Active state of sensory rhodopsin II: structural determinants for signal transfer and proton pumping

The molecular mechanism of transmembrane signal transduction is still a pertinent question in cellular biology. Generally, a receptor can transfer an external signal via its cytoplasmic surface, as found for G-protein-coupled receptors such as rhodopsin, or via the membrane domain, such as that in sensory rhodopsin II (SRII) in complex with its transducer, HtrII. In the absence of HtrII, SRII functions as a proton pump. Here, we report on the crystal structure of the active state of uncomplexed SRII from Natronomonas pharaonis, NpSRII. The problem with a dramatic loss of diffraction quality upon loading of the active state was overcome by growing better crystals and by reducing the occupancy of the state. The conformational changes in the region comprising helices F and G are similar to those observed for the NpSRII-transducer complex but are much more pronounced. The meaning of these differences for the understanding of proton pumping and signal transduction by NpSRII is discussed.     

Sensory rhodopsin II/transducer complex formation in detergent and in lipid bilayers studied with FRET

The photophobic receptor from Natronomonas pharaonis (NpSRII) forms a photo-signalling complex with its cognate transducer (NpHtrII). In order to elucidate the complex formation in more detail, we have studied the intermolecular binding of both constituents (NpSRII and NpHtrII₁₅₇; truncated at residue 157) in detergent buffers, and in lipid bilayers using FRET. The data for hetero-dimer formation of NpSRII/NpHtrII in detergent agrees well with K_D values (∼ 200 nM) described in the literature. In lipid bilayers, the binding affinity between proteins in the NpSRII/NpHtrII complex is at least one order of magnitude stronger. In detergent the strength of binding is similar for both homo-dimers (NpSRII/NpSRII and NpHtrII/NpHtrII) but significantly weaker (K_D  ∼ 16 μM) when compared to the hetero-dimer. The intermolecular binding is again considerably stronger in lipid bilayers; however, it is not as strong as that observed for the hetero-dimer. At a molar transducer/lipid ratio of 1:2000, which is still well above physiological concentrations, only 40% homo-dimers are formed. Apparently, in cell membranes the formation of the assumed functionally active oligomeric 2:2 complex depends on the full-length transducer including the helical cytoplasmic part, which is thought to tighten the transducer-dimer association.     

Functional characterization of sensory rhodopsin II from Halobacterium salinarum expressed in Escherichia coli

Sensory rhodopsin II (SRII) from Halobacterium salinarum is heterologously expressed in Escherichia coli with a yield of 3-4 mg of purified SRII per liter cell culture. UV/Vis absorption spectroscopy display bands characteristic for native SRII. The resonance Raman spectrum provides evidence for a strongly hydrogen-bonded Schiff base like in mammalian rhodopsin but unlike to the homologous pSRII from Natronobacterium pharaonis. Laser flash spectroscopy indicates that SRII in detergent as well as after reconstitution into polar lipids shows its typical photochemical properties with prolonged photocycle kinetics. The first functional heterologous expression of SRII from H. salinarum provides the basis for studies with its cognate transducer HtrII to investigate the molecular processes involved in phototransduction as well as in chemotransduction.     

Light-induced structural changes occur in the transmembrane helices of the Natronobacterium pharaonis HtrII transducer.

The Natronobacterium pharaonis HtrII (NpHtrII) transducer interacts with its cognate photoactive sensory rhodopsin receptor, NpSRII, to mediate phototaxis responses. NpHtrII is predicted to have two transmembrane helices and a large cytoplasmic domain and to form a homodimer. Single cysteines were substituted into an engineered cysteine-less NpHtrII at 38 positions in its transmembrane domain. Oxidative disulfide cross-linking efficiencies of the monocysteine mutants were measured with or without photoactivation of NpSRII. The rapid cross-linking rates at several positions support that NpHtrII is a dimer when functionally expressed in the Halobacterium salinarum membrane. Thirteen positions in the second transmembrane segment (TM2) exhibited significant light-induced increases in cross-linking efficiency, and they define a single face traversing the length of the segment when modeled as an alpha-helix. Four positions in this helix showing light-induced decreases in efficiency are clustered on the cytoplasmic side of the protein. One of the monocysteine mutants, G83C, showed loss of phototaxis responses, and analysis of double mutants showed that the G83C mutation alters the dark structure of the TM2-TM2' region of NpHtrII. In summary, the results reveal conformationally active regions in the second transmembrane segment of NpHtrII and a face along the length of TM2 that becomes more available for TM2-TM2' cross-linking upon receptor photoactivation. The data also establish that one residue in TM2, Gly83, is critical for maintaining the proper conformation of NpHtrII for signal relay from the photoactivated receptor to the kinase-binding region of the transducer.     

