(The FASEB Journal. 1999;13:S191-S194.)
© 1999 FASEB
Molecular structure of proton pump revealed with electron crystallography
YOSHINORI FUJIYOSHI1
Department of Biophysics, Faculty of Science, Kyoto University, Kyoto, Japan
1Correspondence: Department of Biophysics, Faculty of Science, Kyoto University, Oiwake, Kitashirakawa, Sakyo-ku, Kyoto, 606-8502, Japan
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INTRODUCTION
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BACTERIORHODOPSIN(BR) IS a light-driven proton pump found in Halobacterium
salinarium (1)
. Bacteriorhodopsin functions as an
efficient proton pump, and absorption of a single photon by the retinal
suffices to transport a proton from the inside of the cell to the
outside. Many studies have been performed on bR and revealed the
functional intermediates of the bR photocycle (2)
.
Continuing on from the pioneering work with Unwin (3)
,
Henderson et al. (4)
were able to determine an electron
crystallographic density map of bR at a resolution of 3.5 Å. Based on
this 3-dimensional map, Henderson and his co-workers built an atomic
model of the protein including the retinal molecule (4)
and deposited the atomic coordinates in the Brookhaven Protein Data
bank (PDB). They thereafter improved their atomic model by
crystallographic refinement with additional image data as well as
electron diffraction data (5)
. The atomic coordinates of
the resulting new atomic model of bR have also been registered in the
PDB (2BRD).
However, because X-ray crystallography has already achieved structure
analysis at much higher resolutions than 3.5 Å, even with membrane
proteins, we set out to analyze the structure of bR at a higher
resolution, independent of the structure analysis by Henderson et al.
Moreover, confirmation of the reliability of structure analysis by
electron crystallography is still thought to be useful for the method.
While Henderson et al. proposed a model for the proton transport
mechanism within the bR channel (4)
, we were also
interested in the mechanisms by which protons are efficiently guided to
the opening of the bR channel and by which the protons are released
from the other protein surface. Another goal of our work was to confirm
the possibility that electron crystallography could detect the
ionization state of charged amino acid residues. If the ionization
conditions could actually be detected, electron crystallography would
be a very promising technique to trace the proton movement along
essential amino acids in the bR channel. Detection of the ionization
states would tell us exactly the localization of the amino acids to
which the proton is bound in the various intermediates of the bR
photocycle. The discrimination of ionization conditions is also
important to understand the detailed relationship between structure and
function of proteins in general.
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DATA COLLECTION
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Large 2-dimensional crystals of bR were prepared for electron
cryo-microscopy by using a new trehalose embedding technique
(6)
. Electron diffraction patterns were recorded with a
slow scan CCD camera that displays a higher dynamic range than electron
microscopic film (EM-film) (7)
. While electron diffraction
patterns up to a tilt angle of 60° showed reflection spots to a
resolution of 2.5 Å, the resolution of diffraction patterns recorded
at a tilt angle of 70° was limited to ~3.0 Å.
High-resolution images were recorded mainly with a FEG electron
cryo-microscope in which the specimen was kept at a temperature of 4.2
K (8)
. Computational image analysis, which included
crystal unbending, was used to extract the Fourier components, and the
IQ values are a measure for the signal-to-noise ratio of each Fourier
component (9)
. The IQ values indicated that the phase data
extracted from our images were of high quality to a resolution of
better than 3.0 Å.
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STRUCTURE ANALYSIS
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An atomic model of bR including all surface loops was built into
the experimental 3-dimensional density map, which was calculated from
366 electron diffraction patterns and 129 electron micrographs
(10)
. The resulting R-merge and phase residual were 15.5%
and 26.7°, respectively. The resolution of 3.0 Å was only achieved
by using our electron microscope equipped with a liquid helium stage by
which the effects of radiation damage are reduced by a factor of about
2 and 10 compared with liquid nitrogen temperature and room
temperature, respectively (8)
. In practice, it is very
difficult to collect data from higher tilt angles, and thus a
cone-shaped region in the reciprocal space is not sampled. In electron
crystallography, this is known as the missing cone problem. However,
because we could collect data up to a tilt angle of 70°, the residual
missing cone was reduced to <5% of the entire Fourier space. The
reduction of the missing cone dramatically improved the quality of the
3-dimensional map, especially in the direction perpendicular to the
membrane plane. The high quality of our 3-dimensional map enabled us
not only to trace the seven transmembrane 
-helices but also to
interpret all loops in terms of the bR amino acid sequence. The loops
displayed distinct structures, such as an anti-parallel ß-sheet and
quick turns at the end of extended -helices.
