Bacteriorhodopsin mutants containing single substitutions of serine or threonine residues are all active in proton translocation.
To study their role in proton translocation by bacteriorhodopsin, 22 serine and threonine residues presumed to be located within and near the border of the transmembrane segments have been individually replaced by alanine or valine, respectively. Thr-89 was substituted by alanine, valine, and aspartic acid, and Ser-141 by alanine and cysteine. Most of the mutants showed essentially wild-type phenotype with regard to chromophore regeneration and absorption spectrum. However, replacement of Thr-89 by Val and of Ser-141 by Cys caused striking blue shifts of the chromophore by 100 and 80 nm, respectively. All substitutions of Thr-89 regenerated the chromophore at least 10-fold faster with 13-cis retinal than with all-trans retinal. The substitutions at positions 89, 90, and 141 also showed abnormal dark-light adaptation, suggesting interactions between these residues and the retinylidene chromophore. Proton pumping measurements revealed 60-75% activity for mutants of Thr-46, -89, -90, -205, and Ser-226, and about 20% for Ser-141----Cys, whereas the remaining mutants showed normal pumping. Kinetic studies of the photocycle and of proton release and uptake for mutants in which proton pumping was reduced revealed generally little alterations. The reduced activity in several of these mutants is most likely due to a lower percentage of all-trans retinal in the light-adapted state. In the mutants Thr-46----Val and Ser-226----Ala the decay of the photointer-mediate M was significantly accelerated, indicating an interaction between these residues and Asp-96 which reprotonates the Schiff base. Our results show that no single serine or threonine residue is obligatory for proton pumping.
Marti
T
T
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Otto
H
H
Mogi
T
T
Rösselet
S J
SJ
Heyn
M P
MP
Khorana
H G
HG
D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000235:genetics / Q000378:metabolism*; D003624:Darkness; D006217:Halobacterium / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011522:Protons; D015183:Restriction Mapping; D012694:Serine; D013053:Spectrophotometry; D013912:Threonine
D011522:Protons; D013912:Threonine; D012694:Serine; D001436:Bacteriorhodopsins