Bacteriorhodopsin

The protein: bacteriorhodopsin (BR) is a photon-driven ion pump. BR is a seven-helical transmembrane protein with a retinal co-factor. It is found in the bacterium H. salinarium where it converts light to a proton gradient which in turn is used by a second membrane protein called ATP synthase to generate chemical energy in the form of ATP. ATP is then used by the cell to drive a multitude of vital processes.

The method: cubic lipid phase (CLP) crystallization, developed by E. Landau & G. Rosenbusch (PNAS, 1996). The detergent-solubilized membrane protein is added to the CLP matrix where it partitions into the hydrophobic phase. Since the hydrophobic phase has bilayer topology, the protein is assumed to be a lot "happier" in it than in detergent micelles. Addition of a precipitant causes crystal nucleation & growth which is facilitated by controlled lateral diffusion in the three-dimensional bicontinuous matrix.

The crystals: purple crystals appear after a few days, usually hundreds of them in a single set-up. The crystals form small hexagonal plates about 80 um x 80 um x 15 um. They are very stable and can easily be transported in the CLP matrix. The layers in the a/b plane of the 3-dimensional BR crystals formed in CLP are very similar to the naturally occurring 2-D crystals sheets of BR in purple membranes. The space group is P6₃ with a=b=61 Å, c=108 Å and two trimers per unit cell. They diffract to 2.3 Å at the home lab, to 2.05 Å at beam line 9-1 at the Stanford Synchrotron (SSRL), and most recently to 1.40 Å (10 sigma spots) at beamline 5.0.2 at the Advanced Light Source (ALS) and to 1.33 Å at the microfocus beamline (ID13) at the European Synchrotron Research Facility (ESRF). The P6₃ CLP crystals are heavily merohedrally twinned and thus require special consideration during refinement.

The photocycle is initiated by the absorption of a photon by the all-trans retinal co-factor which is linked to the protein by a protonated Schiff base at Lys^216. Photon absorption causes rapid rearrangment of the electronic structure of the extended conjugated retinal pi system which results in trans -> cis isomerization at the C13=C14 double bond (K intermediate). This isomerization in turn reduces the proton affinity of the charged Schiff base nitrogen which loses its proton (deprotonates), while the initial acceptor group, Asp⁸⁵, protonates. This event is probably mediated via water molecule W402, to produce the M intermediate. Simultaneously with the protonation of Asp⁸⁵, a proton is released at the extracellular surface (bottom) of the protein (left panel).

Subsequently, Schiff base reprotonation takes place from the cytoplasmic side. In response to large conformational rearrangements, Asp⁹⁶, which is protonated in the ground state, passes its proton to the deprotonated Schiff base 11 Å away (N intermediate). Asp⁹⁶ is subsequently reprotonated from the cytoplasmic side (top). To complete the cycle, the retinal needs to re-isomerize to all-trans and the proton stored on Asp⁸⁵ moves via waters and Arg⁸² to reprotonate the terminal proton release group (right panel).

500fs

10ps

2us

40us

7ms

4ms

BR₅₇₀

J₆₂₅

K₆₀₀

<->

L₅₅₀

<->

M₄₁₂

<->

N₅₂₀

<->

O₆₄₀

<->

BR₅₇₀

T
NH⁺

twC
NH⁺

C
NH⁺

C
N

C
NH⁺

twT
NH⁺

T
NH⁺

T = all-trans; C = 13=14 cis, 15-anti; twC = twisted 13=14 cis; NH⁺ = protonated Schiff base; N = deprotonated Schiff base

The papers:

Proton Transfer Pathways in Bacteriorhodopsin at 2.3 Angstrom Resolution.
H Luecke, H-T Richter, JK Lanyi (1998) Science 280, 1934-1937.

Structure of Bacteriorhodopsin at 1.55 Angstrom Resolution.
H Luecke, B Schobert, H-T Richter, JP Cartailler, JK Lanyi (1999) J. Mol. Biol. 291, 899-911.

Structural Changes in Bacteriorhodopsin During Ion Transport at 2 Angstrom Resolution.
H Luecke, B Schobert, H-T Richter, JP Cartailler, JK Lanyi (1999) Science 286, 255-260.

Coupling photoisomerization of retinal to directional transport in bacteriorhodopsin.
H Luecke, B Schobert, JP Cartailler, H-T Richter, A. Rosengarth, R. Needleman, JK Lanyi (2000) J. Mol. Biol. 300, 1237-1255.

The atomic models:

1brx.pdb(released Aug 25, 1998): 2.3 Å light-adapted wild-type structure described in the 1998 Science paper.

1c3w.pdb (released Jul 31, 1999): 1.55 Å light-adapted wild-type structure described in the 1999 JMB paper.

1c8r.pdb (released Oct 12, 1999): 1.8 Å light-adapted ground-state structure of the D96N single-site mutant, described in the 1999 Science paper.

1c8s.pdb (released Oct 12, 1999): 2.0 Å late M photocycle-intermediate structure of the D96N single-site mutant, described in the 1999 Science paper.

1F50.pdb (released Aug 9, 2000): 1.7 Å light-adapted ground-state structure of the E204Q single-site mutant, described in the 2000 JMB paper.

1F4Z.pdb (released Aug 9, 2000): 1.8 Å early M photocycle-intermediate structure of the E204Q single-site mutant, described in the 2000 JMB paper.

The intermediate structures: K L early M late M N O