Cytochrome c (cyt c) is a positively charged protein (+8 at neutral pH) that interacts with the inner mitochondrial membrane, which is composed of a large fraction of negatively charged phospholipid, cardiolipin (CL) (-2 at neutral pH). The peroxidation of CL by cyt c initiates the intrinsic pathway of apoptosis. We are investigating the interaction between cyt c and CL and the cyt c-CL complex using nanodiscs (ND). ND are a discoidal phospholipid bilayer model system, for which size and shape are governed by two membrane scaffolding proteins (MSP) belt proteins. The lipid composition can be altered to represent native-like or other experimental conditions. ND preparations are more stable and consistent between preps when compared to liposomes. Fluorescent labeling of the MSP belt proteins provides an effective method to monitor cyt c-CL interactions without perturbing the formation of cyt c-CL complexes. The quenching of the emission from the fluorophore-labeled MSP belt protein by the cyt c heme group is used to measure the binding affinity of cyt c for cardiolipin.Quenching is also used to determine stoichiometry and higher order assembly of cyt c-CL complexes.<br/>

Using fluorescent-labeled nanodiscs to study lipid interactions with yeast Cytochrome C

249th National Meeting (2015)

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