Topography of the membrane-binding domain of cytochrome b5 in lipids by Fourier-transform infrared spectroscopy.
Fourier-transform infrared spectroscopy was used to examine the secondary structure of the membrane-binding domain (nonpolar peptide) of rabbit liver cytochrome b5 in D2O and in the presence of phospholipids and deoxycholate. In all situations, the predominant structure was alpha helix, but an examination of the components of the amide I band in the spectrum of the nonpolar peptide showed that the major peak was shifted from 1655 cm-1 in the lipids to 1650 cm-1 in deoxycholate. This shift to lower frequency, together with a decrease in intensity of the amide II band, is indicative of N-H to N-D exchange of the peptide backbone. A semiquantitative analysis indicated that the alpha helix of the peptide is over 95% exchanged in the presence of deoxycholate but is only 10% exchanged in the presence of lipid. These data suggest that the membrane-inserted portion of the peptide is alpha helical and is largely protected from N-H to N-D exchange by the bilayer. We suggest that this technique appears to provide a general method for determining the type of secondary structure involved in membrane interaction and the percentage of this structure which is involved in the interaction.
Holloway
P W
PW
Department of Biochemistry, University of Virginia School of Medicine, Charlottesville 22908.
Buchheit
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D015786:Cytochromes b5 / Q000737:chemistry / Q000378:metabolism*; D003840:Deoxycholic Acid / Q000494:pharmacology*; D003903:Deuterium; D017666:Deuterium Oxide; D005583:Fourier Analysis; D008099:Liver / Q000378:metabolism; D008566:Membranes / Q000378:metabolism; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000494:pharmacology*; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013055:Spectrophotometry, Infrared / Q000379:methods; D014867:Water
D010743:Phospholipids; D003840:Deoxycholic Acid; D014867:Water; D015786:Cytochromes b5; D003903:Deuterium; D017666:Deuterium Oxide