Membrane structure of voltage-gated channel forming peptides by site-directed spin-labeling.
Three spin-labeled derivatives of the voltage-gated peptide alamethicin were prepared with nitroxides at the C-terminal phenyalaninol, and at positions 9 and 15 in the amino acid sequence. In addition, three spin-labeled derivatives of an analog of alamethicin where alpha-methylalanine residues are replaced by leucine were prepared with nitroxide labels at the same positions. Continuous wave power saturation EPR spectroscopy was used to examine the effect of molecular oxygen and water soluble paramagnetic reagents on the saturation behavior of the labeled peptides. Using the gradients of these species which exist through the membrane-solution interface, distances for these nitroxide derivatives from the membrane-solution interface were estimated. The distances show that alamethicin is inserted along the bilayer normal with the C-terminus of the peptide lying in the aqueous solution 3 or 4 A from the membrane interface. In this configuration alamethicin does not completely cross the bilayer, and the N-terminus of alamethicin is within the membrane hydrocarbon approximately 16 A from the phosphate groups on the opposing interface. The analog where leucines replace alpha-methylalanines shows a similar conformation, except that the entire peptide is translated 3-4 A deeper into the membrane than is native alamethicin. The distances that are measured for alamethicin using EPR are consistent with a linear high resolution NMR structure determined in SDS and the X-ray crystal structure. The membrane position and structure of alamethicin found here limit the likely models for voltage-gating of this peptide.
Barranger-Mathys
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M
Department of Chemistry, University of Virginia, Charlottesville 22901, USA.
Cafiso
D S
DS
D000408:Alamethicin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry*; D000595:Amino Acid Sequence; D004578:Electron Spin Resonance Spectroscopy; D015640:Ion Channel Gating; D007473:Ion Channels / Q000737:chemistry; D008081:Liposomes; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000138:chemical synthesis / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D011487:Protein Conformation; D013113:Spin Labels
D007473:Ion Channels; D008081:Liposomes; D008565:Membrane Proteins; D010455:Peptides; D013113:Spin Labels; D000408:Alamethicin