Light-Driven Chloride and Sulfate Pump with Two Different Transport Modes.

Ion pumps are membrane proteins that actively translocate ions by using energy. All known pumps bind ions in the resting state, and external energy allows ion transport through protein structural changes. The light-driven sodium-ion pump Krokinobacter eikastus rhodopsin 2 (KR2) is an exceptional case in which ion binding follows the energy input. In this study, we report another case of this unusual transport mode. The NTQ rhodopsin from Alteribacter aurantiacus (AaClR) is a natural light-driven chloride pump, in which the chloride ion binds to the resting state. AaClR is also able to pump sulfate ions, though the pump efficiency is much lower for sulfate ions than for chloride ions. Detailed spectroscopic analysis revealed no binding of the sulfate ion to the resting state of AaClR, indicating that binding of the substrate (sulfate ion) to the resting state is not necessary for active transport. This property of the AaClR sulfate pump is similar to that of the KR2 sodium pump. Photocycle dynamics of the AaClR sulfate pump resemble a non-functional cycle in the absence of anions. Despite this, flash photolysis and difference Fourier transform infrared spectroscopy suggest transient binding of the sulfate ion to AaClR. The molecular mechanism of this unusual active transport by AaClR is discussed.

Phototrophy by antenna-containing rhodopsin pumps in aquatic environments.

Energy transfer from light-harvesting ketocarotenoids to the light-driven proton pump xanthorhodopsins has been previously demonstrated in two unique cases: an extreme halophilic bacterium1 and a terrestrial cyanobacterium2. Attempts to find carotenoids that bind and transfer energy to abundant rhodopsin proton pumps3 from marine photoheterotrophs have thus far failed4-6. Here we detected light energy transfer from the widespread hydroxylated carotenoids zeaxanthin and lutein to the retinal moiety of xanthorhodopsins and proteorhodopsins using functional metagenomics combined with chromophore extraction from the environment. The light-harvesting carotenoids transfer up to 42% of the harvested energy in the violet- or blue-light range to the green-light absorbing retinal chromophore. Our data suggest that these antennas may have a substantial effect on rhodopsin phototrophy in the world's lakes, seas and oceans. However, the functional implications of our findings are yet to be discovered.

Functional assay of light-induced ion-transport by rhodopsins.

Microbial rhodopsins are photoreceptive membrane proteins found from diverse microorganisms such as archaea, eubacteria, eukaryotes and viruses. Many microbial rhodopsins possess ion-transport activity by light, such as channels and pumps, and ion-transporting rhodopsins are important tools in optogenetics that control animal behavior by light. Historically, molecular mechanism of rhodopsins has been studied by spectroscopic methods for purified proteins. On the other hand, ion-transport function has to be studied by different methods. This chapter introduces two methods of functional assay of ion-transporting rhodopsins by light. One is a patch clamp method using mammalian cells, and another is an ion-transport assay using pH electrode and microbial cells. These functional assay provides fundamental data of ion-transporting rhodopsins, and thus contributes to evaluation for optogenetic tools.

Unusual Photoisomerization Pathway in a Near-Infrared Light Absorbing Enzymerhodopsin.

Microbial and animal rhodopsins possess retinal chromophores which capture light and normally photoisomerize from all-trans to 13-cis and from 11-cis to all-trans-retinal, respectively. Here, we show that a near-infrared light-absorbing enzymerhodopsin from Obelidium mucronatum (OmNeoR) contains the all-trans form in the dark but isomerizes into the 7-cis form upon illumination. The photoproduct (λmax = 372 nm; P372) possesses a deprotonated Schiff base, and the system exhibits a bistable nature. The photochemistry of OmNeoR was arrested at <270 K, indicating the presence of a potential barrier in the excited state. Formation of P372 is accompanied by protonation changes of protonated carboxylic acids and peptide backbone changes of an α-helix. Photoisomerization from the all-trans to 7-cis retinal conformation rarely occurs in any solvent and protein environments; thus, the present study reports on a novel photochemistry mediated by a microbial rhodopsin, leading from the all-trans to 7-cis form selectively.

