Defining the Apoptotic Trigger: THE INTERACTION OF CYTOCHROME c AND CARDIOLIPIN.
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The interaction between cytochrome c and the anionic lipid cardiolipin has been proposed as a primary event in the apoptotic signaling cascade. Numerous studies that have examined the interaction of cytochrome c with cardiolipin embedded in a variety of model phospholipid membranes have suggested that partial unfolding of the protein is a precursor to the apoptotic response. However, these studies lacked site resolution and used model systems with negligible or a positive membrane curvature, which is distinct from the large negative curvature of the invaginations of the inner mitochondrial membrane where cytochrome c resides. We have used reverse micelle encapsulation to mimic the potential effects of confinement on the interaction of cytochrome c with cardiolipin. Encapsulation of oxidized horse cytochrome c in 1-decanoyl-rac-glycerol/lauryldimethylamine-N-oxide/hexanol reverse micelles prepared in pentane yields NMR spectra essentially identical to the protein in free aqueous solution. The structure of encapsulated ferricytochrome c was determined to high precision (bb ∼ 0.23 Å) using NMR-based methods and is closely similar to the cryogenic crystal structure (bb ∼ 1.2 Å). Incorporation of cardiolipin into the reverse micelle surfactant shell causes localized chemical shift perturbations of the encapsulated protein, providing the first view of the cardiolipin/cytochrome c interaction interface at atomic resolution. Three distinct sites of interaction are detected: the so-called A- and L-sites, plus a previously undocumented interaction centered on residues Phe-36, Gly-37, Thr-58, Trp-59, and Lys-60. Importantly, in distinct contrast to earlier studies of this interaction, the protein is not significantly disturbed by the binding of cardiolipin in the context of the reverse micelle.
author list (cited authors)
O'Brien, E. S., Nucci, N. V., Fuglestad, B., Tommos, C., & Wand, A. J.
complete list of authors
O'Brien, Evan S||Nucci, Nathaniel V||Fuglestad, Brian||Tommos, Cecilia||Wand, A Joshua