Structure, Dynamics and Function of the Proton Pump Bacteriorhodopsin
237
As demonstrated in a previous section, FTIR difference spectroscopy allows
the light-induced changes to be resolved at particular vibrations ofbR. However,
the information content goes beyond the chromophore as compared to the resonance Raman spectrum, and includes bands from the apoprotein. With regard to
the microcrystals being embedded in the lipidic cubic phase the difference
approach is even more advantageous since the strong IR-absorber mono olein
does not respond to light. Its vibrational contribution is thus cancelled. A spectrum of an ensemble of bR crystals within the lipidic cubic phase was recorded
under continuous illumination with yellow light at 5°C (Fig. 16.6, middle spectrum) and ratioed against a spectrum in the dark. The band pattern of the resulting difference spectrum is almost identical to the one obtained with purple membrane (Fig. 16.10, bottom spectrum). The frequencies of the bands indicate that
a mixture of the M with the N intermediate is accumulated under the applied
conditions. Comparison with the intermediate spectra (Fig. 16.3) allows detailed
understanding of the processes during the photo cycle; thus, the chromophore
retinal undergoes the typical isomerization reactions, as detected by the characteristic stretching modes of the C-C single bonds (at 1254, 1200 and 1167 cm- I )
and double bonds (at 1525 cm- I ) of the depleted ground state bR. Retinal bands
due to the photointermediates are observed at around 1560 and at 1186 cm- I ,
respectively. Active, vectorial proton transfer is demonstrated (see the positive
band at 1755 cm- I ) by the protonation of residue D85. The subsequent deprotonation ofD96 is in turn shown from the negative band at 1743 cm- I • Changes in secondary structure of the protein, presumed to allow efficient proton transfer, are
evident from the shift of the amide I vibration to lower frequencies resulting in
a negative difference band at 1670 cm- I and a positive band at 1650 cm- I • The high
light stability of the bR crystals allowed time-resolved FTIR spectroscopy to be
performed. The millisecond kinetics of bR in the 3D crystal tally with those
obtained in the natural 2D arrays of the purple membrane (data not shown).
Taken together, these results demonstrate that the function ofbR is not impaired
in the 3D crystals.
In conclusion, it is shown that vibrational spectroscopy is a powerful technique to assess the functionality of a membrane protein in the crystallized state.
Isomerization of the co-factor retinal and structural changes of the protein backbone are monitored. Finally, active proton transfer which is the physiological task
of bR, was detected on the single residue level. This is clear evidence that the high
resolution structure of bR presented in the next section is biologically relevant.
3.3
The Ground State Crystal Structure of bR
The crystal structure of bR was solved by molecular replacement on the basis of
the electron microscopic structure (Grigorieff et al. 1996) to 2.5 A resolution
(Pebay-Peyroula et aI., 1997). The interactions between the purple membrane like
protein layers are limited. Protein-protein contacts exist only between loops AB
(loop between helix A and helix B) and loop BC. The membrane spanning helices
are numbered from A to G going from the N- to the C-terminal end. Intertrimer
protein contacts were not found. These specific interactions between adjacent
237
As demonstrated in a previous section, FTIR difference spectroscopy allows
the light-induced changes to be resolved at particular vibrations ofbR. However,
the information content goes beyond the chromophore as compared to the resonance Raman spectrum, and includes bands from the apoprotein. With regard to
the microcrystals being embedded in the lipidic cubic phase the difference
approach is even more advantageous since the strong IR-absorber mono olein
does not respond to light. Its vibrational contribution is thus cancelled. A spectrum of an ensemble of bR crystals within the lipidic cubic phase was recorded
under continuous illumination with yellow light at 5°C (Fig. 16.6, middle spectrum) and ratioed against a spectrum in the dark. The band pattern of the resulting difference spectrum is almost identical to the one obtained with purple membrane (Fig. 16.10, bottom spectrum). The frequencies of the bands indicate that
a mixture of the M with the N intermediate is accumulated under the applied
conditions. Comparison with the intermediate spectra (Fig. 16.3) allows detailed
understanding of the processes during the photo cycle; thus, the chromophore
retinal undergoes the typical isomerization reactions, as detected by the characteristic stretching modes of the C-C single bonds (at 1254, 1200 and 1167 cm- I )
and double bonds (at 1525 cm- I ) of the depleted ground state bR. Retinal bands
due to the photointermediates are observed at around 1560 and at 1186 cm- I ,
respectively. Active, vectorial proton transfer is demonstrated (see the positive
band at 1755 cm- I ) by the protonation of residue D85. The subsequent deprotonation ofD96 is in turn shown from the negative band at 1743 cm- I • Changes in secondary structure of the protein, presumed to allow efficient proton transfer, are
evident from the shift of the amide I vibration to lower frequencies resulting in
a negative difference band at 1670 cm- I and a positive band at 1650 cm- I • The high
light stability of the bR crystals allowed time-resolved FTIR spectroscopy to be
performed. The millisecond kinetics of bR in the 3D crystal tally with those
obtained in the natural 2D arrays of the purple membrane (data not shown).
Taken together, these results demonstrate that the function ofbR is not impaired
in the 3D crystals.
In conclusion, it is shown that vibrational spectroscopy is a powerful technique to assess the functionality of a membrane protein in the crystallized state.
Isomerization of the co-factor retinal and structural changes of the protein backbone are monitored. Finally, active proton transfer which is the physiological task
of bR, was detected on the single residue level. This is clear evidence that the high
resolution structure of bR presented in the next section is biologically relevant.
3.3
The Ground State Crystal Structure of bR
The crystal structure of bR was solved by molecular replacement on the basis of
the electron microscopic structure (Grigorieff et al. 1996) to 2.5 A resolution
(Pebay-Peyroula et aI., 1997). The interactions between the purple membrane like
protein layers are limited. Protein-protein contacts exist only between loops AB
(loop between helix A and helix B) and loop BC. The membrane spanning helices
are numbered from A to G going from the N- to the C-terminal end. Intertrimer
protein contacts were not found. These specific interactions between adjacent