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Cite This: Chem. Rev. XXXX, XXX, XXX−XXX

Microbial Halorhodopsins: Light-Driven Chloride Pumps Christopher Engelhard,†,⊥ Igor Chizhov,‡ Friedrich Siebert,§ and Martin Engelhard*,∥ †

Fachbereich Physik, Freie Universität Berlin, 14195 Berlin, Germany Institute for Biophysical Chemistry, Hannover Medical School, OE8830 Carl-Neuberg-Straße 1, 30625 Hannover, Germany § Institut für Molekulare Medizin und Zellforschung, Sektion Biophysik, Albert-Ludwigs-Universität Freiburg, Hermann-Herderstr. 9, 79104 Freiburg, Germany ∥ Max Planck Institute for Molecular Physiology, Otto Hahn Str. 11, 44227 Dortmund, Germany ‡

ABSTRACT: Early research on the four microbial rhodopsins discovered in the archaeal Halobacterium salinarum revealed a structural template that served as a scaffold for two different functions: light-driven ion transport and phototaxis. Bacteriorhodopsin and halorhodopsin are proton and chloride pumps, respectively, while sensory rhodopsin I and II are responsible for phototactic behavior of the archaea. Halorhodopsins have been identified in various other species. Besides this group of archaeal halorhodopsins distinct chloride transporting rhodopsins groups have recently been identified in other organism like Flavobacteria or Cyanobacteria. Halorhodopsin from Natronomonas pharaonis is the best-studied homologue because of its facile expression and purification and its advantageous properties, which was the reason to introduce this protein as neural silencer into the new field of optogenetics. Two other major families of genetically encoded silencing proteins, proton pumps and anion channels, extended the repertoire of optogenetic tools. Here, we describe the functional and structural characteristics of halorhodopsins. We will discuss the data in light of common principles underlying the mechanism of ion pumps and sensors and will review biophysical and biochemical aspects of neuronal silencers.

CONTENTS 1. Introduction 2. Discovery of Halorhodopsins 2.1. Historical Perspectives 2.2. Archetypical Halorhodopsins 2.3. Newly Discovered Halorhodopsins 3. Purification of Halorhodopsins 4. Structure of Halorhodopsins 4.1. General Structure 4.2. Retinal Binding Pocket and Active Site 4.3. Anion Binding Sites 4.4. Chloride Uptake and Release Channels 5. Mechanism of Photocycle and Anion Transport 5.1. General Remarks on Photocycle Measurements and Evaluation 5.2. Photocycle of Halorhodopsin 5.3. Mechanism of Anion Transport 6. Photoreceptors for Optogenetic Silencing of Neuronal Activity 6.1. Properties of Optogenetic Tools: General Aspects 6.1.1. Biophysical and Biochemical Properties 6.1.2. Expression and Targeting 6.1.3. General Requirements for Optogenetic Applications 6.2. New Silencing Optogenetic Tools 7. Concluding Remarks Author Information Corresponding Author © XXXX American Chemical Society

ORCID Present Address Notes Biographies Acknowledgments Abbreviations References

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1. INTRODUCTION Optogenetics has recently drawn considerable attention for developing strategies to control cells like neurons by using light-sensitive proteins.1,2 Different families of photoreceptors have been found to be suitable for optogenetic applications. These light-sensitive proteins include, e.g., phytochromes, LOV domains, or microbial rhodopsins. With the advent of optogenetics the interest into the properties of microbial rhodopsins has been renewed. An additional factor, which stimulated the research, was the discovery that this family of proteins are found in all three kingdoms of life.3−5 There are now over 7000 different such proteins within data banks. All microbial rhodopsins possess a scaffold of seven transmembrane helices (A−G). The C-terminal helix G harbors an anchor, which connects all-trans retinal via a protonated Schiff base to a lysine residue. In general, this family is grouped

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Special Issue: Optogenetics and Photopharmacology Received: November 30, 2017

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DOI: 10.1021/acs.chemrev.7b00715 Chem. Rev. XXXX, XXX, XXX−XXX

