Structural dynamics of membrane-bound BAX oligomers: intramolecular flexibility vs population heterogeneities

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Bax is a executioner member of the Bcl-2 family that plays a key role in the regulation of apoptosis by mediating the permeabilization of the mitochondrial outer membrane (MOM). However, the molecular mechanism of MOM permeabilization remains one of the key questions in the field. For a proper understanding, knowledge about the structure of Bax involved in the process is required. Despite recent advances in the structure of the functional state of Bax for MOM permeabilization, important questions remain open. Our previous data indicate that the C-terminal piercing domain of Bax is highly dynamic and involved in protein/protein interactions within the oligomers. However, precisely this conformational flexibility has so far precluded a proper understanding of the structural organization of full length, active Bax oligomers in the membrane environment. In addition, the clamp model that we proposed requires further validation in terms of protein topology with respect to the membrane. Here we intend to go beyond of the state of the art by combining single molecule imaging and electron paramagnetic resonance in order to tackle these questions. The main strength of this proposal is the use of both optical and magnetic resonance methodologies to the same end.The main aim of this project is to understand the structural organization of active, membrane-embeded Bax oligomers with a focus on the intramolecular flexibility of the piercing domain and the conformational heterogeneities at the population level in the context of oligomeric assemblies. For this purpose, we will use a biophysical approach that combines state-of-the-art electron paramagnetic resonance techniques with single molecule FRET microscopy. We will address the following questions:A) Orientation of Bax dimers with respect to the pore and the membrane planeB) Structural organization of the piercing domain of active Bax: intramolecular dynamics vs population heterogeneities and orientation of helices within the dimerC) Photocaged cysteines as tools for specific orthogonal labeling in one protomer: towards a structural model of Bax oligomers in the membraneThe experiments planned in this proposal include approaches at the forefront of structural research of membrane proteins and will open new possibilities for their characterization in the native environment of the lipid bilayer. The structural organization of Bax during MOM permeabilization and its link to function will be key to achieve a comprehensive understanding of how the Bcl-2 proteins regulate apoptosis, which is a fundamental question in biology.