Computational analysis of the transient movement of helices in sensory rhodopsin II

MD simulation of sensory rhodopsin II was executed for three intermediates (ground-state, K-state, M-state) appearing in its photocycle. We observed a large displacement of the cytoplasmic side of helixF only in M-state among the three intermediates. This displacement was transmitted to TM2, and the cytoplasmic side of TM2 rotated clockwise. These transient movements are in agreement with the results of an EPR experiment. That is, the early stage of signal transduction in a sRII-HtrII complex was successfully reproduced by the in silico MD simulation. By analyzing the structure of the sRII-HtrII complex, the following findings about the photocycle of sRII were obtained: (1) The hydrogen bonds between helixF and other helices determine the direction of the movement of helixF; (2) three amino acids (Arg162, Thr189, Tyr199) are essential for sRII-HtrII binding and contribute to the motion transfer from sRII to HtrII; (3) after the isomerization of retinal, a major conformational change of retinal was caused by proton transfer from Schiff base to Asp75, which, in turn, triggers the steric collision of retinal with Trp171. This is the main reason for the movement of the cytoplasmic side of helixF.     

Transducer binding establishes localized interactions to tune sensory rhodopsin II

In haloarchaea, sensory rhodopsin II (SRII) mediates a photophobic response to avoid photo-oxidative damage in bright light. Upon light activation the receptor undergoes a conformational change that activates a tightly bound transducer molecule (HtrII), which in turn by a chain of homologous reactions transmits the signal to the chemotactic eubacterial two-component system. Here, using single-molecule force spectroscopy, we localize and quantify changes to the intramolecular interactions within SRII of Natronomonas pharaonis (NpSRII) upon NpHtrII binding. Transducer binding affected the interactions at transmembrane alpha helices F and G of NpSRII to which the transducer was in contact. Remarkably, the interactions were distributed asymmetrically and significantly stabilized alpha helix G entirely but alpha helix F only at its extracellular tip. These findings provide unique insights into molecular mechanisms that "prime" the complex for signaling, and guide the receptor toward transmitting light-activated structural changes to its cognate transducer.     

Constitutive activity in chimeras and deletions localize sensory rhodopsin II/HtrII signal relay to the membrane-inserted domain

Halobacterium salinarum sensory rhodopsin II (HsSRII) is a phototaxis receptor for blue-light avoidance that relays signals to its tightly bound transducer HsHtrII (H. salinarum haloarchaeal transducer for SRII). We found that disruption of the salt bridge between the protonated Schiff base of the receptor's retinylidene chromophore and its counterion Asp73 by residue substitutions D73A, N or Q constitutively activates HsSRII, whereas the corresponding Asp75 counterion substitutions do not constitutively activate Natronomonas pharaonis SRII (NpSRII) when complexed with N. pharaonis haloarchaeal transducer for SRII (NpHtrII). However, NpSRII(D75Q) in complex with HsHtrII is fully constitutively active, showing that transducer sensitivity to the receptor signal contributes to the phenotype. The swimming behaviour of cells expressing chimeras exchanging portions of the two homologous transducers localizes their differing sensitivities to the HtrII transmembrane domains. Furthermore, deletion constructs show that the known contact region in the cytoplasmic domain of the NpSRII-NpHtrII complex is not required for phototaxis, excluding the domain as a site for signal transmission. These results distinguish between the prevailing models for SRII-HtrII signal relay, strongly supporting the 'steric trigger-transmembrane relay model', which proposes that retinal isomerization directly signals HtrII through the mid-membrane SRII-HtrII interface, and refuting alternative models that propose signal relay in the cytoplasmic membrane-proximal domain.     

