Summary Apoptosis is crucial for proper development and function of cell populations in tissues, and its dys- regulation is of major relevance for degenerative diseases and cancer. The critical step in triggering apoptosis is the permeabilization of the mitochondrial outer membrane (MOMP). This process is tightly regulated by the Bcl-2 family of proteins, which is subdivided into pro-apoptotic (e.g., Bax), anti-apoptotic (e.g., Bcl-xL), and BH3-only regulator proteins (e.g., Bid). Despite recent advances in the characterization of Bcl-2 proteins, the field lacks a mechanistic understanding of the protein–protein and protein–lipid interactions that mediate MOMP. Such knowledge would be essential for setting the stage for the future development of therapeutic strategies aimed at either suppressing or activating apoptosis. The proposed project is aimed at deciphering the pathways of membrane insertion and refolding of the Bax/Bid/Bcl-xL regulatory triad and characterizing their membrane-modulated interactions within the framework of the Embedded Together model of apoptotic regulation by Bcl-2 proteins. We will draw from our experience with other membrane-inserting proteins, including the diphtheria toxin translocation domain, which has structural similarities to Bcl-2 proteins. Site-specific labeling in combination with a battery of fluorescence (including various types of steady-state and lifetime quenching and FRET) and electron paramagnetic resonance approaches (DEER, O2/NiEDDA accessibility), complemented by Molecular Dynamics computer simulations, will be utilized to obtain structural, dynamic and thermodynamic information necessary for deciphering the mechanism of physiological function. Our preliminary data indicate that conformational switching and activation Bid/Bax/Bcl-xL regulatory triad is modulated by the electrostatic and mechanical properties of the lipid bilayer. We will pursue the following specific aims: (1) Characterize membrane- dependent conformational switching in anti-apoptotic Bcl-xL, (3) Characterize lipid-dependent membrane recruitment of the Bid/Bax/Bcl-xL regulatory triad, (3) Characterize the membrane insertion pathway of pore- forming Bax and its disruption by Bcl-xL. By gaining new insights into molecular mechanisms of protein– protein and protein–lipid interactions in the Bax/Bcl-xL/Bid regulatory triad, we expect to provide a clearer map of the molecular pathways controlling MOMP. In addition, this study will reveal general principles of protein conformational switching on membranes that will inform other regulatory systems of biomedical importance.