Structure, Dynamics and Function of the Proton Pump Bacteriorhodopsin
229
The strongest band in Fig. 16.3 is located at 1526 cm- I and has been assigned
to the C=C double bond of retinal in the unphotolyzed state. The corresponding
bands of the photoproducts are shifted to 1549 cm- I in L, 1555 cm- I in M, 1553
cm- I in N, and 1506 cm- I in 0 but are overlapped by amide II difference bands.
The C-C single bonds of the chromophore are absorbing in the region between
1150 and 1280 cm- I . The negative band at 1639 cm- I has been attributed to the
protonated Schiff base in the unphotolyzed state of bR. It is of equal intensity in
Land M, much smaller in N and most pronounced in O. Other bands involving
the Schiff base can be found at 1302 cm- I (C-H in-plane vibration) and 1400 cm- I
(N-H in-plane). Typical for the 13-cis chromophore, they are strong in Land N.
Conformational changes of the protein backbone can be observed between
1500 and 1600 cm- I (C=N-H, amide II) and between 1600 and 1700 cm- I (C=O,
amide I). They already show up in Land M, reach maximum intensity in Nand
are mostly reversed in O.
Bands above 1700 cm- I are due to the c=o stretching vibration of protonated
carboxylic acids coupled with the in-plane bending vibration of the O-H. The
band at 1761 cm- I in M has been assigned to the primary acceptor of the Schiff
base proton, D85 (Braiman et aI., 1988; Fahmy et aI., 1992). This band shifts to
1755 cm- I in N, interpreted as a change of environment of D85. It shows the same
amplitude in the O-bR difference spectrum. Therefore, it can be concluded that
D85 is still protonated in O. D96, which is the proton donor of the Schiff base, is
protonated in the ground state and transiently deprotonated in N. The corresponding negative band in the N-bR difference spectrum is located at 1741 cm- I •
In conclusion, infrared spectroscopy is capable of monitoring the dynamics of
retinal isomerization, proton transfer as well as structural changes of the protein
backbone in one experiment. Determination of the protonation state of single
residues by crystallographic methods is very difficult. In this respect, IR spectroscopy is superior as it also delivers the dynamics of proton transfer.
2
bR Structure Obtained from 2D-Lattices
2.1
The Electron Microscopic Structure of bR and the Refinement
of this Structure by Neutron and X-Ray Diffraction
The characterization of the aggregation state of bR in the purple membrane was
given by X-ray diffraction on these natural two-dimensional lattices (Blaurock,
1975; Henderson 1975). The basic picture of the tertiary structure, describing the
three-dimensional arrangement of a-helices spanning the membrane, was given
in several papers on low dose electron microscopy (e.g., Unwin and Henderson,
1975; Henderson and Unwin, 1975). The knowledge of the amino acid sequence
(Ovchinnikov et aI., 1979) allowed the prediction of a folding model by determining seven a-helical stretches in the polypeptide chain, which were assigned to the
seven a-helices seen in the electron microscopical structure (Engelman et aI.,
1980). In the following years neutron diffraction was the method of choice to
prove this model using bR molecules containing certain perdeuterated amino
229
The strongest band in Fig. 16.3 is located at 1526 cm- I and has been assigned
to the C=C double bond of retinal in the unphotolyzed state. The corresponding
bands of the photoproducts are shifted to 1549 cm- I in L, 1555 cm- I in M, 1553
cm- I in N, and 1506 cm- I in 0 but are overlapped by amide II difference bands.
The C-C single bonds of the chromophore are absorbing in the region between
1150 and 1280 cm- I . The negative band at 1639 cm- I has been attributed to the
protonated Schiff base in the unphotolyzed state of bR. It is of equal intensity in
Land M, much smaller in N and most pronounced in O. Other bands involving
the Schiff base can be found at 1302 cm- I (C-H in-plane vibration) and 1400 cm- I
(N-H in-plane). Typical for the 13-cis chromophore, they are strong in Land N.
Conformational changes of the protein backbone can be observed between
1500 and 1600 cm- I (C=N-H, amide II) and between 1600 and 1700 cm- I (C=O,
amide I). They already show up in Land M, reach maximum intensity in Nand
are mostly reversed in O.
Bands above 1700 cm- I are due to the c=o stretching vibration of protonated
carboxylic acids coupled with the in-plane bending vibration of the O-H. The
band at 1761 cm- I in M has been assigned to the primary acceptor of the Schiff
base proton, D85 (Braiman et aI., 1988; Fahmy et aI., 1992). This band shifts to
1755 cm- I in N, interpreted as a change of environment of D85. It shows the same
amplitude in the O-bR difference spectrum. Therefore, it can be concluded that
D85 is still protonated in O. D96, which is the proton donor of the Schiff base, is
protonated in the ground state and transiently deprotonated in N. The corresponding negative band in the N-bR difference spectrum is located at 1741 cm- I •
In conclusion, infrared spectroscopy is capable of monitoring the dynamics of
retinal isomerization, proton transfer as well as structural changes of the protein
backbone in one experiment. Determination of the protonation state of single
residues by crystallographic methods is very difficult. In this respect, IR spectroscopy is superior as it also delivers the dynamics of proton transfer.
2
bR Structure Obtained from 2D-Lattices
2.1
The Electron Microscopic Structure of bR and the Refinement
of this Structure by Neutron and X-Ray Diffraction
The characterization of the aggregation state of bR in the purple membrane was
given by X-ray diffraction on these natural two-dimensional lattices (Blaurock,
1975; Henderson 1975). The basic picture of the tertiary structure, describing the
three-dimensional arrangement of a-helices spanning the membrane, was given
in several papers on low dose electron microscopy (e.g., Unwin and Henderson,
1975; Henderson and Unwin, 1975). The knowledge of the amino acid sequence
(Ovchinnikov et aI., 1979) allowed the prediction of a folding model by determining seven a-helical stretches in the polypeptide chain, which were assigned to the
seven a-helices seen in the electron microscopical structure (Engelman et aI.,
1980). In the following years neutron diffraction was the method of choice to
prove this model using bR molecules containing certain perdeuterated amino