Importance of specific hydrogen bonds of archaeal rhodopsins for the binding to the transducer protein

Four rhodopsins, bacteriorhodopsin (bR), halorhodopsin (hR), sensory rhodopsin (sR) and phoborhodopsin (pR) exist in archaeal membranes. bR and hR work as a light-driven ion pump. sR and pR work as a photo-sensor of phototaxis, and form signaling complexes in membranes with their respective cognate transducer proteins HtrI (with sR) and HtrII (with pR), through which light signals are transmitted to the cytoplasm. What is the determining factor(s) of the specific binding to form the complex? Binding of the wild-type or mutated rhodopsins with HtrII was measured by isothermal titration calorimetric analysis (ITC). bR and hR could not bind with HtrII. On the other hand, sR could bind to HtrII, although the dissociation constant (K(D)) was about 100 times larger than that of pR. An X-ray crystallographic structure of the pR/HtrII complex revealed formation of two specific hydrogen bonds whose pairs are Tyr199(pR)/Asn74(HtrII) and Thr189(pR)/Glu43(HtrII)/Ser62(HtrII). To investigate the importance of these hydrogen bonds, the K(D) value for the binding of various mutants of bR, hR, sR and pR with HtrII was estimated by ITC. The K(D) value of T189V(pR)/Y199F(pR), double mutant/HtrII complex, was about 100-fold larger than that of the wild-type pR, whose K(D) value was 0.16 microM. On the other hand, bR and hR double mutants, P200T(bR)/V210Y(bR) and P240T(hR)/F250Y(hR), were able to bind with HtrII. The K(D) value of these complexes was estimated to be 60.1(+/-10.7) microM for bR and to be 29.1(+/-6.1) microM for hR, while the wild-type bR and hR did not bind with HtrII. We concluded that these two specific hydrogen bonds play important roles in the binding between the rhodopsins and transducer protein.     

An archaeal photosignal-transducing module mediates phototaxis in Escherichia coli

Halophilic archaea, such as Halobacterium salinarum and Natronobacterium pharaonis, alter their swimming behavior by phototaxis responses to changes in light intensity and color using visual pigment-like sensory rhodopsins (SRs). In N. pharaonis, SRII (NpSRII) mediates photorepellent responses through its transducer protein, NpHtrII. Here we report the expression of fusions of NpSRII and NpHtrII and fusion hybrids with eubacterial cytoplasmic domains and analyze their function in vivo in haloarchaea and in eubacteria. A fusion in which the C terminus of NpSRII is connected by a short flexible linker to NpHtrII is active in phototaxis signaling for H. salinarum, showing that the fusion does not inhibit functional receptor-transducer interactions. We replaced the cytoplasmic portions of this fusion protein with the cytoplasmic domains of Tar and Tsr, chemotaxis transducers from enteric eubacteria. Purification of the fusion protein from H. salinarum and Tar fusion chimera from Escherichia coli membranes shows that the proteins are not cleaved and exhibit absorption spectra characteristic of wild-type membranes. Their photochemical reaction cycles in H. salinarum and E. coli membranes, respectively, are similar to those of native NpSRII in N. pharaonis. These fusion chimeras mediate retinal-dependent phototaxis responses by Escherichia coli, establishing that the nine-helix membrane portion of the receptor-transducer complex is a modular functional unit able to signal in heterologous membranes. This result confirms a current model for SR-Htr signal transduction in which the Htr transducers are proposed to interact physically and functionally with their cognate sensory rhodopsins via helix-helix contacts between their transmembrane segments.     

Time-resolved detection of sensory rhodopsin II-transducer interaction

The dynamics of protein conformational change of Natronobacterium pharaonis sensory rhodopsin II (NpSRII) and of NpSRII fused to cognate transducer (NpHtrII) truncated at 159 amino acid sequence from the N-terminus (NpSRII-DeltaNpHtrII) are investigated in solution phase at room temperature by the laser flash photolysis and the transient grating methods in real time. The diffusion coefficients of both species indicate that the NpSRII-DeltaNpHtrII exists in the dimeric form in 0.6% dodecyl-beta-maltopyranoside (DM) solution. Rate constants of the reaction processes in the photocycles determined by the transient absorption and grating methods agree quite well. Significant differences were found in the volume change and the molecular energy between NpSRII and NpSRII-DeltaNpHtrII samples. The enthalpy of the second intermediate (L) of NpSRII-DeltaNpHtrII is more stabilized compared with that of NpSRII. This stabilization indicates the influence of the transducer to the NpSRII structure in the early intermediate species by the complex formation. Relatively large molecular volume expansion and contraction were observed in the last two steps for NpSRII. Additional volume expansion and contraction were induced by the presence of DeltaNpHtrII. This volume change, which should reflect the conformational change induced by the transducer protein, suggested that this is the signal transduction process of the NpSRII-DeltaNpHtrII.     

