Abstract Title of dissertation: PROTEIN PERMEABILITY PATHWAYS IN THE MITOCHONDRIAL OUTER MEMBRANE DURING APOPTOSIS – MECHANISTIC INSIGHTS INTO THEIR STRUCTURE AND DYNAMICS

Title of dissertation: PROTEIN PERMEABILITY PATHWAYS IN THE MITOCHONDRIAL OUTER MEMBRANE DURING APOPTOSIS – MECHANISTIC INSIGHTS INTO THEIR STRUCTURE AND DYNAMICS Vidyaramanan Ganesan, Doctor of Philosophy, 2012 Dissertation directed by: Dr. Marco Colombini Department of Biology Apoptosis, a form of programmed cell death is a physiological process that guides the systematic removal of unwanted cells from the body. A key step in apoptosis is the irreversible release of mitochondrial intermembrane space (IMS) proteins into the cytosol by a process called Mitochondrial Outer Membrane Permeabilization (MOMP). MOMP is regulated by a class of proteins called Bcl-2 family and a sphingolipid called ceramide. The pro-apoptotic Bcl-2 proteins, especially Bax and Bak can cooperate with ceramide to form channels in mitochondria that cause protein efflux during MOMP. The ability of ceramide to form protein-permeable channels in MOM is established. Bax and ceramide enhanced MOMP synergistically. The ability of Bax to stimulate ceramide channels was investigated. It was found that the apparent affinity of Bax for a ceramide channel increases with the ceramide channel size. The results indicate that Bax binds a small ceramide channel and drives its growth until the Bax molecule finds the best fit to the channel. This interaction between Bax and a ceramide channel does not require of the presence of other Bcl-2 proteins or mitochondrion-specific factors. The structural features of ceramide were investigated for their role in channel formation. Analogs of ceramide bearing modifications in the functional groups were analyzed for their ability to form channels to assess stability and also to interact with native ceramide to form channels to assess compatibility between interacting groups. The C1-hydroxyl was found to be indispensable for channel formation while the C3-hydroxyl was inconsequential. The amide nitrogen with its ability to donate hydrogen was important for stability. Similarly, converting the carbonyl oxygen to a urea group, now more polar resulted in more stable permeabilization. Phytoceramide, which has a C4 hydroxyl instead of the C4-C5 trans double bond formed stable channels but phytoceramide inhibited channel formation by ceramide suggesting incompatibility in structure. Bax activation involves translocation of Bax from the cytosol to the MOM, conformational changes and subsequent channel formation. All steps involved in Bax activation are not well-understood. We have used ionic strength as a modulating tool to dissect the different steps in Bax mediated MOMP. Increasing the ionic strength was found to delay formation of real-time permeability by Bax. Increasing the ionic strength resulted in smaller channels that grew in size slowly. The high permeability induced by low ionic strength was not reversed by raising the ionic strength suggesting that Bax channels are not in dynamic equilibrium with Bax monomers. Ionic strength also altered the sensitivity of Bax mediated MOMP to inhibition by Bcl-xL. Ionic strength, however did not affect Bax insertion into membranes. Thus, ionic strength presents a good diagnostic tool to modulate Bax mediated channel formation downstream of Bax insertion into membranes. PROTEIN PERMEABILITY PATHWAYS IN THE MITOCHONDRIAL OUTER MEMBRANE DURING APOPTOSIS – MECHANISTIC INSIGHTS INTO THEIR STRUCTURE AND DYNAMICS By Vidyaramanan Ganesan Dissertation submitted to the faculty of the graduate school of the University of Maryland, College Park in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2012 Advisory committee: Dr. Marco Colombini, chair Dr. Heven Sze Dr. Ricardo Araneda Dr. Richard Stewart Dr. Volker Briken