Quantitative characterization of membrane binding of peripheral proteins by spin-label EPR spectroscopy

Key enzymes. involved in transmembrane signaling and lipid metabolism (e.g., protein kinase C and phospholipases A(2), C, and D) are activated by binding to cellular membranes. Elucidation of the molecular mechanisms of these peripheral membrane proteins requires detailed characterization of their interactions with membrane lipids. Previously, EPR studies on protein-membrane interactions have been analyzed using a formalism for integral membrane proteins, permitting determination of the lipid-to-protein stoichiometry (N) and the relative affinity of the labeled versus unlabeled lipids for the protein (K-r). Here, a formalism is developed that permits a comprehensive description of the membrane binding of peripheral proteins. The interaction of an interfacially activated enzyme, secretory phospholipase A(2) (PLA(2)), with membranes containing spin-labeled lipids is studied by EPR, spectroscopy. Under noncatalytic conditions, binding of PLA(2), to fluid membranes (order parameter S-zz approximate to 0.24) causes the formation of a second, immobilized lipid component with S-zz approximate to 0.80. Under catalytic conditions, a third, more mobile component is observed that is evidently generated by the lipid hydrolysis product, lysophospholipid. In addition to N and K-r, the new theory allows the determination of the following parameters: the fraction of protein-accessible lipids (f), the membrane-binding constant of PLA(2) (K), the fraction of the, labeled lipids associated with a membrane-bound protein (n(r)), and the microscopic Gibbs free energies of protein binding of labeled (DeltaG(lab)) and unlabeled lipids (DeltaG(unlab)) The experimental and theoretical approaches described in this work expand the limits of characterization of protein-lipid interactions by EPR spectroscopy.