238
G. BULDT et al.
protein layers result in markedly different conformations in loops AB and Be in
comparison to the EM structure. After improving the crystallization conditions,
larger crystals became available showing a high degree of merohedral twinning
which had to be corrected for (Luecke et aI., 1998). Meanwhile two additional
crystal structures of bR were published resulting from different crystallization
strategies (Essen et aI., 1998, Takeda et aI., 1998). The structure of Essen et al.
nicely shows structural features of how lipids in the protein boundary interact
with amino acid side chains. The most important new features of all these structures are that the conformations of functionally important amino acid side
chains become more and more reliable and that several water molecules were
localized in the proton translocation channel. However, also these structures do
not give a satisfactory answer how a proton is translocated. At least more water
molecules have to be identified in the channel before a more probable model can
be presented. This situation already demonstrates that only the high resolution
structures of a few important intermediates like the L, Ml, M2 and N state will
give the desired information for modelling a more probable mechanism for proton translocation through bR.
4
Summary
bR is an excellent example of the mosaic like picture in the contributions of different methods for the understanding of proton pumping. The following methods were especially fruitful in the case of bR:
- EM and X-ray crystal structure analysis to obtain a structural basis for the
mechanism.
- Time-resolved UV-Vis spectroscopy to establish the photo cycle of bR with
respect to the identification of intermediates and their rise and decay times.
- Time-resolved and steady state FTIR to localize proton transfer steps to certain
amino acids and to characterize intermediate states of the protein and the
chromophore.
- Site-directed mutagenesis to select functionally important amino acids to
assign FTIR difference bands to certain amino acids and to manipulate the
kinetics of the photo cycle for a better analysis of various intermediate states.
References
Althaus T, Eisfeld W, Lohrmann R, Stockburger M (1995) Application of Raman Spectroscopy to Retinal Proteins. Israel J. Chern. 35:227-252.
Blaurock A E (1975) Bacteriorhodopsin: A Trans-membrane Pump Containing a-helix. J. Mol. BioI.
93, 139-158
Braiman M S, Mogi T, Marti T, Stern L J, Khorana H G, Rothschild K J (1988) Vibrational spectroscopy
of bacteriorhodopsin mutants: light-driven proton transport involves protonation changes of
aspartic acid residues 85, 96, and 212. Biochemistry 27:8516-8520.
Biildt G, Konno K, Nakanishi K, Pl6hn H J, Rao B N, Dencher N A (1991) Heavy-atom labelled retinal
analogues located in bacteriorhodopsin by X-ray diffraction. Photo chern. Photobiol. 54, 873-879
Dencher N A , Dresselhaus D, Zaccai G, Biildt,G (1989) Structural changes in bacteriorhodopsin during proton translocation revealed by neutron diffraction. Proc. Nat!. Acad. Sci. USA 86, 7876-7879
Engelman D M, Henderson R, McLachlan A D, Wallace B A (1980) Path of the polypeptide in bacteriorhodopsin. Proc. Natl. Acad. Sci. USA 77, 2023-2027
G. BULDT et al.
protein layers result in markedly different conformations in loops AB and Be in
comparison to the EM structure. After improving the crystallization conditions,
larger crystals became available showing a high degree of merohedral twinning
which had to be corrected for (Luecke et aI., 1998). Meanwhile two additional
crystal structures of bR were published resulting from different crystallization
strategies (Essen et aI., 1998, Takeda et aI., 1998). The structure of Essen et al.
nicely shows structural features of how lipids in the protein boundary interact
with amino acid side chains. The most important new features of all these structures are that the conformations of functionally important amino acid side
chains become more and more reliable and that several water molecules were
localized in the proton translocation channel. However, also these structures do
not give a satisfactory answer how a proton is translocated. At least more water
molecules have to be identified in the channel before a more probable model can
be presented. This situation already demonstrates that only the high resolution
structures of a few important intermediates like the L, Ml, M2 and N state will
give the desired information for modelling a more probable mechanism for proton translocation through bR.
4
Summary
bR is an excellent example of the mosaic like picture in the contributions of different methods for the understanding of proton pumping. The following methods were especially fruitful in the case of bR:
- EM and X-ray crystal structure analysis to obtain a structural basis for the
mechanism.
- Time-resolved UV-Vis spectroscopy to establish the photo cycle of bR with
respect to the identification of intermediates and their rise and decay times.
- Time-resolved and steady state FTIR to localize proton transfer steps to certain
amino acids and to characterize intermediate states of the protein and the
chromophore.
- Site-directed mutagenesis to select functionally important amino acids to
assign FTIR difference bands to certain amino acids and to manipulate the
kinetics of the photo cycle for a better analysis of various intermediate states.
References
Althaus T, Eisfeld W, Lohrmann R, Stockburger M (1995) Application of Raman Spectroscopy to Retinal Proteins. Israel J. Chern. 35:227-252.
Blaurock A E (1975) Bacteriorhodopsin: A Trans-membrane Pump Containing a-helix. J. Mol. BioI.
93, 139-158
Braiman M S, Mogi T, Marti T, Stern L J, Khorana H G, Rothschild K J (1988) Vibrational spectroscopy
of bacteriorhodopsin mutants: light-driven proton transport involves protonation changes of
aspartic acid residues 85, 96, and 212. Biochemistry 27:8516-8520.
Biildt G, Konno K, Nakanishi K, Pl6hn H J, Rao B N, Dencher N A (1991) Heavy-atom labelled retinal
analogues located in bacteriorhodopsin by X-ray diffraction. Photo chern. Photobiol. 54, 873-879
Dencher N A , Dresselhaus D, Zaccai G, Biildt,G (1989) Structural changes in bacteriorhodopsin during proton translocation revealed by neutron diffraction. Proc. Nat!. Acad. Sci. USA 86, 7876-7879
Engelman D M, Henderson R, McLachlan A D, Wallace B A (1980) Path of the polypeptide in bacteriorhodopsin. Proc. Natl. Acad. Sci. USA 77, 2023-2027