Bacteriorhodopsin, Molecular Biology of
577
1
Introduction
Bacteriorhodopsin is a small (26 kDa) in-
tegral membrane protein, the prototype of
seven-helical G-protein-linked receptors.
It is found in extended two-dimensional
hexagonal arrays in the cytoplasmic mem-
brane of halobacteria, the ‘‘purple mem-
brane’’ patches. Upon illumination, this
retinal-containing protein transports pro-
tons against a transmembrane gradient.
Photoisomerization of the retinal from all-
trans to 13-cis,15-anti sets off a sequence
of thermal reactions, the ‘‘photocycle,’’ in
which changes of the retinal cause a cy-
cle of structural changes in the protein,
and these result in vectorial proton trans-
fers between donor and acceptor groups.
Together, the internal transfers add up to
full translocation of a proton from the
cytoplasmic to the extracellular side of
the protein and thus generate a trans-
membrane electrochemical gradient for
protons. This light-driven gradient is uti-
lized for chemiosmotic coupling, in the
same way as in other membranes in which
it is generated by redox reactions, to the
synthesis of ATP, the uptake of K
+
and
nutrients (amino acids), and the transport
of Na
+
out of the cells.
2
Overall Structure of the Protein
Bacteriorhodopsin forms trimers, which
assemble in the two-dimensional hexago-
nal lattice of the purple membrane. This
specialized membrane contains only bac-
teriorhodopsin and speciFc lipids (about
10 lipids/protein), and its regular crys-
talline lattice made it possible to determine
the structure of the protein by cryoelectron
microscopy of single sheets, ultimately to
3
˚
A resolution. Three-dimensional crystals
can be grown from detergent-solubilized
bacteriorhodopsin in cubic lipid phase,
and from X-ray diffraction the structure of
the protein has been described at increas-
ingly better resolutions, most recently to
1.47
˚
A. ±rom these crystallographic stud-
ies, the protein is known to consist of
seven transmembrane helices with short
interhelical loops and short N and C ter-
mini. Three of the helices, B, C, and D,
are normal to the plane of the membrane
and four, A, E, ±, and G, are inclined at
various but small angles to the perpendicu-
lar. The single retinal per protein molecule
is bound to the
ε
-amino group of Lys216
forming a protonated Schiff base near the
m
idd
leofhe
l
ixG
,wh
i
leitspo
lyenecha
in
lies at a small angle from the membrane
plane (±ig. 1).
The Schiff base divides the seven-helical
bundle into extracellular and cytoplasmic
halves. The trajectory of the transported
proton is through the two ‘‘half-channels’’
formed by these regions. IdentiFcation of
the residues and bound water that par-
ticipate in these half-channels has been
the objective of much work in the last
few decades. Their locations and how
they interact are known from crystallogra-
phy, infrared spectroscopy, and solid-state
nuclear magnetic resonance (NMR). The
extracellular half-channel contains numer-
ous polar and hydrogen-bonding residues
and a three-dimensional network of tightly
bound
water.
Mutational
studies
have
shown that the polar side chains play roles
in the release of protons to the extracellular
surface. The anionic Asp85 and Asp212 are
hydrogen-bonded to the centrally located
water402 that receives a hydrogen bond
from the protonated retinal Schiff base.
The Asp85/Asp212/water402 complex is
the counterion to the charge of the Schiff
base, but in the photocycle it is Asp85
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