HISACTOPHILIN-MEDIATED BINDING OF ACTIN TO LIPID LAMELLAE - A NEUTRONREFLECTIVITY STUDY OF PROTEIN MEMBRANE COUPLING

The neutron reflectivity technique is applied to determine the adsorpt ive interaction of the 13.5-kDa actin-binding protein hisactophilin fr om Dictyostelium discoideum with lipid monolayers at a lateral pressur e of 21 mN/m less than or equal to pi less than or equal to 25 mN/m at the air-water interface, We compare binding of natural hisactophilin exhibiting a myristic acid chain membrane anchor at the N-terminus (DI C-HIS) and a fatty acid-deficient genetic product expressed in Escheri chia coli (EG-HIS). It is demonstrated that only the natural hisactoph ilin DIC-HIS is capable of mediating the strong binding of monomeric a ctin to the monolayer, where it forms a layer of about 40 Angstrom thi ckness corresponding to the average diameter of actin monomers, Monola yers composed of pure dimyristoyl phosphatidylcholine with fully deute rated hydrocarbon tails and headgroup (DMPC-d67) and 1:1 mixtures of t his lipid with chain deuterated dimyristoyl phosphatidylglycerol (DMPG -d54) are studied on subphases consisting either of fully deuterated b uffer (D2O) or of a 9:1 H2O/D2O buffer that matches the scattering len gth density of air (CMA buffer). The reflectivity data are analyzed in terms of layer models, consisting of one to three layers, depending o n the contrast of the buffer and the system, We show that both protein species bind tightly to negatively charged 1:1 DMPC-d67/DMPG-d54 mono layers, thereby forming a thin and most probably monomolecular protein layer of 12-15 Angstrom thickness, We find that the natural protein ( DIC-HIS) partially penetrates into the lipid monolayer, in contrast to chain-deficient species (EC-HIS), which forms only an adsorbed layer, The coverage of the monolayer with DIC-HIS strongly depends on the pr esence of anionic DMPG in the monolayer, At a bulk protein concentrati on of 1.5 mu g/ml, the molar ratio of bound protein to lipid is about 1:45 for the 1:1 lipid mixture but only 1:420 for the pure DMPC.