Bacteriorhodopsin & other retinal proteins

Nature efficiently converts light into a chemical energy source useful for the cell. The first step is to harvest photons so as to pump protons across a cell membrane, and the energy thus stored in the transmembrane proton gradient is harvested by ATPsynthase to regenerate ATP. In energy transducing bacterial rhodopsins, such as bacteriorhodopsin and proteorhodopsin, light stimulation leads protons to be pumped directly through the cell membrane. Directional proton transport is achieved by a sequence of proton exchanges events that pass a proton between key residues forming a proton translocation channel spanning the protein.

Upon the absorption of a photon by bacteriorhodopsin, a buried all-trans retinal is isomerised to its 13-cis configuration. This event induces structural changes in the protein that lead to proton pumping. The key groups involved are the protonated Schiff base – which forms a covalent bond connecting the retinal to Lys216 – and Asp85 – which accepts a proton from the Schiff base. Other charged groups – such as aspartate, glutamate and arginine residues, also participate in proton transport but these are not as well conserved as Asp85 across the family of proton pumping rhodopsins and are therefore less central to the core mechanism of proton pumping.

Difference Fourier map revealing green light induced structural changes in bacteriorhodopsin at 100 K. The key event is that Wat402, which connected the Schiff base to Asp85 in the resting structure, becomes dislodged by retinal isomerisation1.

 

Our first intermediate trapping studies used green-light to illuminate crystals of bacteriorhodopsin cooled to 100 K. This work showed that a key water molecule (Wat402) located between the Schiff base and Asp85 is dislodged by light induced retinal isomerisation.1 We then performed similar experiments using green2 and red light3 at 170 K and 150 K respectively. These studies showed that the disruption of Wat402 observed at 100 K triggers further structural changes on the extracellular side of the protein at higher temperature, including an inwards flex of the extracellular portion helix C. From these and other intermediate trapping studies we proposed a structural mechanism for proton pumping by bacteriorhodopsin that unified a great deal of structural and spectroscopic information.4 These findings emphasize the important role played by buried water molecules in triggering protein conformational changes and ensuring that proton pumping occurs only in the correct (up-hill) direction.

Schematic illustration of the mechanism of proton pumping by bacteriorhodopsin to emerge from intermediate trapping studies of protein structural changes in bacteriorhodopsin.4


We later studied conformational changes in bacteriorhodopsin using time resolved wide angle scattering5 (TR-WAXS) – which is an emerging room-temperature structural method that records protein structural changes in solution. Although considerably less details than x-diffraction studies, TR-WAXS has the key advantage that the probed structural changes occur in real-time at room-temperature and are not hindered by crystal contacts. The major findings of our TR-WAXS studies5 were consistent with earlier conclusions based upon intermediate trapping studies.4

We have applied all these biophysical methods: x-ray crystallography; intermediate trapping; and TR-WAXS, to study other bacterial rhodopsins such as proteorhodopsin5-7 and sensory rhodopsin II.8,9 In related work we collaborated in characterising the biological function of proteorhodopsin in oceanic bacteria.10,11


References:

1              Edman, K. et al. High-resolution X-ray structure of an early intermediate in the bacteriorhodopsin photocycle. Nature 401, 822-826 (1999).

2              Royant, A. et al. Helix deformation is coupled to vectorial proton transport in the photocycle of bacteriorhodopsin. Nature 406, 645-648 (2000).

3              Edman, K. et al. Deformation of helix C in the low temperature L-intermediate of bacteriorhodopsin. J Biol Chem 279, 2147-2158 (2004).

4              Neutze, R. et al. Bacteriorhodopsin: a high-resolution structural view of vectorial proton transport. Biochim Biophys Acta 1565, 144-167 (2002).

5              Andersson, M. et al. Structural dynamics of light-driven proton pumps. Structure 17, 1265-1275 (2009).

6              Westenhoff, S. et al. Rapid readout detector captures protein time-resolved WAXS. Nature Methods 7, 775-776 (2010).

7              Malmerberg, E. et al. Time-resolved WAXS reveals accelerated conformational changes in iodoretinal-substituted proteorhodopsin. Biophys J 101, 1345-1353 (2011).

8              Royant, A. et al. X-ray structure of sensory rhodopsin II at 2.1-A resolution. Proc Natl Acad Sci USA 98, 10131-10136 (2001).

9              Edman, K. et al. Early structural rearrangements in the photocycle of an integral membrane sensory receptor. Structure 10, 473-482 (2002).

10           Gómez-Consarnau, L. et al. Light stimulates growth of proteorhodopsin-containing marine Flavobacteria. Nature 445, 210-213 (2007).

11           Gomez-Consarnau, L. et al. Proteorhodopsin phototrophy promotes survival of marine bacteria during starvation. PLoS Biol 8, e1000358 (2010).