Bacteriorhodopsin (bR) from Halobacterium salinarum is a proton pump, which converts the energy of light into a proton gradient that drives ATP synthesis. In vivo, the seven transmembrane helix protein is organised in purple patches, in which bR and lipids form crystalline two dimensional arrays. Upon absorption of a photon, the all trans retinal moiety, covalently bound to Lys216 via a Schiff base, is isomerised to a 13-cis, 15-anti configuration. This initiates a sequence of events, the photocycle, during which a proton is transferred from the Schiff base to Asp85, followed by proton release into the extracellular medium, and reprotonation from the cytoplasmic side of the membrane. The aim of these studies is to understand the structural basis for the proton transfer in bR, the simplest known photon-driven proton pump which as such provides a paradigm for the study of a basic function in bioenergetics.

Following the pioneering work carried out on the microfocus beamline ID13 [1] from crystals grown in lipidic cubic phases, the structure of bR in the ground state was extended to 1.9 Å resolution from larger and non twinned crystals [2]. It revealed eight well ordered water molecules in the extracellular half of the putative proton translocation pathway (Figure 11). The water molecules form a continuous hydrogen bond network from the Schiff base nitrogen (Lys216) to Glu194 and Glu204 and includes residues Asp85, Asp212 and Arg82. This network is involved in both proton translocation occurring during the photocycle as well as in stabilising the structure of the ground state. Nine lipid phytanyl moieties could also be modelled into the electron density maps. From all these elements and further analysis, the atomic resolution structure of bR, lipid and water molecules in the lipidic cubic phase grown crystals could be shown to represent the functional entity of the protein in the purple membrane of the bacteria [2].

The ground state structure of bR is however not sufficient to explain the entire mechanism of the proton translocation. In order to elucidate the initial structural changes coupled to the proton pumping mechanism, an early intermediate of the photocycle was trapped at low temperature within wild type bR crystals and diffraction data measured to 2.1 Å resolution [3]. The experiment was carried out on ID14/3.

The difference Fourier map between this new photoexcited state (Fexc) and the ground state (Fground) described above shows clear movements of the protein, all located in the vicinity of the chromophore (Figure 12). Changes in the orientation of the retinal moiety displace the key water molecule (W402 in Figure 11) which was bridging the Schiff base to the Asp85 in the ground state structure, enabling this latter residue to move towards the retinal. Displacement of main chain Lys216 locally disrupts the hydrogen bonding network of helix G, facilitating structural changes in later stages of the photocycle. Those results give some insights into how the proton is transferred from the Schiff base to the Asp85 and reveal the deformations of the protein related to the photocyle initiation [3].

In order to understand the complete proton transfer mechanism, the structural modifications during the further steps of the photocycle of bR will be studied.

References
[1] E. Pebay-Peyroula, G. Rummel, J.P. Rosenbusch, E.M. Landau, Science, 277, 1676-1681 (1997).
[2] H. Belrhali, P. Nollert, A. Royant, C. Menzel, J.P. Rosenbusch, E.M. Landau, E. Pebay-Peyroula, Structure, 7, 909-917 (1999).
[3] K. Edman, P. Nollert, A. Royant, H. Belrhali, E. Pebay-Peyroula, J. Hajdu, R. Neutze, E.M. Landau, Nature, 401, 822-826 (1999).

Authors
H. Belrhali (a), P. Nollert (b,c), A. Royant (a,d), C. Menzel (e), J.P. Rosenbusch (b), E.M. Landau (b), E. Pebay-Peyroula (d,f) , K. Edman (g), J. Hajdu (g), R. Neutze (g).

(a) ESRF
(b) Biozentrum, University of Basel (Switzerland)
(c) Current address: Department of Biochemistry and Biophysics, UCSF, (USA)
(d) Université Joseph Fourier, Grenoble (France)
(e) University of Münster (Germany)
(f) Institut de Biologie Structurale, CEA-CNRS, Grenoble (France)
(g) Department of Biochemistry, Uppsala University (Sweden)