TOPOLOGY OF AN AMPHIPHILIC MITOCHONDRIAL SIGNAL SEQUENCE IN THE MEMBRANE-INSERTED STATE - A SPIN-LABELING STUDY

To investigate the interaction of the presequence of the precursor of yeast cytochrome C oxidase subunit IV (COX IV) with phospholipid membr anes, a series of single- and double-cysteine-substituted peptide vari ants derived from the 25-residue NH2-terminal presequence has been syn thesized and modified with nitroxide spin labels. The immersion depth, orientation, and secondary structure of the peptide in the POPC bilay er containing 10 mol % POPG were determined using electron paramagneti c resonance (EPR) spectroscopy. EPR saturation analysis of singly labe led variants reveals that the nitroxides attached to the NH2-terminal region of the peptide insert into the acyl chain region of the bilayer , approximately 13 Angstrom deep from the membrane surface. EPR line s hape analysis of doubly labeled variants indicates that the peptide pr edominantly exists as an extended conformation, with little secondary structure. The experimental results, together with the energetic consi deration of peptide-bilayer interactions, suggest that the presequence is located near the interface between the head group region and the a cyl chain region, such that the hydrophobic side chains are solvated b y the acyl chains and the charged side chains extended toward the pola r environment at the bilayer surface.