Retinal analogues with altered conjugated double bond systems or altered stereochemistry were incorporated into the phototaxis receptor sensory rhodopsin (SR) and the light-driven proton pump bacteriorhodopsin (BR) from Halobacterium halobium. Wavelength shifts in absorption ("opsin shifts") due to analogue interaction with the protein microenvironment demonstrate that the same overall electrostatic and steric properties of the retinal binding-site structures exist in both proteins despite their different functions. pi-Electron calculations from the opsin shifts lead to a new description of protein charge distribution that applies to the binding sites of both SR and BR. The new data extends the previously proposed external point charge model for BR to include an ion-pair protein/chromophore interaction near the beta-ionone moiety. The new data modifies the previously proposed external point-charge model, the derivation of which involved an experimentally erroneous opsin shift for one of the BR analogues.

Spontaneous switching of the rotation sense of the flagellar motor of the archaebacterium Halobacterium halobium and modulation of the switch by attractant and repellent photostimuli were analyzed by using a computerized cell-tracking system with 67-ms resolution coupled to electronic shutters. The data fit a three-state model of the switch, in which a Poisson process governs the transition from state N (nonreversing) to state R (reversing). After a reversal, the switch returns to state N, passing through an intermediate state I (inactive), which produces a ca. 2-s period of low reversal frequency before the state N Poisson rate is restored. The stochastic nature of the H. halobium switch reveals a close similarity to Escherichia coli flagellar motor properties as elucidated previously. Sensory modulation of the switch by both photoattractant and photorepellent signals can be interpreted in terms of modulation of the single forward rate constant of the N to R transition. Insight into the mechanism of modulation by the phototaxis receptor sensory rhodopsin I (SR-I) was gained by increasing the lifetime of the principal photointermediate of the SR-I photochemical reaction cycle, S373, by replacing the native chromophore, all-trans-retinal, with the acyclic analog, 3,7,11-trimethyl-2,4,6,8-dodecapentaenal. Flash photolysis of analog-containing cells revealed an eightfold decrease in the rate of thermal decay of S373, and behavioral analysis showed longer periods of reversal suppression than that of cells with the native chromophore over similar ranges of illumination intensities. This indicates that attractant signaling is governed by the lifetime of the S373 intermediate rather than by the frequency of photocycling. In this sense, SR-I is similar to rhodopsin, whose function depends on an active photoproduct (Meta-II).

Ring desmethyl and acyclic analogues of all-trans retinal were incorporated into the apoprotein of the phototaxis receptor sensory rhodopsin I (SR-I) in Halobacterium halobium membranes. All modified retinals generate SR-I analogue pigments which exhibit "opsin shifts," i.e., their absorption spectra are shifted to longer wavelengths compared with model protonated Schiff bases of the same analogues. Each SR-I pigment analogue exhibits cyclic photochemical reactions as monitored by flash spectroscopy, but the analogue photocycles differ from that of native SR-I by exhibiting pronounced biphasic recovery of flash-induced absorption changes and abnormal flash-induced absorption difference spectra. Despite perturbations in the photochemical properties, the SR-I pigment analogues are capable of both attractant (single photon) and repellent (two photon) phototaxis signaling in cells. Our interpretation is that the hydrophobic ring substituents interact with the binding pocket to maintain the correct configuration for native SR-I absorption and photochemistry, but these interactions are not essential for the physiological function of SR-I as a dual attractant/repellent phototaxis receptor. These results support the conclusion emerging from several studies that the photoactivation process that triggers the conformation changes of SR-I and the related proton pump bacteriorhodopsin is conserved despite the different biological functions of their photoactivation.