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LIPID MOLECULES
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Our experimental 3-dimensional map revealed eight lipid
molecules related to one bR molecule. In the crystallographic
refinement, these lipid molecules were modeled as phosphatidyl
glycerophosphate monomethyl ester with dihydrophytol chains, which is a
major phospholipid found in purple membranes (9)
. The
arrangement of the lipid molecules with respect to the bR trimer is
similar to the results obtained by Grigorieff et al. (5)
.
Interactions between the molecules in the bR trimer are mainly formed
by hydrophobic-hydrophobic contacts of the transmembrane helices. The
bR trimer is stabilized by the interaction of the B-helix in one
molecule with helices D and E of an adjacent bR molecule in the
hydrophobic core of the bilayer. One lipid molecule between helices B
and E is only present in the cytoplasmic leaflet and has no counterpart
in the extracellular leaflet of the bilayer.
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SURFACE STRUCTURE OF BR
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Analysis of the potential map calculated from our atomic
model revealed a characteristic distribution of charged amino acids on
both surfaces of the protein. This characteristic distributions of
acidic and basic amino acid residues enabled us to propose hypothetical
mechanisms for efficient guidance of protons to the channel opening on
one side of the protein and for the efficient release of the protons
from the other side.
The program GRASP was used to visualize the structural features of the
cytoplasmic (Fig. 1a
) and the extracellular surface (Fig. 1b
) of the
bR trimer, highlighting the charge distribution. Because of the
illumination direction used to shade the structure in the figure, only
one channel entrance (indicated by an arrowhead) can be seen in the
trimer on the cytoplasmic surface. The entrance is located in a
negatively charged plane (red area) surrounded by mountain-like
protrusions that include positive charges (shown in blue). At the
boundaries of the bR molecules, especially in the inner area of the
trimer, negatively charged grooves can be observed, and the lipid
molecules are caught in the outer area of these grooves. Because the
structure analyzed by electron crystallography represents an intact
trimer, this characteristic charge distribution on the cytoplasmic
surface of the bR trimer suggests an attractive mechanism for efficient
channeling of the protons to the cytoplasmic entrance of the
transmembrane channel. In our proposed mechanism, the positively
charged protons are initially adsorbed on the negatively charged
surfaces of the lipid bilayer, inside as well as outside of the bR
trimer. Protons adsorbed on the lipid surfaces outside of the bR trimer
would migrate along the grooves formed between the individual bR
monomers into the center of the bR trimer. Protons that have initially
adsorbed to the lipid surface inside the trimer as well as those that
were guided into the center of the trimer are trapped there until they
finally enter the channel because, as shown in Fig. 1a
,
positively charged mountain-like protrusions enclose the plane in which
the channel entrance is located.
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Figure 1. Surface rendered views of (a) the cytoplasmic side and
(b) the extracellular side of the bR trimer. Blue and
red areas indicate positive and negative surface charges, respectively.
Due to the unidirectional illumination used for the calculation of the
surface representation with the program GRASP, only one opening of the
three proton channels can clearly be seen (marked by arrowhead).
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On the extracellular surface of the bR trimer, a completely different
surface structure and charge distribution is observed. The center of
the trimer is dominated by positive charges, and thus protons leaving
the negatively charged channel (red area in Fig. 1b
) tend to
be released from the surface of the bR trimer.
Thus, our electron crystallographic analysis of the intact trimer
structure of bR led to a very attractive explanation for the mechanisms
governing the efficient accumulation of protons on the cytoplasmic side
and the release of protons from the extracellular side.
