PROTONATABLE RESIDUES AT THE CYTOPLASMIC END OF TRANSMEMBRANE HELIX-2IN THE SIGNAL TRANSDUCER HTRI CONTROL PHOTOCHEMISTRY AND FUNCTION OF SENSORY RHODOPSIN-I

Neutral residue replacements were made of 21 acidic and basic residues within the N-terminal half of the Halobacterium salinarium signal tra nsducer HtrI [the halobacterial transducer for sensory rhodopsin I (SM I)] by site specific mutagenesis, The replacements are all within the region of HtrI that we previously concluded from deletion analysis to contain sites of interaction with the phototaxis receptor SRI, Immunob lotting shows plasmid expression of the htrI-sopI operon containing th e mutations produces SRI and mutant HtrI in cells at near wild-type le vels, Six of the HtrI mutations perturb photochemical kinetics of SRI and one reverses the phototaxis response, Substitution with neutral am ino acids of Asp-86, Glu-87, and Glu-108 accelerate, and of Arg-70, Ar g-84, and Arg-99 retard, the SRI photocycle. Opposite effects on photo cycle rate cancel in double mutants containing one replaced acidic and one replaced basic residue, Laser flash spectroscopy shows the kineti c perturbations are due to alteration of the rate of reprotonation of the retinylidene Schiff base, All of these mutations permit normal att ractant and repellent signaling, On the other hand, the substitution o f Glu-56 with the Isosteric glutamine converts the normally attractant effect of orange light to a repellent signal in vivo at neutral pH (i nverted signaling), Low pH corrects the inversion due to Glu-56 --> Gl n and the apparent pK of the inversion is increased when arginine is s ubstituted at position 56, The results indicate that the cytoplasmic e nd of transmembrane helix-2 and the initial part of the cytoplasmic do main contain interaction sites with SRI, To explain these and previous results, we propose a model in which (i) the HtrI region identified h ere forms part of an electrostatic bonding network that extends throug h the SRI protein and includes its photoactive site; (ii) alteration o f this network by photoisomerization-induced Schiff base deprotonation and reprotonation shifts HtrI between attractant and repellent confor mations; and (iii) HtrI mutations and extracellular pH alter the equil ibrium ratios of these conformations.