Cytochrome c and Apoptosis

University Of North Carolina At Chapel Hill, Chapel Hill NC

Investigators

Abstract

Cytochrome c and apoptosis activating factor-1 (apaf-1) are the judge and jury of mitochondrially-mediated programmed cell death (apoptosis). Their interaction seals a cell's fate by launching a tightly controlled program that ends in cellular disintegration, and imbalances in this regulation are associated with cancer, neurological diseases, and heart disease. Given the biological and medical relevance of this protein-protein reaction, it is surprising that its molecular basis is unknown. The broad long-term goal of this project is to determine the molecular basis for the interaction between cytochrome c and apaf-1. The main hypotheses are: a) Cytochrome c uses the same cationic patch to interact with apaf-1 and its redox partners. b) Apaf-1 uses specific anionic patches to interact with cytochrome c. The specific aims are 1) Quantify apaf-1 / cytochrome c binding, 2) Identify probable binding sites on apaf-1 and cytochrome c, 3) Test the binding sites. Sedimentation equilibrium methods will be used to quantify binding, but alternative methods are also discussed. Probable binding sites on the WD repeat domain of apaf-1 have been identified in modeling studies, and the redox-partner binding site on cytochrome c has been known for many years. The sites will be tested by using site directed mutagenesis. To ensure that biologically significant data are obtained, the variants will first be assessed by using a quick and biologically relevant caspase activation assay. Once the biologically relevant residues have been identified, the biophysics of the variants will be characterized. Circular dichroism spectropolarimetry will be used to assess their integrity. Sedimentation velocity experiments on apaf-1 variants will be used to assess their effects on intraprotein interactions. Sedimentation equilibrium techniques from aim 1 will be used to quantify their effects on the free energy and stoichiometry of complex formation.