Lipid-protein multiple binding equilibriums in membranes

Publisher Website
Google Scholar
Abstract
Phospholipids at the lipid-protein interface of membrane proteins are in dynamic equilibrium with fluid bilayer. In order to express the number of binding sites (N) and the relative binding constants (K) in terms of measurable quantities, the equilibrium is formulated as an exchange reaction between lipid molecules competing for hydrophobic sites on the protein surface. Experimental data are reported on 2 integral membrane proteins, cytochrome oxidase and (Na,K)-ATPase, reconstituted into defined phospholipids. ESR measurements on reconstituted preparations of beef heart cytochrome oxidase in 1,2-dioleoyl-sn-3-phosphatidylcholine containing small quantities of the spin-labeled phospholipid 1-palmitoyl-2-(14-proxylstearoyl)-sn-3-phosphatidylcholine (PC*) gave a linear plot of bilayer/bound PC* vs. the lipid/protein ratio as predicted by the theory, with K .simeq. 1 and N = 40 (normalized to heme aa3). This demonstrates that the spin-label moiety attached to the hydrocarbon chain does not significantly perturb the binding equilibria. In the 2nd experimental system, (Na,K)-ATPase purified from rectal glands of Squalus acanthias was reconstituted with defined phosphatidylcholines as the lipid solvent and spin-labeled phospholipids with choline or serine head groups (PC*, PS*) as the solute. The (Na,K)-ATPase has a larger number of lipid binding or contact sites (N = 60-65 per .alpha.2.beta.2 dimer) and exhibits a detectably larger average binding constant for the negatively charged phosphatidylserine than for the corresponding phosphatidylcholine. These results show that a multiple equilibria, noninteracting site binding treatment can account for the behavior of lipids exchanging between the protein surface and the lipid bilayer. Selective sites among a background of nonselective sites are experimentally detectable as a change in the measured relative binding constant.