Rhodopsin

from Halobacterium halobium

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Bacteriorhodopsin (bR) is a proton pump located in the cytoplasmic membrane of purple bacteria. The data presented here originate from different experiments, e.g. the analysis of two- and threedimensional crystals. bR is synthesized as a protein of 262 amino acids, of which a propeptide of 13 amino acids is cleaved off. Atomic coordinates for amino acids 7 - 227 could be modelled for the membrane integrated protein.

bR is a primary transport system. Light energy (wave length 568 nm) is used to pump protons against a concentration gradient. Both the cofactor retinal and the protein change their conformation during the reaction cycle causing different absorption maxima of the complex according to its state. The intermediate steps of the reaction cycle are:

The data bank atom coordinates used here describe the ground state bR₅₆₈.

The amino acid chain of bR is folded into seven transmembrane helices, with the amino terminus located in the periplasm and the carboxy terminus in the cytoplasm .

A -

B -

C -

D -

E -

F -

NH₂ - bR - COOH

rotate to view from the periplasm to the cytoplasm

Each of the helices B, C and F contain a (phylogenetically concerved) proline , which interrupt the intrhelical hydrogen bond networks. That sets free the carbonyl oxygen of Thr46 to form a hydrogen bridge to water molecules during proton transport. Helix C is bent at the position of Pro91 which results in a tighter packing od the helices on both sides of the retinal (use the mouse to get a better view).

The structure of the protein reveals some hollow parts. These 'empty' spaces may accomodate one or more water molecules, some of which are neccessary to form hydrogen bridges to the proton to be transported. In the following the amino acids surrounding the spaces are in light blue, the positions of the water molecules are indicated by blue spheres.

click! # H₂O

I 1

II 1

III 7

IV 1

V 3-4

VI 1

VII 1

VIII 1

IX 1

X 1-2

XI 1

XII 2

The retinal is bound to Lys216 in the form of a Schiff's base . The first step of the photo cycle is the isomerization of the cofactor from the all-trans conformation to the 13-cis conformation:

This is the step to the state J₆₁₀, which within nanoseconds looses some energy to form K₅₉₀. A microsecond later L₅₅₀ is formed, from which the first proton transfer is initiated.
In the ground state of the protein both the nitrogen of the Schiff base and Asp85 are ionized

. This zwitterion is stabilized by hydrogen bonds: one water molecule forms bridges to both ionized groups; another water hydrogen bonds one carboxyl oxygen of Asp85, the other carboxyl oxygen is bridged to the hydroxylgroup of Thr 89

. The isomerization of the retinal causes a conformational change of the protein, which breaks this hydrogen bond network and more hydrogen bond down to Asp96

. Now one proton is able to change position from the Schiff base to Asp85 (M₄₁₂, 100-microsecond time scale). Experiments with amino acid replacement mutants indicate that the transport of the proton is further mediated by Tyr57, Arg82, Glu194 and Glu204

. Most probabely the proton is set free on the extracellular side of the membrane via a bound water molecule, this occurs at about the same time as the protonation of Asp85 in a chain reaction.

The next step is the reprotonation of the Schiff base, while the retinal is still in the cis-conformation (N₅₂₀). The donor is Asp96 . Asp96 is surrounded by a cage of hydrophobic amino acids preventing a direct transfer of a proton. From other investigations it is known that in this state the part of the protein facing the cytoplasm assumes a different conformation. Therefore water molecules may permeate into the channel between the helices. This enables the formation of hydrogen bridges by which a proton may be passed toward Lys216. The state O₆₄₀ is arrived by reprotonation of Asp96 and isomerisation of the retinal back to the all-trans form (facilitated by protonation of Lys216-Nz). All steric obstacles introduced by the light-driven isomerization at C₁₃ are reversed and the ground state bR₅₆₈ with the linear polyene chain of the retinal is gained after Asp85 gave off a proton to the hydrogen bond chain to the surface of the protein.

Literature:
N Grigorieff et al, Electron-crystallographic refinement of the structure of bacteriorhodopsin, J. Mol. Biol. 259 (1996) 393-421
H Luecke et al, Proton transfer pathways in bacteriorhodopsin at 2.3 Å resolution, Science 280 (1998) 1934-1937
JK Lanyi, Understanding structure and function in the light-driven proton pump bacteriorhodopsin, J. Struct. Biol. 124 (1998) 164-178

6-99 - R Bergmann