Date of Award
Master of Science (MSc)
David W. Andrews
Bid and Bax are proteins that play a key role in mitochondrial outer membrane permeabilization during apoptosis. Elucidating the molecular mechanisms regulating the function of these proteins is essential to understanding how apoptosis is altered in diseases and for developing therapies. In this thesis, a mitochondria-like supported lipid bilayer (SLB) system is established and used to characterize the membrane binding behaviour of cBid and Bax with single molecule resolution. The formation of SLBs on mica substrate was characterized and lipid diffusion in the plane of the membrane was measured by fluorescence correlation spectroscopy to be 2-6 µm2/s depending on the fluorescent probe used. The binding of cBid to lipids in supported bilayers occurred with an apparent KD of 121 µM. Combining fluorescence intensity distribution analysis and confocal imaging, cBid binding to SLBs revealed 2 populations of molecules in the membrane: monomers which primarily diffuse in the plane of the membrane and higher order oligomers which are predominantly immobile. In the presence of Bax, the distribution of cBid molecules shifted towards more mobile protein and smaller complexes, while in the presence of Bcl-XL an opposite shift towards immobile protein and larger complexes occurred. This suggests a mechanism for cBid function where the mobility of cBid in the membrane and its oligomeric state can alter its propensity to activate Bax and influence the progression of apoptosis. Bax binding to SLBs was also observed as two populations of mobile and immobile protein. Both populations comprised mostly Bax monomers. Immobile Bax oligomers up to decamers were also observed, and the formation of higher Bax oligomers appeared to be a cooperative processes once a dimer was formed. In comparison to Bax binding to liposomal membranes, its affinity for SLBs appeared to be 40-fold less with a measured KD of 381 µM.
Shivakumar, Sanjeevan, "CHARACTERIZING CBID AND BAX BINDING TO SUPPORTED LIPID BILAYERS WITH SINGLE MOLECULE RESOLUTION" (2012). Open Access Dissertations and Theses. Paper 7514.
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