Residue replacements of buried aspartyl and related residues in sensory rhodopsin I: D201N produces inverted phototaxis signals.
Residue replacements were made at five positions (Arg-73, Asp-76, Tyr-87, Asp-106, and Asp-201) in the Halobacterium salinarium phototaxis receptor sensory rhodopsin I (SR-I) by site-specific mutagenesis. The sites were chosen for their correspondence in position to residues of functional importance in the homologous light-driven proton pump bacteriorhodopsin found in the same organism. This work identifies a residue in SR-I shown to be of vital importance to its attractant signaling function: Asp-201. The effect of the substitution with the isosteric asparagine is to convert the normally attractant signal of orange light stimulation to a repellent signal. In contrast, similar neutral substitution of the four other ionizable residues near the photoactive site allows essentially normal attractant and repellent phototaxis signaling. Wild-type two-photon repellent signaling by the receptor is intact in the Asp-201 mutant, genetically separating the wild-type attractant and repellent signal generation processes. A possible explanation and implications of the inverted signaling are discussed. Results of neutral residue substitution for Asp-76 confirm our previous evidence that proton transfer reactions involving this residue are not important to phototaxis but that Asp-76 functions as the Schiff base proton acceptor in proton translocation by transducer-free SR-I.
Olson
K D
KD
Department of Microbiology and Molecular Genetics, University of Texas Medical School, Houston 77030, USA.
Zhang
X N
XN
Spudich
J L
JL
D001436:Bacteriorhodopsins / Q000235:genetics / Q000502:physiology* / Q000528:radiation effects; D001483:Base Sequence; D002465:Cell Movement / Q000235:genetics / Q000502:physiology* / Q000528:radiation effects; D019617:Halobacterium salinarum / Q000235:genetics / Q000502:physiology* / Q000528:radiation effects; D025602:Halorhodopsins; D006863:Hydrogen-Ion Concentration; D008027:Light; D008566:Membranes / Q000502:physiology / Q000528:radiation effects; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010782:Photolysis; D011984:Sensory Receptor Cells / Q000502:physiology* / Q000528:radiation effects; D025603:Sensory Rhodopsins; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology* / Q000528:radiation effects; D013329:Structure-Activity Relationship
D025602:Halorhodopsins; C061497:SRI protein, Halobacterium; D025603:Sensory Rhodopsins; D001436:Bacteriorhodopsins