Proton-transporting heliorhodopsins from marine giant viruses.

Rhodopsins convert light into signals and energy in animals and microbes. Heliorhodopsins (HeRs), a recently discovered new rhodopsin family, are widely present in archaea, bacteria, unicellular eukaryotes, and giant viruses, but their function remains unknown. Here, we report that a viral HeR from Emiliania huxleyi virus 202 (V2HeR3) is a light-activated proton transporter. V2HeR3 absorbs blue-green light, and the active intermediate contains the deprotonated retinal Schiff base. Site-directed mutagenesis study revealed that E191 in TM6 constitutes the gate together with the retinal Schiff base. E205 and E215 form a PAG of the Schiff base, and mutations at these positions converted the protein into an outward proton pump. Three environmental viral HeRs from the same group as well as a more distantly related HeR exhibited similar proton-transport activity, indicating that HeR functions might be diverse similarly to type-1 microbial rhodopsins. Some strains of E. huxleyi contain one HeR that is related to the viral HeRs, while its viruses EhV-201 and EhV-202 contain two and three HeRs, respectively. Except for V2HeR3 from EhV-202, none of these proteins exhibit ion transport activity. Thus, when expressed in the E. huxleyi cell membranes, only V2HeR3 has the potential to depolarize the host cells by light, possibly to overcome the host defense mechanisms or to prevent superinfection. The neuronal activity generated by V2HeR3 suggests that it can potentially be used as an optogenetic tool, similarly to type-1 microbial rhodopsins.

Mechanism of the Irreversible Transition from Pentamer to Monomer at pH 2 in a Blue Proteorhodopsin.

Proteorhodopsin (PR) is a light-driven proton pump found in marine bacteria, and thousands of PRs are classified as blue-absorbing PRs (BPR; λmax ∼ 490 nm) and green-absorbing PRs (GPR; λmax ∼ 525 nm). We previously converted BPR into GPR using an anomalous pH effect, which was achieved by an irreversible process at around pH 2. Recent size-exclusion chromatography (SEC) and atomic force microscopy (AFM) analyses of BPR from Vibrio califitulae (VcBPR) revealed the anomalous pH effect owing to the irreversible transition from pentamer to monomer. Different pKa values of the Schiff base counterion between pentamer and monomer lead to different colors at the same pH. Here, we incorporate systematic mutation into VcBPR and examine the anomalous pH effect. The anomalous pH effect was observed for the mutants of key residues near the retinal chromophore such as D76N, D206N, and Q84L, indicating that the Schiff base counterions and the L/Q switch do not affect the irreversible transition from pentamer to monomer at pH ∼ 2. We then focus on the two specific interactions at the intermonomer interface in a pentamer, E29/R30/D31 and W13/H54. Single mutants such as E29Q, R30A, W13A, and H54A and the wild type (WT) exhibited an anomalous pH effect. In contrast, the anomalous pH effect was lost for E29Q/H54A, R30A/H54A, and W13A/E29Q. Size-exclusion chromatography (SEC) and atomic force microscopy (AFM) measurements showed monomer forms in the original states of the double mutants, being a clear contrast to the pentamer forms of all single mutants in the original states. It was concluded that the pentamer structure of VcBPR was stabilized by an electrostatic interaction in the E29/R30/D31 region and a hydrogen-bonding interaction in the W13/H54 region, which was disrupted at pH 2 and converted into monomers.

Optogenetic reprogramming of carbon metabolism using light-powering microbial proton pump systems.

In microbial fermentative production, ATP regeneration, while crucial for cellular processes, conflicts with efficient target chemical production because ATP regeneration exhausts essential carbon sources also required for target chemical biosynthesis. To wrestle with this dilemma, we harnessed the power of microbial rhodopsins with light-driven proton pumping activity to supplement with ATP, thereby facilitating the bioproduction of various chemicals. We first demonstrated a photo-driven ATP supply and redistribution of metabolic carbon flows to target chemical synthesis by installing already-known delta rhodopsin (dR) in Escherichia coli. In addition, we identified novel rhodopsins with higher proton pumping activities than dR, and created an engineered cell for in vivo self-supply of the rhodopsin-activator, all-trans-retinal. Our concept exploiting the light-powering ATP supplier offers a potential increase in carbon use efficiency for microbial productions through metabolic reprogramming.

