aggre gates. These effects may be important for both lateral and transbilayer com munication in the membrane.