Lipid Composition, Protonation, and Divalent Cations as Modulators of Protein-Membrane Interactions

Abstract

Every tissue, cell, and organelle have specific electrochemical properties that arise from differences in pH, ion concentration, and/or the presence of charged macromolecules. As proteins move among these different environments, changes in the electrochemical environment alter the protonation state of their side chains and, in turn, trigger functionally-relevant structural rearrangements in proteins. A Notable example of such conformational switching resulting in the transition of a soluble protein into a lipid membrane includes apoptotic regulation by the Bcl-2 protein family (e.g., Bid and Bcl-xL). Another example is the pH-Low Insertion Peptide (pHLIP), characterized by its pH-induced membrane insertion and refolding, and capable of selectively targeting tumors. We have characterized and compared the complex interplay between membrane lipid composition and divalent cations (Mg2+ and Ca2+) on the protonation-dependent membrane insertion of pHLIP and apoptotic regulators Bcl-xL and Bid. Our model studies demonstrate that the presence of 1.0 mM of Mg2+ can change the free energy of partitioning of a regulatory helical segment into a lipid bilayer by over 10 kcal/mol. Using a combination of cellular and spectroscopic techniques, we demonstrate that physiological concentrations of Mg2+ and Ca2+ dramatically change the thermodynamics of peptide/protein membrane interactions. Physiological concentrations of Mg2+ and Ca2+ presented two alternative effects on protonation events: (1) a dose-dependent shift in the pH-dependent insertion pKa to a more neutral range or (2) additional protonation-independent insertion, happening at neutral or even basic pH. Both of these effects are modulated by lipid composition. Our results highlight the importance proper lipid composition and divalent cation conditions on the study of protein membrane interaction. They also suggest that physiologically-relevant changes in lipid composition can act as cation-coupled regulatory factor in other cellular processes.