234
G. BULDT et aJ.
(Fig. 16.4A). The largest differences between the differently hydrated samples
were found in the amide regions. It appears that samples at hydration levels
greater than 60 % r.h. display the structural changes in the diffraction experiment
and at the same time show in the amide I region a larger difference band at 1670
cm-' than at 1660 cm-'. On the other hand the samples which do not show the
changes in the tertiary structure (r.h. less than 60 %) display a larger difference
band at 1660 cm-' than at 1670 cm-'. These differences in the amide I region are
also found in the amide II region, where the difference band at 1556 cm-' is much
larger in the more hydrated samples.
The concept of two M states was brought up from the evaluation of timeresolved spectroscopy data in the visible wavelength region (Varo and Lanyi,
1990). An irreversible step was assumed at this position in the photo cycle
between M, and M2 acting as a switch by changing the proton accessibility of the
Schiff base from the extracellular to the cytoplasmic side and thus creating the
vectoriality of the proton pump.
With respect to the function ofbR, Thiedemann et al. (1992) were able to show
that proton pumping was only found in samples with hydration levels above 60 %
r.h. These results indicate that the observed structural changes are necessary for
proton translocation and that at least part of these changes may form the switch
which changes the accessibility to the Schiff's base.
2.2.5
Charge-Controlled Conformational Changes
Further support for this interpretation of the proton translocation mechanism
was given through the investigation on the bR mutant Asp38Arg. X-ray diffracton
experiments on samples at pH 6.7 do not show changes in the tertiary structure
of bR whereas measurements at pH 9.6 display a diffraction pattern, showing the
characteristic large structural changes (Sass et al., 1998). The interpretation of
these experiments is, that at pH 6.7 the M,-state is trapped under illumination
whereas at pH 9.6 the Mrstate is accumulated. Assuming a sequential appearence
of M, and M2 independent of pH, but with individual pH-dependent relaxation
times, it seems that also for this mutant the observed large structural changes are
necessary for vectorial proton pumping. In addition, these results give a clear
indication that the changes in the tertiary structure are driven by alterations in
the charge distribution of the protein, which follow photo isomerization.
The substitution of an aspartic acid by an arginine makes the charge pattern at
the cytoplasmic side more positive either directly by the positive charge of the
arginine or indirectly but more effectively since another positive charge is no longer compensated by the interaction with the aspartate. This new charge pattern,
more positive at the cytoplasmic side than in wt bR, could interfere with the charge
variation resulting from the deprotonation of the Schiff base and therefore slows
down the large structural rearrangements. This would result in the accumulation
of the M, state. Since no large structural changes are detectable under this condition and if one assumes a sequential order of M, and M2 the general conclusion for
wt bR can be drawn that a charge redistribution around the Schiff base results in
an altered force field within bR which drives the large structural changes.
G. BULDT et aJ.
(Fig. 16.4A). The largest differences between the differently hydrated samples
were found in the amide regions. It appears that samples at hydration levels
greater than 60 % r.h. display the structural changes in the diffraction experiment
and at the same time show in the amide I region a larger difference band at 1670
cm-' than at 1660 cm-'. On the other hand the samples which do not show the
changes in the tertiary structure (r.h. less than 60 %) display a larger difference
band at 1660 cm-' than at 1670 cm-'. These differences in the amide I region are
also found in the amide II region, where the difference band at 1556 cm-' is much
larger in the more hydrated samples.
The concept of two M states was brought up from the evaluation of timeresolved spectroscopy data in the visible wavelength region (Varo and Lanyi,
1990). An irreversible step was assumed at this position in the photo cycle
between M, and M2 acting as a switch by changing the proton accessibility of the
Schiff base from the extracellular to the cytoplasmic side and thus creating the
vectoriality of the proton pump.
With respect to the function ofbR, Thiedemann et al. (1992) were able to show
that proton pumping was only found in samples with hydration levels above 60 %
r.h. These results indicate that the observed structural changes are necessary for
proton translocation and that at least part of these changes may form the switch
which changes the accessibility to the Schiff's base.
2.2.5
Charge-Controlled Conformational Changes
Further support for this interpretation of the proton translocation mechanism
was given through the investigation on the bR mutant Asp38Arg. X-ray diffracton
experiments on samples at pH 6.7 do not show changes in the tertiary structure
of bR whereas measurements at pH 9.6 display a diffraction pattern, showing the
characteristic large structural changes (Sass et al., 1998). The interpretation of
these experiments is, that at pH 6.7 the M,-state is trapped under illumination
whereas at pH 9.6 the Mrstate is accumulated. Assuming a sequential appearence
of M, and M2 independent of pH, but with individual pH-dependent relaxation
times, it seems that also for this mutant the observed large structural changes are
necessary for vectorial proton pumping. In addition, these results give a clear
indication that the changes in the tertiary structure are driven by alterations in
the charge distribution of the protein, which follow photo isomerization.
The substitution of an aspartic acid by an arginine makes the charge pattern at
the cytoplasmic side more positive either directly by the positive charge of the
arginine or indirectly but more effectively since another positive charge is no longer compensated by the interaction with the aspartate. This new charge pattern,
more positive at the cytoplasmic side than in wt bR, could interfere with the charge
variation resulting from the deprotonation of the Schiff base and therefore slows
down the large structural rearrangements. This would result in the accumulation
of the M, state. Since no large structural changes are detectable under this condition and if one assumes a sequential order of M, and M2 the general conclusion for
wt bR can be drawn that a charge redistribution around the Schiff base results in
an altered force field within bR which drives the large structural changes.