Behaviour of bovine phosphatidylethanolamine-binding protein with model membranes - Evidence of affinity for negatively charged membranes

The ability of phosphatidylethanolamine-binding protein (PEBP) to bind memb ranes was tested by using small and large unilamellar vesicles and monolaye rs composed Of L-alpha -1,2-dimyristoylphosphatidylcholine, L-alpha -1,2-di myristoylphosphatidylglycerol and L-alpha -1,2-dimyristoylphosphatidylethan olamine. PEBP only bound to model membranes containing L-alpha -1,2-dimyris toylphosphatidylglycerol; the interaction was primarily due to electrostati c forces between the basic protein and the acidic phospholipids. Further ex periments indicated that the interaction was not dependent on the length an d unsaturation of the phospholipid acyl chains and was not modified by the presence of cholesterol in the membrane. PEBP affinity for negatively charg ed membranes is puzzling considering the previous identification of the pro tein as a phosphatidylethanolamine-binding protein, and suggests that the a ssociation of PEBP with phospholipid membranes is driven by a mechanism oth er than its binding to solubilized phosphatidylethanolamine. An explanation was suggested by its three-dimensional structure: a small cavity at the pr otein surface has been reported to be the binding site of the polar head of phosphatidylethanolamine, while the N-terminal and C-terminal parts of PEB P, exposed at the protein surface, appear to be involved in the interaction with membranes. To test this hypothesis, we synthesized the two PEBP termi nal regions and tested them with model membranes in parallel with the whole protein. Both peptides displayed the same behaviour as whole PEBP, indicat ing that they could participate in the binding of PEBP to membranes. Our re sults strongly suggest that PEBP directly interacts with negatively charged membrane microdomains in living cells.