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Figure 1. First photocycle models of a microbial rhodopsin: fundamental ancestors of future photocycle models. Already in this early paper, lightand dark-adapted photocycles were introduced. The models are in principle in accordance with modern descriptions. With the data available in the early 1970s a correct assignment of the release of a proton was not yet possible (proton release occurs before M412). In addition the determination of the trans−cis isomerization of retinal during the photocycle became available only later.37 Reproduced with permission from ref 34. Copyright 1975 Elsevier. k0 to k4 are first order rate constants. LA and DA are abbriviations for light adated and dark adapted BR.

into ion pumps, channels, and sensors (for more general reviews about the family of microbial rhodopsins, see refs 3 and 5−9). The first microbial rhodopsin, which they named bacteriorhodopsin (BR), was discovered by Stoeckenius and Oesterhelt in 1971 as the main component of the purple membrane.10 They also were able to determine the function of BR as a lightdriven proton pump.11 They argued that BR plays the central role in energy coupling, a theory which was put forward by Mitchell. For detailed information on Mitchell’s chemiosmotic hypothesis refer to his Nobel lecture.12 Racker and Stoeckenius, who prepared vesicles containing ATP synthase and purple membrane, later conclusively proved this hypothesis.13 The proton gradient generated on light excitation was sufficient for the formation of ATP from ADP. A couple of years later, Mukohata and his co-worker Matsuno-Yagi14 observed in a Halobacterium salinarum strain lacking BR an increase of pH as well as ATP upon light excitation of the cells. The authors concluded the presence of a bacteriorhodopsin different from that in purple membrane, which Mukohata and Kaji subsequently named halorhodopsin (HR).15 Interestingly, already in the mid-1970s hints for another, a third rhodopsin like pigment, were described. Hildebrandt and Dencher noticed a sensory response of the archaea with a maximum of its action potential at 565 nm,16 which was later recognized as sensory rhodopsin I (SRI).17,18 Subsequently, a second sensory rhodopsin (SRII), which triggers a photophobic response of the archaea was identified.19−22 These early experiments proved the existence of at least two different functionalities found in H. salinarum: ion transport and sensory response. Two decades later, a third distinct function was reported in Chlamydomonas reinhardtii. Nagel and colleagues demonstrated that channel-rhodopsin-1 (ChR1) and channelrhodopsin-2 (ChR2) from the green algae C. reinhardtii function as light gated cation channels.23,24 These publications marked the onset of the field of optogenetics.1 It was immediately recognized that this light-activated protein, once expressed in neurons, can be used for fast cellular depolarization. Subsequently, halorhodopsin from Natronomonas pharaonis (NpHR) was also expressed in neuronal cells,25 and upon light excitation the cells were hyperpolarized. Experiments that were performed together with channelrhodopsin enabled the bidirectional control of neural activity.26

Besides this group of archaeal halorhodopsins two other distinct chloride transporting rhodopsins groups have most recently been identified in flavobacteria27,28 and in cyanobacteria.29,30 Phylogenetic analysis and sequence comparison suggest that these three groups of anion pumps are distinct from each other forming their own branch of the phylogenetic tree.31 Knowledge about the molecular mechanism of light-activated anion transfer in halorhodopsins from different species is not only of significance for precise manipulation of neural activity but also for a general understanding of the diverse functions of microbial rhodopsins. In this review, we provide an overview about the structural and functional properties of halorhodopsins and discuss general mechanism of ion transfer. An additional chapter is dedicated to the use of ion pumps and anion channels as optogenetic tools.

2. DISCOVERY OF HALORHODOPSINS 2.1. Historical Perspectives

With the discovery of BR10,11 intensive research on its functional and structural properties started. In these early years some important data were assembled, which became a paradigm for the description of all subsequently discovered microbial rhodopsins. Early obtained spectroscopic data indicated that BR undergoes a light/dark adaptation cycle, which was attributed to all-trans → 13-cis retinal thermal isomerization,32 a property which has been demonstrated for other microbial rhodopsins as well. The first experiments describing a reversible photolysis revealed the spectral nature of photocycle components. For example, an intermediate was identified absorbing at 412 nm.33 In a more detailed analysis Lozier at al. proposed a comprehensive photocycle model34 (Figure 1). This prototype already included archetypical intermediates that are still applied to describe photocycles of newly discovered microbial rhodopsins. This paper also coined the nomenclature for the intermediates: based on the photoreaction of Iodopsin, which included an intermediate, Metaiodopsin II (Meta II was also found in rhodopsin), absorbing below 400 nm,35 Lozier and co-worker named the intermediate absorbing at 412 nm M 412 . The other intermediates were then labeled in sequence of their appearance K590 → L550 → M412 → N520 → O640. In this paper, the uptake of the proton was placed in the second half of the photocycle. Later photocurrent kinetic experiments of B