A Unified View on Varied Ultrafast Dynamics of the Primary Process in Microbial Rhodopsins.

All-trans to 13-cis photoisomerization of the protonated retinal Schiff base (PRSB) chromophore is the primary step that triggers various biological functions of microbial rhodopsins. While this ultrafast primary process has been extensively studied, it has been recognized that the relevant excited-state relaxation dynamics differ significantly from one rhodopsin to another. To elucidate the origin of the complicated ultrafast dynamics of the primary process in microbial rhodopsins, we studied the excited-state dynamics of proteorhodopsin, its D97N mutant, and bacteriorhodopsin by femtosecond time-resolved absorption (TA) spectroscopy in a wide pH range. The TA data showed that their excited-state relaxation dynamics drastically change when pH approaches the pKa of the counterion residue of the PRSB chromophore in the ground state. This result reveals that the varied excited-state relaxation dynamics in different rhodopsins mainly originate from the difference of the ground-state heterogeneity (i.e., protonation/deprotonation of the PRSB counterion).

Molecular Origin of the Anomalous pH Effect in Blue Proteorhodopsin.

Proteorhodopsin (PR) is a light-driven proton pump found in marine bacteria, and thousands of PRs are classified into blue-absorbing PR (BPR; λmax ∼ 490 nm) and green-absorbing PR (GPR; λmax ∼ 525 nm). We previously presented conversion of BPR into GPR using the anomalous pH effect. When we lowered the pH of a BPR to pH 2 and returned to pH 7, the protein absorbs green light. This suggests the existence of the critical point of the irreversible process at around pH 2, but the mechanism of anomalous pH effect was fully unknown. The present size exclusion chromatography (SEC) and atomic force microscope (AFM) analysis of BPR from Vibrio califitulae (VcBPR) revealed the anomalous pH effect because of the conversion from pentamer to monomer. The different pKa of the Schiff base counterion between pentamer and monomer leads to different colors at the same pH.

Acid-base equilibrium of the chromophore counterion results in distinct photoisomerization reactivity in the primary event of proteorhodopsin.

Proteorhodopsin (PR) is a proton-pumping rhodopsin, and it is known to exhibit a multi-phasic decay of the excited-state population in the primary process. So far, this complex excited-state decay has been attributed to the branching of the relaxation pathway on the excited-state potential energy surface. However, a recent ultrafast spectroscopic study on a sodium-pumping rhodopsin suggested that such a complex decay may originate from the heterogeneity in the ground state due to the acid-base equilibrium of the counterion of the protonated retinal Schiff base (PRSB). In this study, we studied the excited-state dynamics of PR at pH 11 and 4, in which the counterion of the PRSB, Asp97, is completely deprotonated and protonated, respectively. The obtained time-resolved absorption data revealed that the excited-state lifetime is decisively governed by the protonation state of Asp97, and the photoisomerization of the PRSB chromophore proceeds faster and more efficiently when Asp97 is deprotonated. This conclusion was further supported by high similarity of the excited-state dynamics between PR at pH 4 and the D97N mutant in which Asp97 is replaced with neutral Asn. The results of this study suggest that the protonation state of the PRSB counterion plays a decisive role in determining the excited-state dynamics and the photoisomerization reactivity of rhodopsins in general, by making a significant influence on the exited-state potential energy surface of the PRSB chromophore.

Crystal structure of heliorhodopsin.

