Membrane location of apocytochrome c and cytochrome c determined from lipid-protein spin exchange interactions by continuous wave saturation electron spin resonance.
Apocytochrome c derived from horse heart cytochrome c was spin-labeled on the cysteine residue at position 14 or 17 in the N-terminal region of the primary sequence, and cytochrome c from yeast was spin-labeled on the single cysteine residue at sequence position 102 in the C-terminal region. The spin-labeled apocytochrome c and cytochrome c were bound to fluid bilayers composed of different negatively charged phospholipids that also contained phospholipid probes that were spin-labeled either in the headgroup or at different positions in the sn-2 acyl chain. The location of the spin-labeled cysteine residues on the lipid-bound proteins was determined relative to the spin-label positions in the different spin-labeled phospholipids by the influence of spin-spin interactions on the microwave saturation properties of the spin-label electron spin resonance spectra. The enhanced spin relaxation observed in the doubly labeled systems arises from Heisenberg spin exchange, which is determined by the accessibility of the spin-label group on the protein to that on the lipid. It is found that the labeled cysteine groups in horse heart apocytochrome c are located closest to the 14-C atom of the lipid acyl chain when the protein is bound to dimyristoyl- or dioleoyl-phosphatidylglycerol, and to that of the 5-C atom when the protein is bound to a dimyristoylphosphatidylglycerol/dimyristoylphosphatidylcholine (15:85 mol/mol mixture. On binding to dioleoylphosphatidylglycerol, the labeled cysteine residue in yeast cytochrome c is located closest to the phospholipid headgroups but possibly between the polar group region and the 5-C atom of the acyl chains. These data determine the extent to which the different regions of the proteins are able to penetrate negatively charged phospholipid bilayers.
Snel
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Max-Planck-Institut für biophysikalische Chemie, Abteilung Spektroskopie, Göttingen, Germany.
Marsh
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D000818:Animals; D001059:Apoproteins / Q000737:chemistry* / Q000378:metabolism; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism; D045304:Cytochromes c; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D006736:Horses; D007700:Kinetics; D008051:Lipid Bilayers; D008433:Mathematics; D008929:Mitochondria, Heart / Q000378:metabolism; D010743:Phospholipids; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013113:Spin Labels; D013329:Structure-Activity Relationship
D001059:Apoproteins; D003574:Cytochrome c Group; D008051:Lipid Bilayers; D010743:Phospholipids; D013113:Spin Labels; D045304:Cytochromes c