Binding of peripheral proteins to mixed lipid membranes: Effect of lipid demixing upon binding

Binding isotherms have been determined for the association of horse heart c ytochrome c with dioleoyl phosphatidylglycerol (DOPG)/dioleoyl phosphatidyl choline (DOPC) bilayer membranes over a range of lipid compositions and ion ic strengths. In the absence of protein, the DOPG and DOPC lipids mix nearl y ideally. The binding isotherms have been analyzed using double layer theo ry to account for the electrostatics, either the Van der Waals or scaled pa rticle theory equation of state to describe the protein surface distributio n, and a statistical thermodynamic formulation consistent with the mass-act ion law to describe the lipid distribution. Basic parameters governing the electrostatics and intrinsic binding are established from the binding to me mbranes composed of anionic lipid (DOPG) alone. Both the Van der Waals and scaled particle equations of state can describe the effects of protein dist ribution on the DOPG binding isotherms equally well, but with different val ues of the maximum binding stoichiometry (13 lipids/protein for Van der Waa ls and 8 lipids/protein for scaled particle theory). With these parameters set, it is then possible to derive the association constant, K-r, of DOPG r elative to DOPC for surface association with bound cytochrome c by using th e binding isotherms obtained with the mixed lipid membranes. A value of K-r (DOPG:DOPC) = 3.3-4.8, depending on the lipid stoichiometry, is determined that consistently describes the binding at different lipid compositions an d different ionic strengths. Using the Value of K-r obtained it is possible to derive the average in-plane lipid distribution and the enhancement in p rotein binding induced by lipid redistribution using the statistical thermo dynamic theory.