Heliorhodopsins (HeRs) are a family of rhodopsins that was recently discovered using functional metagenomics1. They are widely present in bacteria, archaea, algae and algal viruses2,3. Although HeRs have seven predicted transmembrane helices and an all-trans retinal chromophore as in the type-1 (microbial) rhodopsin, they display less than 15% sequence identity with type-1 and type-2 (animal) rhodopsins. HeRs also exhibit the reverse orientation in the membrane compared with the other rhodopsins. Owing to the lack of structural information, little is known about the overall fold and the photoactivation mechanism of HeRs. Here we present the 2.4-Å-resolution structure of HeR from an uncultured Thermoplasmatales archaeon SG8-52-1 (GenBank sequence ID LSSD01000000). Structural and biophysical analyses reveal the similarities and differences between HeRs and type-1 microbial rhodopsins. The overall fold of HeR is similar to that of bacteriorhodopsin. A linear hydrophobic pocket in HeR accommodates a retinal configuration and isomerization as in the type-1 rhodopsin, although most of the residues constituting the pocket are divergent. Hydrophobic residues fill the space in the extracellular half of HeR, preventing the permeation of protons and ions. The structure reveals an unexpected lateral fenestration above the ß-ionone ring of the retinal chromophore, which has a critical role in capturing retinal from environment sources. Our study increases the understanding of the functions of HeRs, and the structural similarity and diversity among the microbial rhodopsins.

Point Mutation of Anabaena Sensory Rhodopsin Enhances Ground-State Hydrogen Out-of-Plane Wag Raman Activity.

The interaction between the retinal protonated Schiff base (RPSB) and surrounding protein residues inside the retinal pocket is believed to play a major role in the ultrafast isomerization of the former. Coherent time-resolved vibrational spectroscopic techniques are applied to reveal the effect of changes in the protein architecture by point mutations (V112N and L83Q) close to the RPSB in Anabaena sensory rhodopsin (ASR). Our study reveals that such point mutations have a minor effect on the low-frequency (<400 cm-1) torsional modes but dramatically influence the ground-state vibrational Raman activity of the C14-H out-of-plane (HOOP) wag mode (800-820 cm-1). In mutated ASR, the increase of HOOP Raman activity in the ground state is experimentally observed for the all- trans RPSB, which has shorter excited-state lifetime than in wild-type ASR. This indicates that predistortion of the RPSB inside the mutated retinal pocket is a major factor in the acceleration of the isomerization rate.

Fluorescence Enhancement of a Microbial Rhodopsin via Electronic Reprogramming.

The engineering of microbial rhodopsins with enhanced fluorescence is of great importance in the expanding field of optogenetics. Here we report the discovery of two mutants (W76S/Y179F and L83Q) of a sensory rhodopsin from the cyanobacterium Anabaena PCC7120 with opposite fluorescence behavior. In fact, while W76S/Y179F displays, with respect to the wild-type protein, a nearly 10-fold increase in red-light emission, the second is not emissive. Thus, the W76S/Y179F, L83Q pair offers an unprecedented opportunity for the investigation of fluorescence enhancement in microbial rhodopsins, which is pursued by combining transient absorption spectroscopy and multiconfigurational quantum chemistry. The results of such an investigation point to an isomerization-blocking electronic effect as the direct cause of instantaneous (subpicosecond) fluorescence enhancement.

Mapping the ultrafast vibrational dynamics of all-trans and 13-cis retinal isomerization in Anabaena Sensory Rhodopsin.

Discrepancies in the isomerization dynamics and quantum yields of the trans and cis retinal protonated Schiff base is a well-known issue in the context of retinal photochemistry. Anabaena Sensory Rhodopsin (ASR) is a microbial retinal protein that comprises a retinal chromophore in two ground state (GS) conformations: all-trans, 15-anti (AT) and 13-cis, 15-syn (13C). In this study, we applied impulsive vibrational spectroscopic techniques (DFWM, pump-DFWM and pump-IVS) to ASR to shed more light on how the structural changes take place in the excited state within the same protein environment. Our findings point to distinct features in the ground state structural conformations as well as to drastically different evolutions in the excited state manifold. The ground state vibrational spectra show stronger Raman activity of the C14-H out-of-plane wag (at about 805 cm-1) for the 13C isomer than that for the AT isomer, which hints at a pre-distortion of 13C in the ground state. Evolution of the Raman frequency after interaction with the actinic pulse shows a blue-shift for the C[double bond, length as m-dash]C stretching and CH3 rocking mode for both isomers. For AT, however, the blue-shift is not instantaneous as observed for the 13C isomer, rather it takes more than 200 fs to reach the maximum frequency shift. This frequency blue-shift is rationalized by a decrease in the effective conjugation length during the isomerization reaction, which further confirms a slower formation of the twisted state for the AT isomer and corroborates the presence of a barrier in the excited state trajectory previously predicted by quantum chemical calculations.

