The Influence of Electric Field on the Ordering of Lipid Monolayer and Lipid-Protein Binding

This thesis is devoted to studies of the influence of an externally controlled electric potential difference on Gibbs monolayers of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), as well as its influence on the binding of the C2 domain of cytosolic phospholipase-A2 (cPLA2alpha-C2) protein to the SOPC monolayer. X-ray reflectivity, molecular dynamics (MD) simulations and electrochemical methods are used to gather microscopic and macroscopic information about the ordering, orientation, and binding configuration of the molecules on the electric potential difference or membrane potential.;Gibbs monolayers of SOPC assemble at the interface between a bulk aqueous electrolyte solution and a bulk organic electrolyte solution with 1,2-dichloroethane (DCE) as the solvent. The interfacial area per lipid for different values of electric potential difference between the aqueous and organic phases is determined from interfacial tension measurements. The area per lipid increases when the electric potential difference is greater than 0.18 V. Cyclic voltammetry confirms the result and provides evidence that the interfacial behavior of SOPC monolayers is reversible. X-ray reflectivity measurements are analyzed to determine the interfacial electron density profile, including the thicknesses of the phosphocholine (PC) head group region and the SOPC hydrocarbon chain regions of the monolayer. An increase of electron density in the layer of hydrocarbon chains with increasing electric potential difference is consistent with the penetration of DCE molecules into the hydrocarbon layer, as a result of the increase of area per lipid. The projected total length of SOPC molecules decreases with increasing electric potential difference, where the length is projected onto the direction of the electric field (i.e., the z direction perpendicular to the interface). MD simulations at fixed area per lipid show small differences in the rotation of SOPC molecules when the imposed electric field is varied. This indicates that the primary effect in the experiments is due to the changing area per lipid as a function of electric potential difference, and the subsequent re-orientation of the lipid to accommodate the change in interfacial density.;The analysis of X-ray reflectivity on cPLA2alpha-C2 domains bound to the SOPC monolayer on the water|DCE interface provides information on the angular orientation and penetration depth of the domains under external electric field. The best-fit orientations for the X-ray reflectivity curves for potential difference Delta&phis;w-o =- 0.07 V, 0.03 V, 0.13V, 0.18V are very similar. The best-fit configuration at an electric potential difference of 0.03 V is given by orientation angles theta = 84°, ϕ = 70°, and a penetration of 11.5 +/- 1.7 A. Under this orientation, the calcium binding loop CBL3 penetrates into the hydrocarbon chains of the SOPC monolayer, and the hydrophobic interaction between the chains and the hydrophobic residues is believed to be important for binding. In addition, the Ca2+ ions on the CBLs are located within 1 A of the lipid phosphate group, which provides a favorable electrostatic attraction between cPLA2alpha-C2 and lipids. This configuration generally agrees with MD simulations and EPR measurements carried out by other groups.;Under low electric potential differences (-0.07 V to 0.18 V), the electron density profile shows an increase of electron density in the lipid tail group region after protein binding. This increase is due to both protein and DCE penetration into this region. When the electric potential difference is increased to 0.38 V, the reflectivity curves change significantly. Our study of SOPC monolayers shows that the lipid density will decrease at these high potentials, thus reducing the number of binding sites for proteins. It is also likely that the highly negative charged (-7.6 at pH=7) cPLA2alpha-C2 domains are removed from the interface by the large positive electric potential difference. Future research is required to distinguish between these two possibilities.