Apoptosis provides a critical mechanism to destroy damaged cells. Failures in the apoptosis pathway (e.g. p53 mutations) commonly lead to the unregulated cell growth of cancer. Recent work has identified the well studied protein, cytochrome c, (Cc) as a critical intermediate in the apoptosis pathway. When released from mitochondria, Cc binds to an apoptotic regulator protein, Apaf-1. The Cc: Apaf-1 complex activates the protease caspase-9, which, in turn, activates additional proteases and nucleases. In this way the cytochrome c: Apaf-1 interaction plays a critical role in committing the cell to apoptosis. This complex thereby provides an attractive therapeutic target for cancer (and other diseases) where apoptosis plays a critical role. We propose to carry out detailed studies of the Apaf-1: Cc interaction. Studies include binding stoichiometry, binding free energies, environmental effects and regulator effects (pH, ionic strength, dATP). Effects of point mutations on recognition and binding will be assessed using a large library of Cc variants. Finally, mass spectrometric amide hydrogen exchange experiments are proposed to localize the binding interface by which Cc recognizes Apaf-1 within the Cc: Apaf-1 complex.