Probing the sensory rhodopsin II binding domain of its cognate transducer by calorimetry and electrophysiology

Sensory rhodopsin II, a repellent phototaxis receptor from Natronobacterium pharaonis (NpSRII) forms a tight complex with its cognate transducer (NpHtrII). Light excitation of the receptor triggers conformational changes in both proteins, thereby activating the cellular two-component signalling cascade. In membranes, the two proteins form a 2:2 complex, which dissociates to a 1:1 heterodimer in micelles. Complexed to the transducer sensory rhodopsin II is no longer capable of light-driven proton pumping. In order to elucidate the dimerisation and the size of the receptor-binding domain of the transducer, isothermal titration calorimetry and electrophysiological experiments have been carried out. It is shown, that an N-terminal sequence of 114 amino acid residues is sufficient for tight binding (K(d)=240nM; DeltaH=-17.6kJmol(-1)) and for inhibiting the proton transfer. These data and results obtained from selected site-directed mutants indicate a synergistic interplay of transducer transmembrane domain (1-82) and cytoplasmic peptide (83-114) leading to an optimal and specific interaction between receptor and transducer.     

Structural insights into the early steps of receptor-transducer signal transfer in archaeal phototaxis

Electron paramagnetic resonance-based inter-residue distance measurements between site-directed spin-labelled sites of sensory rhodopsin II (NpSRII) and its transducer NpHtrII from Natronobacterium pharaonis revealed a 2:2 complex with 2-fold symmetry. The core of the complex is formed by the four transmembrane helices of a transducer dimer. Upon light excitation, the previously reported flap-like movement of helix F of NpSRII induces a conformational change in the transmembrane domain of the transducer. The inter-residue distance changes determined provide strong evidence for a rotary motion of the second transmembrane helix of the transducer. This helix rotation becomes uncoupled from changes in the receptor during the last step of the photocycle.     

Salt-driven equilibrium between two conformations in the HAMP domain from Natronomonas pharaonis: the language of signal transfer?

HAMP domains (conserved in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) perform their putative function as signal transducing units in diversified environments in a variety of protein families. Here the conformational changes induced by environmental agents, namely salt and temperature, on the structure and function of a HAMP domain of the phototransducer from Natronomonas pharaonis (NpHtrII) in complex with sensory rhodopsin II (NpSRII) were investigated by site-directed spin labeling electron paramagnetic resonance. A series of spin labeled mutants were engineered in NpHtrII157, a truncated analog containing only the first HAMP domain following the transmembrane helix 2. This truncated transducer is shown to be a valid model system for a signal transduction domain anchored to the transmembrane light sensor NpSRII. The HAMP domain is found to be engaged in a "two-state" equilibrium between a highly dynamic (dHAMP) and a more compact (cHAMP) conformation. The structural properties of the cHAMP as proven by mobility, accessibility, and intra-transducer-dimer distance data are in agreement with the four helical bundle NMR model of the HAMP domain from Archaeoglobus fulgidus.     

Signal transmission through the HtrII transducer alters the interaction of two alpha-helices in the HAMP domain

A conformational change of the transducer HtrII upon photoexcitation of the associated photoreceptor sensory rhodopsin II (SRII) was investigated by monitoring the kinetics of volume changes and the diffusion coefficient (D) of the complex during the photochemical reaction cycle. To localize the region of the transducer responsible, we truncated it at various positions in the cytoplasmic HAMP (histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) domain. The truncations do not alter receptor binding, which is dependent primarily on membrane-embedded domain interactions. We found that the light-induced reduction in D occurs in transducers of lengths 120 and 157 residues (Tr120 and Tr157), which are both predicted to contain a HAMP domain consisting of two amphipathic α-helices (AS-1 and AS-2). In contrast, the change in D was abolished in a transducer of 114 amino acid residues (Tr114), which lacks a distal portion of the second α-helix AS-2. The volume changes in SRII-Tr114 are comparable in amplitude and kinetics with those in SRII-Tr120 and SRII-Tr157, confirming the integrity of the complex, which was previously concluded from the similar SRII binding affinity and similar blocking of SRII proton transport by full-length HtrII and Tr114. Our results indicate that a substantial conformational change occurs in the HAMP domain during SRII-HtrII signaling. The data presented here are the first demonstration of stimulus-induced conformational changes of a HAMP domain and provide evidence that the presence of AS-2 is crucial for the conformational alterations. The reduction in diffusion coefficient is likely to due to structural changes in the AS-1 and AS-2 helices such that hydrogen bonding with the surrounding water molecules is increased, thereby increasing friction with the solvent. Similar structural changes may be a general feature in HAMP domain switching, which occurs in diverse signaling proteins, including sensor kinases, taxis receptors/transducers, adenylyl cyclases, and phosphatases.     