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Further progress was attained when HR from Natronomonas pharaonis was discovered.53,54 This HR homologue (NpHR, the terms NpHR and HsHR were coined by Sato al. in 200555) from an alkalophilic bacterium turned out to have favorable properties in relation to expression, purification, and stability. This is a reason why up to now most data on HR were gathered from NpHR and why it was directly introduced as a neural silencer tool into the field of optogenetics (for a recent review see ref 5).

purple-membrane sheets bound to planar bilayer membranes indicated that proton release occurs during the formation of M412.36 This timing of release and uptake of the proton indicates that during the M-state a switch has to occur, which regulates the accessibility of the Schiff base from the extracellular and cytoplasmic side of the membrane. This property is essential for the vectorial transport of ions and is also of importance for the function of other microbial rhodopsins. Even though their functions are quite diverse, comprising light-driven ion pumping, phototaxis, and ion-channels, two other primary findings seem to be common for all microbial rhodopsins. First, for all pigments so far analyzed the primary photo event is the isomerization of the all-trans retinylidene Schiff base to its 13-cis configuration at a time scale in the ps range. This reaction was first described by Braiman and Mathies in 1982.38 The second observation concerns the sites and nature of functional amino acids. Functionally important amino acids are located on helix C and a lysine residue as retinal binding site on helix G. The special pattern of these amino acids determine the function of the microbial rhodopsin. For example, mutating D 85 in BR to Asn (as in HR) turns the proton pump into a halide pump.39 From a historical point of view, it is noteworthy that emerging new biophysical methods were applied to the analysis not only of BR but also to animal rhodopsin. In the seventies these two membrane proteins were most easily accessible possessing a chromophore suitable for UV/vis and vibrational spectroscopy like FTIR40 and Resonance Raman41,42 methods. Another biophysical method, solid state NMR spectroscopy, also made its debut in the analysis of membrane proteins. One of the first NMR papers on BR was published by the Griffin group, in which the isomeric nature of retinal was determined.43,44 In later work it was shown that the method can also be applied to specifically labeled amino acids incorporated into BR.45 These developments of new techniques and methods were implemented in order to gain more detailed information on pertinent problems like, e.g., the elucidation of membrane proteins. Papers from this period also showed the synergistic advantage of combining biophysics with biological chemistry, the latter providing tailor-made samples.

2.3. Newly Discovered Halorhodopsins

Metagenomic analyses of marine organisms revealed a new family of proton pumps (proteorhodopsins).56 Further investigations showed that some marine bacteria possess a novel rhodopsin functionality, an outward Na+-pumping rhodopsin (KR2).57 Comparing the amino acid composition at functionally important sites on helix C one can correlate the function to distinct amino acid patterns. In BR these sites are D85, T89, and D96 for which a DTD motif has been coined. Accordingly, proteorhodopsins are characterized by a DTE, sodium pumps like KR2 by a NDQ, and archaeal HR by a TSA motif (see Table 1).57 Nonlabens marinus S1-08T encodes three Table 1. Amino Acid Motifs of Microbial Rhodopsinsa microbial rhodopsins

motif

archaeal proton pumps proteorhodopsins sensory rhodopsins sodium pumps archaeal halide pumps eubacterial chloride pumps cyanobacterial anion pumps

DTD DTE DTF NDQ TSA NTQ TSDb

a

For a recent phylogenetic tree of selected functional groups of microbial rhodopsins see Harris and co-workers.31 bThe third position could also be V, L, or I.