Time-resolved FTIR study of light-driven sodium pump rhodopsins.

Light-driven sodium ion pump rhodopsin (NaR) is a new functional class of microbial rhodopsin. A previous flash photolysis study of Krokinobacter eikastus rhodopsin 2 (KR2) revealed the presence of three kinetically distinct intermediates: K, L/M, and O. Previous low-temperature Fourier-transform infrared (FTIR) spectroscopy of KR2 showed that photoisomerization from the all-trans to the 13-cis form is the primary event of the Na+ pumping photocycle, but structural information on the subsequent intermediates is limited. Here, we applied step-scan time-resolved FTIR spectroscopy to KR2 and Nonlabens dokdonensis rhodopsin 2 (NdR2). Both low-temperature static and time-resolved FTIR spectra resolved a K-like intermediate, and the corresponding spectra showed few differences. Strong hydrogen-out-of-plane (HOOP) vibrations, which appeared in the K intermediate, are common among other rhodopsins. It is, however, unique for NaR that such HOOP bands are persistent in late intermediates, such as L and O intermediates. This observation strongly suggests similar chromophore structures for the K, L, and O intermediates. In fact, an isotope-labeled study that used 12,14-D2 retinal revealed that the chromophore configuration of the O intermediate in NaR is 13-cis. In contrast to the vibrations of the chromophore, those of the protein differ among intermediates, and this is related to the sodium-pumping function. The molecular mechanism of the light-driven sodium pump is discussed on the basis of the present time-resolved FTIR results.

Oligomeric states of microbial rhodopsins determined by high-speed atomic force microscopy and circular dichroic spectroscopy.

Oligomeric assembly is a common feature of membrane proteins and often relevant to their physiological functions. Determining the stoichiometry and the oligomeric state of membrane proteins in a lipid bilayer is generally challenging because of their large size, complexity, and structural alterations under experimental conditions. Here, we use high-speed atomic force microscopy (HS-AFM) to directly observe the oligomeric states in the lipid membrane of various microbial rhodopsins found within eubacteria to archaea. HS-AFM images show that eubacterial rhodopsins predominantly exist as pentamer forms, while archaeal rhodopsins are trimers in the lipid membrane. In addition, circular dichroism (CD) spectroscopy reveals that pentameric rhodopsins display inverted CD couplets compared to those of trimeric rhodopsins, indicating different types of exciton coupling of the retinal chromophore in each oligomer. The results clearly demonstrate that the stoichiometry of the fundamental oligomer of microbial rhodopsins strongly correlate with the phylogenetic tree, providing a new insight into the relationship between the oligomeric structure and function-structural evolution of microbial rhodopsins.

Origin of the Reactive and Nonreactive Excited States in the Primary Reaction of Rhodopsins: pH Dependence of Femtosecond Absorption of Light-Driven Sodium Ion Pump Rhodopsin KR2.

KR2 is the first light-driven Na+-pumping rhodopsin discovered. It was reported that the photoexcitation of KR2 generates multiple S1 states, i.e., "reactive" and "nonreactive" S1 states at physiological pH, but their origin remained unclear. In this study, we examined the S1 state dynamics of KR2 using femtosecond time-resolved absorption spectroscopy at different pH's in the range from 4 to 11. It was found that the reactive S1 state is predominantly formed at pH >9, but its population drastically decreases with decreasing pH while the population of the nonreactive S1 state(s) increases. The pH dependence of the relative population of the reactive S1 state correlates very well with the pH titration curve of Asp116, which is the counterion of the protonated retinal Schiff base (PRSB) in KR2. This strongly indicates that the deprotonation/protonation of Asp116 is directly related to the generation of the multiple S1 states in KR2. The quantitative analysis of the time-resolved absorption data led us to conclude that the reactive and nonreactive S1 states of KR2 originate from KR2 proteins having a hydrogen bond between Asp116 and PRSB or not, respectively. In other words, it is the ground-state inhomogeneity that is the origin of the coexistence of the reactive and nonreactive S1 states in KR2. So far, the generation of multiple S1 states having a different photoreactivity of rhodopsins has been mainly explained with the branching of the relaxation pathway in the Franck-Condon region in the S1 state. The present study shows that the structural inhomogeneity in the ground state, in particular that of the hydrogen-bond network, is the more plausible origin of the reactive and nonreactive S1 states which have been widely observed for various rhodopsins.

