Aspartic acid-212 of bacteriorhodopsin is ionized in the M and N photocycle intermediates: an FTIR study on specifically 13C-labeled reconstituted purple membranes.
Purple membrane was regenerated from the denatured proteolytic (protease V8) fragments V-1 and V-2 of bacteriorhodopsin (BR), native membrane lipids, and all-trans-retinal. FTIR difference spectra of M and N intermediates of the reconstituted system are in close correspondence to those obtained from native BR. Asp-212 is the only internal aspartic acid in the V-2 fragment (helices F and G). Reconstituting a V-2 fragment from a [4-13C]Asp-labeled BR preparation with an unmodified V-1 fragment and vice versa have allowed us to assign IR bands to either Asp-212 or any of the remaining aspartic acids on V-1 (helices A-E). A carboxylate vibration at 1392 cm-1 has been identified in the M and N intermediates and assigned to Asp-212. Since no contribution of this residue to C = O stretches of protonated carboxyl groups was detected, Asp-212 must be ionized in light-adapted BR as well. The effect of [4-13C]Asp labeling of V-1 revealed a carboxylate vibration at 1385 cm-1 in light-adapted BR. Since Asp-96 and Asp-115 are protonated, this band is caused by Asp-85. All absorption changes of C = O stretches of protonated carboxyl groups are due to Asp residues on V-1. Correspondingly, the proton acceptor for Schiff base deprotonation in M is located on V-1, and must be Asp-85 (the only ionized Asp on V-1). The band assignments are compared with those reported for BR mutants, and the potential role of Asp-212 for proton translocation is discussed.
Institut für Biophysik und Strahlenbiologie, Albert-Ludwigs-Universität, Freiburg, FRG.
K
K
Fahmy
O
O
Weidlich
M
M
Engelhard
H
H
Sigrist
F
F
Siebert
D001224:Aspartic Acid / Q000737:chemistry*; D001436:Bacteriorhodopsins / Q000737:chemistry*; D002247:Carbon Isotopes; D005583:Fourier Analysis; D007553:Isotope Labeling; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010777:Photochemistry; D012545:Schiff Bases / Q000737:chemistry; D012697:Serine Endopeptidases / Q000378:metabolism; D013055:Spectrophotometry, Infrared
D002247:Carbon Isotopes; D010446:Peptide Fragments; D012545:Schiff Bases; D001224:Aspartic Acid; D001436:Bacteriorhodopsins; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase