Multiple equilibria binding treatment of lipid and detergent interactions with membrane proteins. Application to cytochrome c oxidase solubilized in cholate.
A modified multiple binding equilibria treatment is presented that allows determination of thermodynamic parameters of the interaction of phospholipids with integral membrane proteins solubilized in excess detergent. Lipid binding is modeled as a series of exchange reactions between lipid molecules and detergent molecules at the hydrophobic protein surface. A general equation is derived which expresses a relative association constant (K) and the total number of contact sites at the lipid-protein interface (N) in terms of experimentally measurable variables. A useful simplification of the general equation occurs when the amount of detergent is high relative to the total number of lipid binding sites in the sample. Computer simulations show that in cases we have examined there appears to be an experimentally accessible range of detergent to protein molar ratios where the approximation at high detergent is useful for analyzing experimental data. This model is used to examine the competition between cholate and spin-labeled phospholipids for the hydrophobic surfaces of bovine heart cytochrome c oxidase. We find, for example, that K = 12 +/- 2 for phosphatidylcholine relative to cholate (i.e., the cholate molecules are relatively easily displaced by membrane lipids). This helps to explain the experimental observation that cholate is an effective detergent both for solubilizing cytochrome c oxidase and for reconstituting this protein into a defined lipid bilayer environment. An excess of cholate readily displaces almost all of the native phospholipids, and the protein is dispersed in cholate micelles. However, when phospholipids are added back, the cholate molecules at the protein surface are replaced because of the higher relative binding of the phospholipids. Observed differences between the behavior of phosphatidylcholine and phosphatidylglycerol suggest that reconstitution in cholate is a selective process in which detergent molecules in localized areas on the protein surface are more readily displaced by certain phospholipids.
D000818:Animals; D002417:Cattle; D019826:Cholic Acid; D002793:Cholic Acids / Q000494:pharmacology*; D003902:Detergents; D003576:Electron Transport Complex IV / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008565:Membrane Proteins / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000201:enzymology; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding; D012995:Solubility; D013367:Submitochondrial Particles / Q000201:enzymology; D013816:Thermodynamics
D002793:Cholic Acids; D003902:Detergents; D008565:Membrane Proteins; D010743:Phospholipids; D003576:Electron Transport Complex IV; D019826:Cholic Acid