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 cytochrome c with dioleoyl phosphatidylglycerol (DOPG)/dioleoyl phosphatidylcholine (DOPC) bilayer membranes over a range of lipid compositions and ionic strengths. In the absence of protein, the DOPG and DOPC lipids mix nearly ideally. The binding isotherms have been analyzed using double layer theory to account for the electrostatics, either the Van der Waals or scaled particle theory equation of state to describe the protein surface distribution, and a statistical thermodynamic formulation consistent with the mass-action law to describe the lipid distribution. Basic parameters governing the electrostatics and intrinsic binding are established from the binding to membranes composed of anionic lipid (DOPG) alone. Both the Van der Waals and scaled particle equations of state can describe the effects of protein distribution on the DOPG binding isotherms equally well, but with different values of the maximum binding stoichiometry (13 lipids/protein for Van der Waals 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 relative to DOPC for surface association with bound cytochrome c by using the 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 and 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 protein binding induced by lipid redistribution using the statistical thermodynamic theory.