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 r eceptor sensory rhodopsin I (SR-I) by site-specific mutagenesis. The s ites were chosen for their correspondence in position to residues of f unctional importance in the homologous light-driven proton pump bacter iorhodopsin found in the same organism. This work identifies a residue in SR-I shown to be of vital importance to its attractant signaling f unction: Asp-201. The effect of the substitution with the isosteric as paragine is to convert the normally attractant signal of orange light stimulation to a repellent signal. In contrast, similar neutral substi tution of the four other ionizable residues near the photoactive site allows essentially normal attractant and repellent phototaxis signalin g. 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 neut ral 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 accept or in proton translocation by transducer-free SR-I.