Magic angle sample spinning 13C nuclear magnetic resonance of isotopically labeled bacteriorhodopsin.
Bacteriorhodopsin (bR), the light-driven proton pump protein from Halobacterium halobium, was biosynthetically labeled with [4-13C]Asp. The incorporation yield was 48%. The magic angle sample spinning (MASS) 13C nuclear magnetic resonance (NMR) spectrum of this sample revealed six different peaks superimposed on a broad band of naturally abundant peptide-bond 13C. Two of the six carbonyl signals can be attributed to internal-protonated Asp carboxyl groups, one of which might be Asp115. An additional resonance at 110 ppm can be associated with the C-11 carbon of Trp, indicating an unusual biosynthetic pathway of this amino acid in Halobacterium halobium. Similar measurements performed on papain-treated purple membrane which lacks the C-terminal tail display two new intense signals at 178 and 178.9 ppm. If the same spectrum is taken without cross-polarization, these signals do not decrease or disappear. On the basis of their intensities and their chemical shifts, one can assign in addition to the C-terminal Asp four Asp residues facing the cytoplasmic phase. In native bR, at least two of these form a salt-bridge-like bond which also might include the C-terminal tail. These experiments not only provide data about the chemical environment of the Asp residues within the hydrophobic core of bacteriorhodopsin but also yield information about the interactions between surface components.
Engelhard
M
M
Max-Planck-Institut für Ernährungsphysiologie, Dortmund, FRG.
Hess
B
B
Emeis
D
D
Metz
G
G
Kreutz
W
W
Siebert
F
F
D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001436:Bacteriorhodopsins / Q000378:metabolism*; D002247:Carbon Isotopes; D002250:Carbon Radioisotopes; D006217:Halobacterium / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D009842:Oligopeptides; D011487:Protein Conformation
D002247:Carbon Isotopes; D002250:Carbon Radioisotopes; D009842:Oligopeptides; D001224:Aspartic Acid; D001436:Bacteriorhodopsins