Probing the proton channel and the retinal binding site of Natronobacterium pharaonis sensory rhodopsin II.
The sensory rhodopsin II from Natronobacterium pharaonis (NpSRII) was mutated to try to create functional properties characteristic of bacteriorhodopsin (BR), the proton pump from Halobacterium salinarum. Key residues from the cytoplasmic and extracellular proton transfer channel of BR as well as from the retinal binding site were chosen. The single site mutants L40T, F86D, P183E, and T204A did not display altered function as determined by the kinetics of their photocycles. However, the photocycle of each of the subsequent multisite mutations L40T/F86D, L40T/F86D/P183E, and L40T/F86D/P183E/T204A was quite different from that of the wild-type protein. The reprotonation of the Schiff base could be accelerated approximately 300- to 400-fold, to approximately two to three times faster than the corresponding reaction in BR. The greatest effect is observed for the quadruple mutant in which Thr-204 is replaced by Ala. This result indicates that mutations affecting conformational changes of the protein might be of decisive importance for the creation of BR-like functional properties.
Klare
Johann P
JP
Max-Planck-Institut für Molekulare Physiologie, Otto Hahn Strasse 11, D-44227 Dortmund, Germany.
Schmies
Georg
G
Chizhov
Igor
I
Shimono
Kazumi
K
Kamo
Naoki
N
Engelhard
Martin
M
D000409:Alanine / Q000737:chemistry; D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002338:Carotenoids / Q000737:chemistry* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D025602:Halorhodopsins; D006863:Hydrogen-Ion Concentration; D007834:Lasers; D008027:Light; D050356:Lipid Metabolism; D008958:Models, Molecular; D009154:Mutation; D019633:Natronobacterium / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011522:Protons; D025604:Rhodopsins, Microbial / Q000737:chemistry* / Q000378:metabolism*; D025603:Sensory Rhodopsins; D013912:Threonine / Q000737:chemistry
D019843:Archaeal Proteins; D025602:Halorhodopsins; D011522:Protons; D025604:Rhodopsins, Microbial; D025603:Sensory Rhodopsins; C061508:sensory rhodopsin II protein, archaeal; D013912:Threonine; D002338:Carotenoids; D004247:DNA; D000409:Alanine