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  Proteorhodopsin is a light-driven proton pump with variable vectoriality

Friedrich, T., Geibel, S., Kalmbach, R., Chizhov, I., Ataka, K., Heberle, J., et al. (2002). Proteorhodopsin is a light-driven proton pump with variable vectoriality. Journal of Molecular Biology (London), 321(5), 821-821. doi:10.1016/S0022-2836(02)00696-4.

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Friedrich, Thomas1, Author           
Geibel, Sven1, Author           
Kalmbach, Rolf2, Author
Chizhov, Igor2, Author
Ataka, Kenichi3, Author
Heberle, Joachim3, Author
Engelhard, Martin4, Author           
Bamberg, Ernst1, Author           
1Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society, ou_2068289              
2Max-Planck-Institute of Molecular Physiology, Department of Physical Biochemistry, 44227 Dortmund, Germany, ou_persistent22              
3Research Center Jülich, IBI-2: Structural Biology, 52425 Jülich, Germany, ou_persistent22              
4Abt. III: Physikalische Biochemie, Max Planck Institute of Molecular Physiology, Max Planck Society, ou_1753289              


Free keywords: Schiff base proton acceptor; photocurrents; photocycle; pH dependence; two-photon process
 Abstract: Proteorhodopsin, a homologue of archaeal bacteriorhodopsin (BR), belongs to a newly identified family of retinal proteins from marine bacteria, which could play an important role in the energy balance of the biosphere. We cloned the cDNA sequence of proteorhodopsin by chemical gene synthesis, expressed the protein in Escherichia coli cells, purified and reconstituted the protein in its functional active state. The photocycle characteristics were determined by time-resolved absorption and Fourier transform infrared (FT-IR) spectroscopy. The pH-dependence of the absorption spectrum indicates that the pKa of the primary acceptor of the Schiff base proton (Asp97) is 7.68. Generally, the photocycle of proteorhodopsin is similar to that of BR, although an L-like photocycle intermediate was not detectable. Whereas at pH>7 an M-like intermediate is formed upon illumination, at pH 5 no M-like intermediate could be detected. As the photocycle kinetics do not change between the acidic and alkaline state of proteorhodopsin, the only difference between these two forms is the protonation status of Asp97. This is corroborated by time-resolved FT-IR spectroscopy, which demonstrates that proton transfer from the retinal Schiff base to Asp97 is observed at alkaline pH, but the other vibrational changes are essentially pH-independent. After reconstitution into proteoliposomes, light-induced proton currents of proteorhodopsin were measured in a compound membrane system where proteoliposomes were adsorbed to planar lipid bilayers. Our results show that proteorhodopsin is a light-driven proton pump with characteristics similar to those of BR at alkaline pH. However, at acidic pH, the direction of proton pumping is inverted. Complementary experiments were carried out on proteorhodopsin expressed heterologously in Xenopus laevis oocytes under voltage clamp conditions. The following results were obtained. (1) At alkaline pH, proteorhodopsin mediates outwardly directed proton pumping like BR. (2) The direction of proton pumping can be inverted, when Asp97 is protonated. (3) The current can be inverted by changes of the polarity of the applied voltage. (4) The light intensity-dependence of the photocurrents leads to the conclusion that the alkaline form of proteorhodopsin shows efficient proton pumping after sequential excitation by two photons.


Language(s): eng - English
 Dates: 2002-07-042002-04-122002-07-042002-08-272002-08-30
 Publication Status: Published in print
 Pages: 18
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1016/S0022-2836(02)00696-4
PMID: 12206764
 Degree: -



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Title: Journal of Molecular Biology (London)
  Alternative Title : J Mol Biol
Source Genre: Journal
Publ. Info: London : Academic Press
Pages: - Volume / Issue: 321 (5) Sequence Number: - Start / End Page: 821 - 821 Identifier: n.a.: /journals/resource/954922646042