| Programmed cell death, or apoptosis, is a fundamental biological mechanism necessary for the development, maintenance, and defense of multicellular organisms. The cell death process balances the cell survival pathways and is essential for homeostasis. Any shift in this balance can lead to disease states such as cancer or neurodegeneration. Therefore, understanding the molecular level details of cell death pathways is essential for understanding many human diseases.; This dissertation focuses on a key protein-protein interaction necessary for initiating cell death. Specifically, the interaction between cytochrome c and apoptotic protease activating factor-1 (apaf-1) is studied using both biochemical and biophysical techniques. Towards the study of this complex, a bacterial expression system for recombinant horse cytochrome c was developed. This system yields soluble heme-bound cytochrome c whose structure and stability is comparable to that of authentic horse cytochrome c. Furthermore, the recombinant protein was shown to be an active inducer of apoptosis in a cell-free apoptosis assay. This expression system, along with a related expression system for recombinant human cytochrome c, was used to produce cytochrome c variants to study the cytochrome c binding epitope. We show evidence for a conserved, yet expanded, cytochrome c apoptotic binding epitope.; Biophysical studies were used to understand the binding affinity and stoichiometry for this complex. Sedimentation equilibrium analysis indicated that the binding of cytochrome c to apaf-1 is tight with a submicromolar affinity. Furthermore, we also show that the binding stoichiometry is 1:1. Based on the biochemical and biophysical evidence, we propose a “clam-shell” model to explain the interaction between these two proteins. The large cytochrome c-binding domain on apaf-1 surrounds cytochrome c like a clam and binds over a large surface area. Furthermore, we show that this complex in the presence of dATP forms a large 15 S oligomeric complex in vitro which has previously been termed the apoptosome. Our results indicate that the apoptosome exists as one species and not multiple forms as previously suggested. These results also shed light on a biologically relevant protein-protein interaction. |