Structural characteristics around the β-ionone ring of the retinal chromophore in Salinibacter sensory rhodopsin I

Organisms sense and respond to environmental stimuli through membrane-embedded receptors and transducers. Sensory rhodopsin I (SRI) and sensory rhodopsin II (SRII) are the photoreceptors for the positive and negative phototaxis in microorganisms, respectively. They form signaling complexes in the membrane with their cognate transducer proteins, HtrI and HtrII, and these SRI-HtrI and SRII-HtrII complexes transmit a light signal through their cytoplasmic sensory signaling system, inducing opposite effects (i.e., the inactivation or activation of the kinase CheA). Here we found, by using Fourier transformed infrared spectroscopy, that a conserved residue, Asp102 in Salinibacter SRI (SrSRI), which is located close to the β-ionone ring of the retinal chromophore, is deprotonated upon formation of the active M-intermediate. Furthermore, the D102E mutant of SrSRI affects the structure and/or structural changes of Cys130. This mutant shows a large spectral shift and is comparably unstable, especially in the absence of Cl(-). These phenomena have not been observed in the wild-type, or the N105Q and N105D mutants of Natronomonas pharaonis SRII (NpSRII), indicating differences in the structure and structural changes between SrSRI and NpSRII around the β-ionone ring. These differences could also be supported by the measurements of the reactivity with the water-soluble reagent azide. On the basis of these results, we discuss the structure and structural changes around the retinal chromophore in SrSRI.     

Structural analysis of a HAMP domain: the linker region of the phototransducer in complex with sensory rhodopsin II

Sensory rhodopsin II, the photophobic receptor from Natronomonas pharaonis (NpSRII)5, forms a 2:2 complex with its cognate transducer (N. pharaonis halobacterial transducer of rhodopsins II (NpHtrII)) in lipid membranes. Light activation of NpSRII leads to a displacement of helix F, which in turn triggers a rotation/screw-like motion of TM2 in NpHtrII. This conformational change is thought to be transmitted through the membrane adjacent conserved signal transduction domain in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases (HAMP domain) to the cytoplasmic signaling domain of the transducer. The architecture and function of the HAMP domain are still unknown. In order to obtain information on the structure and dynamics of this region, EPR experiments on a truncated transducer (NpHtrII(157)) and NpSRII, site-directed spin-labeled and reconstituted into purple membrane lipids, have been carried out. A nitroxide scanning involving residues in the transducer helix TM2, in the predicted AS-1 region, and at selected positions in the following connector and AS-2 regions of the HAMP domain has been performed. Accessibility and dynamics data allowed us to identify a helical region up to residue Ala(94) in the AS-1 amphipathic sequence, followed by a highly dynamic domain protruding into the water phase. Additionally, transducer-transducer and transducer-receptor proximity relations revealed the overall architecture of the AS-1 sequences in the 2:2 complex, which are suggested to form a molten globular type of a coiled-coil bundle.     

Association of pharaonis phoborhodopsin with its cognate transducer decreases the photo-dependent reactivity by water-soluble reagents of azide and hydroxylamine

pharaonis phoborhodopsin (ppR; also pharaonis sensory rhodopsin II, psRII) is a receptor of the negative phototaxis of Natronobacterium pharaonis. In halobacterial membrane, ppR forms a complex with its transducer pHtrII, and this complex transmits the light signal to the sensory system in the cytoplasm. In the present work, the truncated transducer, t-Htr, was used which interacts with ppR [Sudo et al. (2001) Photochem. Photobiol. 74, 489-494]. Two water-soluble reagents, hydroxylamine and azide, reacted both with the transducer-free ppR and with the complex ppR/t-Htr (the complex between ppR and its truncated transducer). In the dark, the bleaching rates caused by hydroxylamine were not significantly changed between transducer-free ppR and ppR/t-Htr, or that of the free ppR was a little slower. Illumination accelerated the bleach rates, which is consistent with our previous conclusion that the reaction occurs selectively at the M-intermediate, but the rate of the complex was about 7.4-fold slower than that of the transducer-free ppR. Azide accelerated the M-decay, and its reaction rate of ppR/t-Htr was about 4.6-fold slower than free ppR. These findings suggest that the transducer binding decreases the water accessibility around the chromophore at the M-intermediate. Its implication is discussed.     