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DETECTION OF THE IONIZATION STATE
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While the scattering factor of a neutral and a charged oxygen atom
are similar for X rays, they differ greatly for electrons in the low
resolution range. The electron scattering factor of a negatively
charged oxygen atom even has a negative value in the low-resolution
range. Therefore, the density of a negatively charged atom in the
simulation of a potential map appears weak or even to have a negative
contrast, while a neutral oxygen atom creates a clear positive contrast
in the potential map. However, such effects should disappear in the
potential map if the low-resolution data is omitted. This is helpful
because it allows discrimination between a negatively charged atom and
an atom that is neutral but disordered, both of which would appear as
weak densities in the map. For example, when we recalculated a map
using only data from 7 to 3 Å, the initially weak density of a
negatively charged atom became much stronger, while the density of a
neutral but disordered atom remained weak.
In our 3-dimensional map of bR including the low-resolution data, we
saw no density for Asp 85 or Asp 212; however, when we recalculated the
map using only data from 7 to 3Å, the densities for these two residues
became visible. On the other hand, the same densities for Asp 96 and
Asp 115 were observed in maps calculated with and without the
low-resolution data. By these examinations, we confirmed that electron
crystallography is capable of discriminating between charged and
uncharged Asp residues, because it was already spectroscopically shown
that Asp 85 and Asp 212 are charged while Asp 96 and Asp 115 are
uncharged (11)
. This result shows that high-resolution
electron crystallography, taking advantage of the refined electron
cryo-microscope (6)
and the improved specimen preparation
technique (6)
, can be used to detect protons in the
structure of a membrane protein.
The various intermediate states of bR during the photocycle are well
characterized (2)
and helped Henderson to propose the
model for proton translocation across the channel in bR based on the
atomic model (4)
. However, to really understand the proton
pumping mechanism in every detail, we must determine the
high-resolution 3-dimensional structure of all the intermediates of the
bR photocycle. If the electron crystallographic analysis of the
intermediates could really be achieved in practice, we would be able to
create a movie of the proton translocation. Because electron
crystallography can discriminate between the charged and uncharged
state of the key amino acid residues in the transmembrane helices of bR
based on the characteristic difference of the electron scattering
factors, we were able to follow the proton on its passage through the
bR channel. Therefore, high-resolution electron crystallography will be
able to provide us with new and exciting insights into the structure
and function of bR as well as many other membrane proteins.
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ACKNOWLEDGMENTS
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These studies were performed in collaboration with K. Mitsuoka, T.
Hirai, K. Murata, A. Miyazawa, A. Kidera, and Y. Kimura. This work was
supported by the Japan Society for the Promotion of Science
(JSPS-RFTF96L00502).
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REFERENCES
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-
Oesterhelt, D., Stoeckenius, W. (1971) Rhodopsin-like protein from the purple membrane of Halobactoterium halobium. Nature (London) New Biol. 233,149
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Lanyi, J. K. (1993) Proton translocation mechanism and energetics in the light-driven pump bacteriorhodopsin. Biochimica at Biophysica Acta 118,241
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Henderson, R., Unwin, P. N. T. (1975) Three-dimensional model of purple membrane obtained by electron microscopy. Nature (London) 257,28[Medline]
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Henderson, R., Baldwin, J. M., Ceska, T., Zemlin, F., Beckmann, E., Downing, K. H. (1990) A model for the structure of bacteriorhodopsin based on high resolution electron cryo-microscopy. J. Mol. Biol. 213,899[Medline]
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Grigorieff, N., Ceska, T. A., Downing, K. H., Baldwin, J. M., Henderson, R. (1996) Electron-crystallographic refinement of the structure of bacteriorhodopsin. J. Mol. Biol. 259,393[Medline]
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Kimura, Y., Vassylyev, D. G., Miyazawa, A., Kidera, A., Matsushima, M., Mitsuoka, K., Murata, K., Hirai, T., . Fujiyoshi (1997) Surface of bacteriorhodopsin revealed by high-resolution electron crystallography. Nature (London) 389,206[Medline]
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INTRODUCTION
DATA COLLECTION
