Conformational switching associated with the conversion of a protein structure from a water-soluble to a membrane-inserted form is a critical step in several cellular processes; our ability to predict and manipulate such switching can be beneficial to human health. Notable examples of conformational switching include tumor targeting by the pH Low Insertion Peptide (pHLIP), cellular entry of bacterial toxins, and membrane-induced activation of the Bcl-2 family of apoptotic regulators. While these processes are of fundamental biomedical importance, the mechanism of conformational switching needs to be better understood. Specifically, the role of lipid composition and divalent cations in modulating membrane insertion remains largely unexplored, despite the mounting evidence of their physiological importance. We will test our hypothesis that changes in lipid composition, Ca2+ and Mg2+ concentrations, and pH play key roles in modulating conformational switching on membrane interfaces. In Aim 1, we will decipher the thermodynamic rules for predicting protein-membrane interactions by characterizing the protonation- and Ca2+/Mg2+-dependent bilayer partitioning of model peptides. We will use our findings to advance the existing tools for sequence-based predictions of membrane interactions and make them readily applicable to more realistic descriptions of the complex cellular environment. Specifically, we strive to predict the effects of divalent cations and the changes in pKas of titratable amino acid residues on membrane interfaces, which are critical for understanding membrane-dependent conformational rearrangements that underlie the functioning of many systems of biomedical importance. In Aim 2, we will determine the role of lipid composition in modulating the pH-dependent and Ca2+/Mg2+-dependent folding and transmembrane insertion of pHLIP. This will include (a) deciphering the coupled effect of Ca2+/Mg2+ and lipid composition on pHLIP targeting to model membranes, (b) determining the conformation of pHLIP-lipid complexes by molecular dynamics (MD) simulations and spectroscopic experiments, (c) establish the role of individual acidic residues in the conformational switching of pHLIP and (d) determining the protonation of anionic residues in membrane- inserted pHLIP by NMR spectroscopy. In Aim 3, we will test whether the molecular determinants identified in Aim 2 are sufficient to describe the behaviors of pHLIP in complex lipid systems containing de-mixed liquid domains, and in mammalian cells (i.e., we will determine the effect of pH, divalent cations, PS, and cholesterol on the interaction of pHLIP with cellular membranes).