Transmembrane signal transduction in archaeal phototaxis: the sensory rhodopsin II-transducer complex studied by electron paramagnetic resonance spectroscopy

Archaeal photoreceptors, together with their cognate transducer proteins, mediate phototaxis by regulating cell motility through two-component signal transduction pathways. This sensory pathway is closely related to the bacterial chemotactic system, which has been studied in detail during the past 40 years. Structural and functional studies applying site-directed spin labelling and electron paramagnetic resonance spectroscopy on the sensory rhodopsin II/transducer (NpSRII/NpHtrII) complex of Natronomonas pharaonis have yielded insights into the structure, the mechanisms of signal perception, the signal transduction across the membrane and provided information about the subsequent information transfer within the transducer protein towards the components of the intracellular signalling pathway. Here, we provide an overview about the findings of the last decade, which, combined with the wealth of data from research on the Escherichia coli chemotaxis system, served to understand the basic principles microorganisms use to adapt to their environment. We document the time course of a signal being perceived at the membrane, transferred across the membrane and, for the first time, how this signal modulates the dynamic properties of a HAMP domain, a ubiquitous signal transduction module found in various protein classes.     

Crystal structure of the Anabaena sensory rhodopsin transducer

We present crystal structures of the Anabaena sensory rhodopsin transducer (ASRT), a soluble cytoplasmic protein that interacts with the first structurally characterized eubacterial retinylidene photoreceptor Anabaena sensory rhodopsin (ASR). Four crystal structures of ASRT from three different spacegroups were obtained, in all of which ASRT is present as a planar (C4) tetramer, consistent with our characterization of ASRT as a tetramer in solution. The ASRT tetramer is tightly packed, with large interfaces where the well-structured beta-sandwich portion of the monomers provides the bulk of the tetramer-forming interactions, and forms a flat, stable surface on one side of the tetramer (the beta-face). Only one of our four different ASRT crystals reveals a C-terminal alpha-helix in the otherwise all-beta protein, together with a large loop from each monomer on the opposite face of the tetramer (the alpha-face), which is flexible and largely disordered in the other three crystal forms. Gel-filtration chromatography demonstrated that ASRT forms stable tetramers in solution and isothermal microcalorimetry showed that the ASRT tetramer binds to ASR with a stoichiometry of one ASRT tetramer per one ASR photoreceptor with a K(d) of 8 microM in the highest affinity measurements. Possible mechanisms for the interaction of this transducer tetramer with the ASR photoreceptor via its flexible alpha-face to mediate transduction of the light signal are discussed.     

Mechanism divergence in microbial rhodopsins

A fundamental design principle of microbial rhodopsins is that they share the same basic light-induced conversion between two conformers. Alternate access of the Schiff base to the outside and to the cytoplasm in the outwardly open “E” conformer and cytoplasmically open “C” conformer, respectively, combined with appropriate timing of pKa changes controlling Schiff base proton release and uptake make the proton path through the pumps vectorial. Phototaxis receptors in prokaryotes, sensory rhodopsins I and II, have evolved new chemical processes not found in their proton pump ancestors, to alter the consequences of the conformational change or modify the change itself. Like proton pumps, sensory rhodopsin II undergoes a photoinduced E → C transition, with the C conformer a transient intermediate in the photocycle. In contrast, one light-sensor (sensory rhodopsin I bound to its transducer HtrI) exists in the dark as the C conformer and undergoes a light-induced C → E transition, with the E conformer a transient photocycle intermediate. Current results indicate that algal phototaxis receptors channelrhodopsins undergo redirected Schiff base proton transfers and a modified E → C transition which, contrary to the proton pumps and other sensory rhodopsins, is not accompanied by the closure of the external half-channel. The article will review our current understanding of how the shared basic structure and chemistry of microbial rhodopsins have been modified during evolution to create diverse molecular functions: light-driven ion transport and photosensory signaling by protein–protein interaction and light-gated ion channel activity.