236
G. BiiLDT et al.
~
purple membrane
1800 1700 1600 1500 1400 1300 1200 1100 1000
wavenumber / em - 1
Fig. 16.6. Upper spectrum: Resonance Raman microspectrum of a single BR crystal at room temperature. The high power density of the exciting laser leads to accumulation of intermediate states during
the measurement. The frequency of the strong bands at 1528 and 1563 cm'! are assigned to the C=C
stretching vibration of retinal in the ground and M state, respectively.
Middle spectrum: Light-induced FTIR difference spectrum of an ensemble of BR microcrystals. Spectra were obtained under continuous illumination with yellow light at 5°C with the light -adapted
ground state as reference. Marker bands are indicated by dotted lines and are discussed in the text.
Bottom spectrum: FTIR difference spectrum of BR within the native purple membrane. The spectrum was scaled by a factor of 0.07 to facilitate comparison with the spectrum of the microcrystals
obtained under identical conditions (middle spectrum)
Microcrystals within the crystallization matrix of the lipidic cubic phase were
used directly from the crystallization batch. Since the diameter of the probing
laser spot is about 1 flm a single crystal can be investigated. With the wavelength
of the Ar-Iaser used (488 nm) the Raman spectrum ofbR is resonance enhanced
(Fig. 16.6, top spectrum). Bands of the chromophore retinal are selectively
observable. However, the expected Raman spectrum of retinal in the ground state
of bR is overlaid by additional bands. The frequencies of those bands argue for
the presence of the M intermediate. This mixture of states is due to the high
power density of the laser. Directing the exciting laser into the lipidic phase
reveals only small (non-resonant) bands from the lysolipid mono olein (data not
shown) which was used as crystallization matrix. Yet, no vibrations from retinal
are discernable. This provides evidence that the lipidic phase is completely
devoid of bR.
G. BiiLDT et al.
~
purple membrane
1800 1700 1600 1500 1400 1300 1200 1100 1000
wavenumber / em - 1
Fig. 16.6. Upper spectrum: Resonance Raman microspectrum of a single BR crystal at room temperature. The high power density of the exciting laser leads to accumulation of intermediate states during
the measurement. The frequency of the strong bands at 1528 and 1563 cm'! are assigned to the C=C
stretching vibration of retinal in the ground and M state, respectively.
Middle spectrum: Light-induced FTIR difference spectrum of an ensemble of BR microcrystals. Spectra were obtained under continuous illumination with yellow light at 5°C with the light -adapted
ground state as reference. Marker bands are indicated by dotted lines and are discussed in the text.
Bottom spectrum: FTIR difference spectrum of BR within the native purple membrane. The spectrum was scaled by a factor of 0.07 to facilitate comparison with the spectrum of the microcrystals
obtained under identical conditions (middle spectrum)
Microcrystals within the crystallization matrix of the lipidic cubic phase were
used directly from the crystallization batch. Since the diameter of the probing
laser spot is about 1 flm a single crystal can be investigated. With the wavelength
of the Ar-Iaser used (488 nm) the Raman spectrum ofbR is resonance enhanced
(Fig. 16.6, top spectrum). Bands of the chromophore retinal are selectively
observable. However, the expected Raman spectrum of retinal in the ground state
of bR is overlaid by additional bands. The frequencies of those bands argue for
the presence of the M intermediate. This mixture of states is due to the high
power density of the laser. Directing the exciting laser into the lipidic phase
reveals only small (non-resonant) bands from the lysolipid mono olein (data not
shown) which was used as crystallization matrix. Yet, no vibrations from retinal
are discernable. This provides evidence that the lipidic phase is completely
devoid of bR.