Elucidation of Electrostatic Determinants in Cytochrome C-Cardiolipin Binding

Abstract

Recently, there has been great interest in understanding the role of cytochrome c (Cytc) as it pertains to the intrinsic apoptotic pathway. Positively charged domains on Cytc interact with the anionic phospholipid cardiolipin. Although the exact mechanism of this interaction is still under investigation, Cytc undergoes a conformational change upon association with cardiolipin. In the presence of reactive oxygen species, these conformational changes lead to increased peroxidase activity of Cytc. Cytc is capable of oxidizing cardiolipin acyl chains leading to Cytc dissociation from the membrane, and downstream initiation of apoptosis through apoptosome formation. Using circular dichroism, fluorescence and UV-visible spectroscopy methods, we investigate the cytochrome c/cardiolipin interaction. To date, three potential cardiolipin interaction sites have been proposed on cytochrome c. Through environmental manipulation, we are able to isolate and study the cytochrome c/cardiolipin interaction at anionic binding site A. Using mutagenesis we are able to investigate the role of specific positively charged amino acids in site A during electrostatic cardiolipin binding. We utilize optimized titration techniques that generate reproducible quantitative data that can then be fit to extrapolate the binding contribution to cardiolipin for each amino acid investigated. Monitoring the binding of Cytc variants to cardiolipin vesicles allows us to investigate the role of individual amino acids in the protein-lipid interaction and elucidate the contribution of different sites on cytochrome c to cardiolipin binding.