Defining the Apoptotic Trigger

Abstract

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 examd. 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 resoln. and used model systems with negligible or a pos. membrane curvature, which is distinct from the large neg. curvature of the invaginations of the inner mitochondrial membrane where cytochrome c resides. Here, the authors 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 prepd. in pentane yielded NMR spectra essentially identical to the protein in free aq. soln. The structure of encapsulated ferricytochrome c was detd. to high precision ( bb = ∼0.23 Å) using NMR-based methods and was closely similar to the cryogenic crystal structure ( bb = ∼1.2 Å). Incorporation of cardiolipin into the reverse micelle surfactant shell caused localized chem. shift perturbations of the encapsulated protein, providing the 1st view of the cardiolipin/cytochrome c interaction interface at at. resoln. Three distinct sites of interaction were 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 was not significantly disturbed by the binding of cardiolipin in the context of the reverse micelle. [on SciFinder(R)]