Proton Transfer in Bacteriorhodopsin

DFG Forschergruppe: Retinal Protein Action


The Bacteriorhodopsin is a trans-membrane helical protein found in bacteria Halobacterium Salinarium and works as a proton pump. The retinal chromophore is bound to the protein via a Lysine sidechain and is in all-trans configuration in the ground state.The chromophore absorbs light in the ground state and isomerizes to 13-cis configuration and initiates a photocycle comprising of spectrally distinguishable intermediates designated as bR, K, L, M, N and O.


The first proton transfer step, the deprotonation of the Schiff base to Asp 85 during L to M transition, causes a large blue shift in the spectrum. Coupled to this, a proton is transferred to the extracellular surface of the protein from the so-called protein release group (PRG) located near the extracellular surface (Step 2) within the M state. The third step consists of the reprotonation of the Schiff base from Asp 96 located towards to the cytoplasmic side during M to N. In step 4, Asp 96 is re-protonated from the cytoplasmic side during the N to O transition and the retinal re-isomerizes back to all-trans configuration, however, being twisted in O state. Finally during the O to bR step, Asp 85 deprotonates to the proton release group completing the photocycle and the twist in the retinal structure is released.
At the end of the photocycle, a proton is transferred from the cytoplasmic side to the extracellular side of the protein.  (for reviews see [1], [2]). We use a hybrid Quantum Mechanics/Molecular Mechanics approach alongwith Generalized Solvent Boundary Potential(GSBP) for modelling the solvation effects to investigate the proton transfer pathways in the protein. We calculate minimum energy pathways to calculate the energy-barrier for the proton transfer reaction. The QM region is treated with an approximate SCC-DFTB[3] method while the MM part is treated with CHARMM force field[4].


1. Proton Transfer at the extracellular side of Bacteriorhodopsin:

 The last proton transfer takes place during O-to-bR step where a proton is transferred from the Asp85 to the extracellular proton release group forming the ground state thereby completing the photocycle. The FTIR studies have indicated a possibility of a transient [O] state characterized by anionic Asp85 and neutral Asp212.

Our minimum energy path analysis indicate that the proton transfer from Asp85 to Asp212 is possible and depends cruicially upon the retinal geometry and the number and orientations of active-site waters.


2.Investigation of the Proton Release Group at the extracellular side of Bacteriorhodopsin:
Concomitant with the first proton transfer from the Schiff base to a newaby aspartic acid 85 residue, a proton is released to the surface of the protein from the so-called Proton Release Group(PRG) located at the extracellular side of the protein. The identity of this group has been controversial. The original idea of Glu204 being the PRG was revised to combination of Glu204 and Glu194 forming PRG. The FTIR experiments indicated that an IR continuum absorption band present in the ground state dissapears during the formation of the M state. Protonated water clusters are known to generate such continuum bands. Based on this argument, In 2001, it was proposed the PRG is composed of protonated water cluster such as zundel ion.
We are currently simulating such water clusters with M and L states with the Infra-red(IR) analysis of the simulation to follow. We are also investigating the role of the schiff base deprotonation and orientation of Arg82 sidechain on the PRG.

3. Investigation of the Proton Transfer Pathways from the O to bR-ground state of Bacteriorhodopsin:

 The last proton transfer takes place from the O intermediate on a millisecond timescale to recover the bR ground state. This proton transfer is one of slowest during the photocycle and occurs over 11 Angstrom distance and is therefore challenging to investigate. Several features are unknown for this transfer. For example: the orientation and the role of the Arg82 sidechain or number and location of the waters.
We are currently performing minimum energy QM/MM path calculations to investigate the energy-barriers for this proton transfer reaction. We will model the effect of the solvent using the implicit solvent model called GSBP.


[1] Lanyi, J., Annu. Rev. Physiol. 2004, 66, 665.
[2] Neutze, R. et al, Biochim. Biophys. Acta 2002, 1565, 144.
[3] Elstner, M. et al, Phys. Rev. B 1998, 58, 7260.
[4] MacKerell, A.  et al, J. Phys. Chem. B 1998, 102, 3586.