different types of rhodopsins: Yoshizawa et al. analyzed the function of three flavobacterial rhodopsins from this organism.27 N. marinus rhodopsin 1 (NM-R1) and N. marinus rhodopsin 2 (NM-R2) are light-driven outward-translocating H+ and Na+ pumps, respectively. N. marinus rhodopsin 3 (NMR3; also named ClR27) with a novel NTQ motif functions as an inward directed Cl− pump. In analogy to the nomenclature of archaeal rhodopsins, we propose to name new pigments according to their original organism and their functionality, i.e., NM-R3 (ClR) should be renamed to NmHR. In 2017, two papers were published describing members of a new branch of anion pumps.29,30 MrHR from the cyanobacterium Mastigocladopsis repens functions as an inward directed chloride pump. Its amino acid motif TSD is quite close to that of the archaeal halide pumps (TSA).29 In the same year a second anion pump from a Synechocystis species has been characterized,30 which possesses also a TSD motif. Interestingly, SyHR turned out to be the first example as a transporter not only for chloride but also for the divalent sulfate. It is intriguing that the connecting element of the three groups is their function as light-driven anion pumps, although the motifs are quite different and the sequence homology between them is not so high. A phylogenetic analysis places the cyanobacterial branch of light-driven anion pumps close to those of archaeal origin.31 Molecular details and functional studies of these three groups of anion pumps have been and will be important to

2.2. Archetypical Halorhodopsins

The first hint for an archaeal rhodopsin other than BR was published by Mukohata and his co-worker Matsuno-Yagi in 1977.14 They concluded from experiments using a H. salinarum strain lacking purple membrane on a second BR-like membrane protein, capable to drive light-activated ATP synthesis. Its action spectrum was red-shifted from that of BR and its heat stability as well as NH2OH sensitivity also differed. In a later publication, Mukohata named this protein halorhodopsin (HR).15 Soon it was recognized that HR also possesses a photocycle.46 Interestingly, different to BR, an M-intermediate seemed to be lacking.47,48 At first, this new halobacterial rhodopsin was described as a sodium pump49,50 but was later unequivocally identified as an inward-directed chloride pump by Schobert and Lanyi.51 The authors demonstrated, using vesicles containing HR, that a chloride inward transport arises against both electrical and concentrations gradients. In 1984, an unambiguous proof was presented by Bamberg and colleagues by using black lipid membranes to which HR-containing vesicles were added.52 The authors demonstrated that photocurrents could only be observed in the presence of chloride. C


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BR prompting the question of how the same scaffold can serve as template for diverse functionalities, i.e., those of a proton versus a chloride pump. It is of interest to note that this work is again a good example for the congenial interplay between method development (improved electron microscopy) and the scientific demand for better data (high-resolution structure). In the same year, Kolbe et al. published the first highresolution structure of HsHR using crystals grown in a cubic lipidic phase.74 Landau and Rosenbusch had introduced this now frequently used new approach for the crystallization of membrane proteins.75 Despite the facts that the proteinchemical properties of NpHR are more favorable than those of HsHR and that it can be easily expressed in E. coli,63,76 it took ten years before a crystal structure of NpHR was finally obtained.77 With the crystal structure of NpHR available, a comparison between the two homologues NpHR and HsHR on the one hand and between HR and BR on the other hand provided further insight on similarities and differences in the general architecture of the three proteins. As it is evident from Figure 2A, the traces of the backbone are almost identical. This

deepen further our understanding of the molecular mechanism of anion transport.

3. PURIFICATION OF HALORHODOPSINS The purification of HR was originally performed from its natural hosts H. salinarum and N. pharaonis.58−60 Ihara et al.61 introduced an N. pharaonis mutant, overproducing NpHR that was used for the crystallographic work of the group of Kouyama. Subsequently expression of microbial rhodopsins in E. coli became available,62 which was adapted also to NpHR63 as well. The advantage of this new approach was 2-fold. Larger amounts of proteins could be obtained in shorter times and second C-terminal His-tags allowed NpHR to be purified in high and excellent yields. The purification protocol for membrane proteins demanded to solubilize the proteins from their membrane. In order to obtain conditions that are more natural NpHR had to be reconstituted into lipid membranes.64 It has not been possible so far to express HsHR in E. coli. However, an efficient expression system for His-tagged HsHR has been established in the H. salinarum strain L33.65 In general, the expression of newly discovered halorhodopsins have been accomplished using an E. coli expression system.28−30 Hosaka and co-workers66 used a cell free protein synthesis approach in their work on the crystallization of NmHR. This method had already been successfully applied for the synthesis of BR.67 4. STRUCTURE OF HALORHODOPSINS 4.1. General Structure

