Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Computational and Molecular Biophysics

Language

English (en)

Date of Award

January 2010

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Paul Schlesinger

Abstract

Bax is a pro-apoptotic member of the BCL-2 protein family and its paramount function during apoptosis is to form pores in the mitochondrial outer membrane: MOM). In a simplified apoptosis model, upon apoptotic stimulation, Bax is activated via an unresolved mechanism, translocates to the MOM, where it changes conformation, inserts into the membrane as a monomer, and finally undergoes in-membrane oligomerization to form a pore. Once the Bax pore is formed, cytochrome c and other mitochondria-resident proteins escape into the cytoplasm where they activate the cascade of caspases, which dismantle the cell. To date, the structure of the inactive cytoplasmic form of Bax is known; however, the structure of the membrane-integrated, active form of Bax remains unsolved. The absence of membrane structure for Bax leaves the mechanism of Bax pore formation poorly understood. To investigate the mechanism of Bax pore formation, we developed and used approaches to study fluorescently labeled Bax in solution and in lipid membranes using single-molecule sensitivity fluorescence techniques based on fluorescence correlation spectroscopy - FCS. These single-molecule fluorescence techniques provide us with a non-invasive method to study the mechanism of Bax pore formation reconstituted in giant unilamellar vesicles: GUVs). Our results show, that in the environment of a GUV lipid membrane, Bax can form a complex distribution of coexisting pore sizes ranging from 1 nm to 20 ┬╡m in diameter. Evidence is provided by directly examining oligomerization and mobility changes of Bax molecules in GUVs by fluorescence cross-correlation spectroscopy: FCCS) and observing the large complexes by confocal microscopy. We show that in the presence of Bcl-xL, an inhibitor of Bax pore formation, membrane-bound Bax was primarily monomeric. We also show, that in the large length-scale format of the GUV, Bax forms mega-pores - structures that reveal its affinity for highly curved membranes. Analysis of the line tension in the rim of Bax mega-pores indicates that Bax dramatically reduces line tension while stabilizing these lipidic pores. Furthermore, our results demonstrate that Bax forms pores by increasing membrane surface energy and by changing the curvature of lipid membranes, thus manifesting an ability to sculpt lipid bilayers. In addition to studying Bax in lipid membranes, we have investigated the in vitro mechanism of Bax activation in detergent micelles, where, due to the formation of large hydrodynamic volume protein-detergent micelles, Bax has been proposed to form homo-oligomers upon activation prior to membrane insertion, which is in contrast to the in vivo studies which reported that Bax binds MOM as a monomer and then undergoes homo-oligomerization. Our studies using fluorescence intensity distribution analysis: FIDA) have shown that Bax is a monomer before, during and after interaction with detergent micelles; however, it does form large hydrodynamic volume complexes with detergent micelles. The results of our FIDA study establish the connection between the in vitro and in vivo mechanism of Bax activation, showing that, prior to the formation of an oligomeric pore in a lipid membrane, Bax binds lipid membranes as a monomer.

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Permanent URL: http://dx.doi.org/10.7936/K7JD4TS3

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