Structure, Dynamics and Function of the Proton Pump Bacteriorhodopsin
231
the resolution region of 60 to 7 A. By comparing diffraction patterns between films
in H20 and DzO it was derived that at least 80 % of the differences resulted from
changes in the protein structure and only a minor contribution could originate
from a redistribution of water molecules. These observations indicate that changes
in the tertiary structure of bR take place during the photocycle. These results are
in contradiction to the electron diffraction experiments of Glaser et al. (1986).
2.2.2
Trapping the M-State in the Mutant Asp96Asn
It was observed by optical spectroscopy that the bR-mutant Asp96Asn is characterized by a large decrease in the decay rate of the M-state with increasing pH.
Koch et al. (1991) performed X-ray diffraction experiments on films of this
mutant under continuous illumination at room temperature and pH 9.6. They
found similar intensity changes between the bRs68 and M412 state structures
(Fig.16.4B, 100 % r.h.) as observed in the neutron diffraction measurements.
2.2.3
Time-Resolved X-Ray Diffraction Experiment on the bR Mutant Asp96Asn
In order to examine how these structural changes correlate with relaxation processes in the photo- and pumping-cycle of bR, the structural transition from the
M-state to the ground state was followed by time-resolved X-ray diffraction using
intense synchrotron radiation (Koch et al., 1991). The time course of flashinduced changes for three reflections at neutral pH is illustrated in Fig. 16.5. The
changes in individual reflections before and immediately after the light flash are
consistent both in amplitude and in direction with the steady-state experiments.
A comparison of these structural relaxation times with optical decay rates of
intermediate states in the photo cycle indicated that the observed structural
changes decay with the transition from the N state to the ground state. In functional terms this means that the structural changes relax after the reprotonation
of the Schiff's base.
2.2.4
M Splits into Two States M, and M2
So far the relaxation of tertiary structural changes was followed by time-resolved
X-ray experiments. The onset of these changes could not precisely be attributed
to photo cycle intermediates. A first hint for an answer to this problem was
obtained from X-ray diffraction experiments on bR mutant Asp96Asn (pH 9.6) at
different hydration levels (Sass et al., 1997). PM-films equilibrated at different relative humidities (15, 57, 75 and 100 % r.h.) were transformed to the M-state by
continuous illumination. Films, equilibrated at relative humidities above 60 %
showed the known changes in the tertiary structure, whereas films below 60 %
r.h. displayed only very small changes in their diffraction pictures (Fig. 16.4B).
The corresponding difference density maps show at high humidity positive difference density at helices B, F and G (Fig. 16.4C). No significant difference peaks
231
the resolution region of 60 to 7 A. By comparing diffraction patterns between films
in H20 and DzO it was derived that at least 80 % of the differences resulted from
changes in the protein structure and only a minor contribution could originate
from a redistribution of water molecules. These observations indicate that changes
in the tertiary structure of bR take place during the photocycle. These results are
in contradiction to the electron diffraction experiments of Glaser et al. (1986).
2.2.2
Trapping the M-State in the Mutant Asp96Asn
It was observed by optical spectroscopy that the bR-mutant Asp96Asn is characterized by a large decrease in the decay rate of the M-state with increasing pH.
Koch et al. (1991) performed X-ray diffraction experiments on films of this
mutant under continuous illumination at room temperature and pH 9.6. They
found similar intensity changes between the bRs68 and M412 state structures
(Fig.16.4B, 100 % r.h.) as observed in the neutron diffraction measurements.
2.2.3
Time-Resolved X-Ray Diffraction Experiment on the bR Mutant Asp96Asn
In order to examine how these structural changes correlate with relaxation processes in the photo- and pumping-cycle of bR, the structural transition from the
M-state to the ground state was followed by time-resolved X-ray diffraction using
intense synchrotron radiation (Koch et al., 1991). The time course of flashinduced changes for three reflections at neutral pH is illustrated in Fig. 16.5. The
changes in individual reflections before and immediately after the light flash are
consistent both in amplitude and in direction with the steady-state experiments.
A comparison of these structural relaxation times with optical decay rates of
intermediate states in the photo cycle indicated that the observed structural
changes decay with the transition from the N state to the ground state. In functional terms this means that the structural changes relax after the reprotonation
of the Schiff's base.
2.2.4
M Splits into Two States M, and M2
So far the relaxation of tertiary structural changes was followed by time-resolved
X-ray experiments. The onset of these changes could not precisely be attributed
to photo cycle intermediates. A first hint for an answer to this problem was
obtained from X-ray diffraction experiments on bR mutant Asp96Asn (pH 9.6) at
different hydration levels (Sass et al., 1997). PM-films equilibrated at different relative humidities (15, 57, 75 and 100 % r.h.) were transformed to the M-state by
continuous illumination. Films, equilibrated at relative humidities above 60 %
showed the known changes in the tertiary structure, whereas films below 60 %
r.h. displayed only very small changes in their diffraction pictures (Fig. 16.4B).
The corresponding difference density maps show at high humidity positive difference density at helices B, F and G (Fig. 16.4C). No significant difference peaks