title: Reconstitution of bacteriorhodopsin from the apoprotein and retinal studied by Fourier-transform infrared spectroscopy.
abstract: The reconstitution of the retinal-containing protein bacteriorhodopsin (BR) from the apoprotein and retinal has been studied by Fourier-transform infrared (FTIR) difference spectroscopy. 9-cis-Retinal which occupies the binding site but does not reconstitute the chromophore was used as "caged retinal". Photoisomerization to the all-trans isomer triggers the reconstitution reaction. Absorption bands in the FTIR difference spectra of the educt and product of the reaction could be assigned by comparison with a 9-cis-retinal FTIR spectrum or an FT-Raman spectrum of BR and due to band shifts observed upon deuterium exchange. Specific difference bands were assigned to the protonated carboxyl groups of D96 and D115 by use of the mutants D115N and D96N. Both aspartic acids are protonated also in the apoprotein with pKa values above 10 and undergo a frequency shift toward higher wavenumbers indicating a more hydrophobic environment in the reconstituted protein. No indication was found for protonation changes of carboxyl groups or other protonatable residues when carrying out the reaction at pH values between 4 and 10. The pH-dependent protonation changes reported earlier [Fischer & Oesterhelt (1980) Biophys. J. 31, 139-146] therefore may be caused by protons in a hydrogen-bonded network. Mutations of E204, but not of D38 or E9, cancel proton uptake during reconstitution at high pH as well as proton release at low pH. It is concluded, that E204, without changing its protonation state itself, is part of a protonatable hydrogen-bonded network which changes its pKa during reconstitution thereby causing the observed protonation changes.
authors.affiliation: Max-Planck-Institut für Biochemie, Martinsried, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Rüdiger
authors.forename: J
authors.initials: J
authors.lastname: Tittor
authors.forename: K
authors.initials: K
authors.lastname: Gerwert
authors.forename: D
authors.initials: D
authors.lastname: Oesterhelt
mesh_terms: D001436:Bacteriorhodopsins / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D012172:Retinaldehyde / Q000378:metabolism*; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D001436:Bacteriorhodopsins; D012172:Retinaldehyde