Unique Hydrogen Bonds in Membrane Protein Monitored by Whole Mid-IR ATR Spectroscopy in Aqueous Solution.

Protein function is coupled to its structural changes, for which stimulus-induced difference Fourier-transform infrared (FTIR) spectroscopy is a powerful method. By optimizing the attenuated total reflection (ATR)-FTIR analysis on sodium-pumping rhodopsin KR2 in aqueous solution, we first measured the accurate difference spectra upon sodium binding in the whole IR region (4000-1000 cm-1). The new spectral window allows the analysis of not only the fingerprint region (1800-1000 cm-1) but also the hydrogen-bonding donor region (4000-1800 cm-1), revealing an unusually strong hydrogen bond of Tyr located in the sodium binding site of KR2. Progress in ATR-FTIR difference spectroscopy provides an approach to investigating stimulus-induced structural changes of membrane proteins under physiological aqueous conditions.

Functional characterization of sodium-pumping rhodopsins with different pumping properties.

Sodium pumping rhodopsins (NaRs) are a unique member of the microbial-type I rhodopsin family which actively transport Na+ and H+ depending on ionic condition. In this study, we surveyed 12 different NaRs from various sources of eubacteria for their electrophysiological as well as spectroscopic properties. In mammalian cells several of these NaRs exhibited a Na+ based pump photocurrent and four interesting candidates were chosen for further characterization. Voltage dependent photocurrent amplitudes revealed a membrane potential-sensitive turnover rate, indicating the presence of an electrically-charged intermediate(s) in the photocycle reaction. The NaR from Salinarimonas rosea DSM21201 exhibited a red-shifted absorption spectrum, and slower kinetics compared to the first described sodium pump, KR2. Although the ratio of Na+ to H+ ion transport varied among the NaRs we tested, the NaRs from Flagellimonas sp_DIK and Nonlabens sp_YIK_SED-11 showed significantly higher Na+ selectivity when compared to KR2. All four further investigated NaRs showed a functional expression in dissociated hippocampal neuron culture and hyperpolarizing activity upon light-stimulation. Additionally, all four NaRs allowed optical inhibition of electrically-evoked neuronal spiking. Although efficiency of silencing was 3-5 times lower than silencing with the enhanced version of the proton pump AR3 from Halorubrum sodomense, our data outlines a new approach for hyperpolarization of excitable cells without affecting the intracellular and extracellular proton environment.

Domain-based biophysical characterization of the structural and thermal stability of FliG, an essential rotor component of the Na+-driven flagellar motor.

Many bacteria move using their flagellar motor, which generates torque through the interaction between the stator and rotor. The most important component of the rotor for torque generation is FliG. FliG consists of three domains: FliGN, FliGM, and FliGC. FliGC contains a site(s) that interacts with the stator. In this study, we examined the physical properties of three FliG constructs, FliGFull, FliGMC, and FliGC, derived from sodium-driven polar flagella of marine Vibrio. Size exclusion chromatography revealed that FliG changes conformational states under two different pH conditions. Circular dichroism spectroscopy also revealed that the contents of α-helices in FliG slightly changed under these pH conditions. Furthermore, we examined the thermal stability of the FliG constructs using differential scanning calorimetry. Based on the results, we speculate that each domain of FliG denatures independently. This study provides basic information on the biophysical characteristics of FliG, a component of the flagellar motor.