Reconstitution of Transferrin Receptor in Mixed Lipid Vesicles. An Example of the Role of Elastic and Electrostatic Forces for Protein/Lipid Assembly

We studied the interaction of transferrin receptors (of cell line Molt-4) with mixed model membranes as a function of lipid chain length (phospholipids with C14:0 and C18:1 hydrocarbon chains) and of the surface charge of the membrane using mixtures of C14:0 lecithin (DMPC) with C14:0 phosphatidylglycerol (DMPG) and C14:Q phosphatidylserine (DMPS). Spontaneous self-assembly of receptors and lipids was achieved by freeze-thaw cycles of a codispersion of mixed vesicles and receptors in buffer and subsequent separation of receptor-loaded and receptor-free vesicles by density gradient centrifugation. Information on specific lipid/protein interaction mechanisms was obtained by evaluation of protein-induced shifts of phase boundaries of lipid mixtures by calorimetry and by FTIR spectroscopy of partially deuterated lipid mixtures. The important role (1) of minimizing the elastic forces caused by the mismatch of the lengths of hydrophobic cores of the protein (l_p) and the bilayer (l_L) and (2) of the electrostatic coupling of protein head groups with the charged membrane/water interface for the lipid/protein self-assembly is established. The electrostatic interaction energy per receptor is about 10³ k_BT (by coupling to about 1000 charged lipids) which is sufficient to overcompensate the elastic energy associated with a mismatch of l_p – l_L ≈ 1.0 nm. The maximum receptor concentration incorporated was measured as a function of membrane surface charge and lipid chain length. The maximum receptor molar fraction varied from x_p^max = 5 × 10⁻⁵ for DMPC to x_p^max = 4 × 10⁻⁴ for 1:1 DMPC/DMPG; moreover x_p^max is higher for DMPS than for DMPG as charged component. For the long-chain lipids, x_p^max is higher for a 9:1 DEPE/DEPC mixture [(4.2–9) × 10⁻⁴] than for pure DEPC (ca. 3.5 × 10⁻⁴). By decomposition of reconstituted receptors with proteases, we demonstrated the homogeneous orientation of the receptor with its extracellular head group pointing to the convex side of the vesicles. Finally, binding curves of FITC-labeled Fe-transferrin to reconstituted receptors were recorded. The dissociation constant (about K_D ≈ 10⁻⁸ M) is reduced in bilayers with unmatched thickness, pointing to elastic distortion of the receptor. The binding curves of the (acidic) ligand to the charged membranes exhibit sigmoidal shape owing to electrostatic repulsion causing a ligand depletion zone on the membrane surface.