Abstract
The proapoptotic protein Bax is an essential component of stress-induced apoptosis. When activated, Bax binds to the mitochondrial outer membrane (MOM), dimerizes, oligomerizes, and forms a pore. Since the formation of this pore is irreversible and is the point of no return for apoptosis, recent research has focused on determining a model of Bax at the MOM and has proposed many models of membrane embedded Bax. There is no agreed upon model, however, since there is no clear data set that sets one model apart from the others. A data-confirmed model of Bax at the MOM, however, would be valuable to developing therapeutics for cancers and neurological diseases which are a result of irregular apoptosis. The goal of this research was to determine an active model of Bax dimerization at the MOM through a clear data set obtained from Double Electron Electron Resonance (DEER) spectroscopy. To do this, a T135C mutation was successfully introduced through site directed mutagenesis into the 5 helix of Bax. However, because the Bax construct lacked a stop codon, research focused on digesting the Bax gene out of the pET15b plasmid and ligating it into the pTXB1 plasmid. Failed ligations of Bax into pTXB1 led research to discover a pre-existing Bsrg1 cut site which was complimentary to the Sap1 primer used to cut Bax out of pET15b. This Bsrg1 cut site led to incomplete digestion of Bax from pET15b and meant Bax could not be successfully ligated into pTXB1 and studied with EPR. While the research outlined in this paper did not find data to determine a model of active Bax at the MOM, it did provide clear next steps for future research to do so.