Membrane location of spin-labeled cytochrome c determined by paramagnetic relaxation agents.
The mitochondrial protein horse heart cytochrome c was specifically spin-labeled with succinimidyl-2,2,5, 5-tetramethyl-3-pyrroline-1-oxyl-carboxylate on different lysine residues at positions 86, 87, 72, 8, or 25, respectively. Site-specifically labeled species were separated chromatographically and identified by peptide sequencing of tryptic digests. The monolabeled protein was bound to negatively charged phospholipid membranes composed of dioleoylphosphatidylglycerol, and the accessibility of the spin-labeled lysine residues to lipid-soluble molecular oxygen and to lipid-impermeant chromium maltolate was determined from the saturation properties of the ESR spectra. The accessibilities of the spin-labeled proteins relative to those obtained for phospholipids spin-labeled in the headgroup region, in the presence of unlabeled protein, identify the position of the spin-labeled lysine residues relative to the phospholipid bilayer surface. We have found that cytochrome c does not penetrate into the membrane interior and that the active side of cytochrome c in the protein-membrane interaction is the side on which lys86, lys87, and lys72 are located.
Kostrzewa
A
A
Max-Planck-Institut für biophysikalische Chemie, Abt. Spektroskopie, D-37070 Göttingen, Germany.
Páli
T
T
Froncisz
W
W
Marsh
D
D
D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D003497:Cyclic N-Oxides; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006736:Horses; D008024:Ligands; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D009942:Organometallic Compounds / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry; D010712:Phosphatidic Acids / Q000737:chemistry; D010715:Phosphatidylglycerols / Q000737:chemistry; D011753:Pyrones / Q000737:chemistry; D013113:Spin Labels
D003497:Cyclic N-Oxides; D003574:Cytochrome c Group; D008024:Ligands; D008051:Lipid Bilayers; D009942:Organometallic Compounds; D010712:Phosphatidic Acids; D010715:Phosphatidylglycerols; D011753:Pyrones; D013113:Spin Labels; C037657:dioleoylphosphatidic acid; C088206:chromium maltolate; C094286:succinimidyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylate; C051388:1,2-dioleoyl-sn-glycero-3-phosphoglycerol; D010100:Oxygen; C003959:TEMPO