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 A2, 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 (Kr). 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 A2 (PLA2), with membranes containing spin-labeled lipids is studied by EPR spectroscopy. Under noncatalytic conditions, binding of PLA2 to fluid membranes (order parameter Szz ≈ 0.24) causes the formation of a second, immobilized lipid component with Szz ≈ 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 Kr, the new theory allows the determination of the following parameters: the fraction of protein-accessible lipids (f), the membrane-binding constant of PLA2 (K), the fraction of the labeled lipids associated with a membrane-bound protein (nr), and the microscopic Gibbs free energies of protein binding of labeled (ΔGlab) and unlabeled lipids (ΔGunlab). The experimental and theoretical approaches described in this work expand the limits of characterization of protein-lipid interactions by EPR spectroscopy.

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Journal of Physical Chemistry B





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0037194968 (Scopus)

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