2Fo-Fc electron density map of retinal at 1.55 A resolution

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.

Schematic of proton pump mechanism
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 Lys216.  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

NH+
 
 
 
twC 
NH+
 

NH+
 

N
 

NH+
 
 twT 
NH+
 

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