The primary structures of HsHR and NpHR show an amino acid sequence identity of 65.7%.68,69 This decreases to about 24% when compared to BR representing their functional differences as Cl− or H+ pumps. Despite the seemingly high homology between HsHR and NpHR there are interesting differences, which might contribute to the observed distinctive differences of the functional properties of these two proteins. Generally, NpHR contains fewer buried positively charged residues than HsHR, and the interhelical loop between helices B and C is comparatively larger. On the other hand, NpHR does not possess an arginine (R103 in HsHR) at the extracellular end of helix C. Following the numbering of the BR sequence, one can deduce that proton pumps are characterized by D-85 and D-96, sensors by D-85 and Y-96 or F-96, and chloride pumps by T-85 and A-96.70 The classification for ion-pumping microbial rhodopsins has been further refined by Kandori and his colleagues (reviewed in ref 6; see above). The first structural information about HsHR was gained from cryo-electron microscopy on 2D crystals.71−73 In contrast to BR, which forms trimers in a two-dimensional lattice, HsHR crystallizes by forming a tetragonal lattice.72 However, this arrangement seemed to be physiologically irrelevant because of the alternating inside-out organization of the monomers. In a later publication, Henderson and colleagues73 improved the resolution considerably by using a new generation of electron microscopes and by introducing a novel procedure of data evaluation, which led to corrections of translational disorders that were more accurate. The effective resolution was 5 Å in the plane of the membrane and 12 Å perpendicular to it. The authors could discern the position of the retinal chromophore as well those of aromatic residues. The results clearly showed that the overall architecture of HsHR is quite similar to that of

Figure 2. Structural comparison between HsHR (green, PDB IE12), NpHR (blue, PDB 3A7K), and BR (orange, PDB 3NS0). Structure Bfactors are mapped to color saturation and loop thickness. The full view of the structures (A) demonstrates that the three proteins show very similar structures in the transmembrane helices A−G, while structures are both more dissimilar and much more flexible in the extra- and intracellular loops. Residues D85 and D96, critical for pumping in BR, are replaced by threonine and alanine, respectively, in HR (B).

congruence is also reflected in the overlap of the retinal chromophore and the side chain positions of BRD85 and BRD96 with the corresponding amino acids threonine and alanine in HsHR and NpHR (Figure 2B). At the position of the carboxyl group from BRD85, a chloride is located in both HRs. It is interesting to note that the side chain of threonine in HsHR is rotated by about 60° as compared to that in NpHR, placing its OH group away from the chloride while forming a hydrogen bond with a palmitic acid. Palmitate, which is not synthesized by H. salinarum but is ubiquitous in its natural environment or growth media, is at the center around which the trimer of HsHR is assembled.74 Similarly, NpHR possesses also a cofactor, bacterioruberin, which is bound in a crevice between neighboring subunits of the trimer but is not involved in the chloride-binding site.77 D


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Converse to the core part of the proteins, the extracellular and cytoplasmic surfaces are quite different in their dynamic properties. The B-factors of the loops are considerably larger displaying a mobility to accommodate chloride release and uptake. The cytoplasmic (CP) and extra cellular (EC) surfaces are quite different in size. The extracellular side, being the uptake side for chloride, is covered in NpHR by the larger BC loop and the α-helical N-terminus. Compared to HsHR the BC loop carries an insertion of ten amino acids.68 This extension together with the helix of the N-terminus forms a hydrophobic cap, which is absent in HsHR. Kouyama et al. argue that these differences between NpHR and HsHR might be significant for their differing properties in the mechanism of chloride transport77 (see below). Two crystal structures of NmHR have been published in 2016.66,78 Both structures revealed a closer similarity to the sodium pump KR2 from K. eikastus than to the archaeal halorhodopsins,79,80 which indicates that NmHR and KR2 evolved from each other. This observation is somewhat surprising because of the opposite transport directions and charges.66 The overall structures of BR, HsHR, and NpHR are quite similar to that of NmHR although there are differences in the loop and N- and C-terminal helix regions. A well-defined Cl−1 binding site was close to the protonated Schiff base. A second Cl−1 binds to the A-B loop,78 which is strengthened by a positive patch.

Figure 3. Counterion complex of bacteriorhodopsin (A: HsBR, PDB 1C3W83) and those of halorhodopsins from H. salinarum (B: HsHR, PDB 1E1265), N. pharaonis (C: NpHR, PDB 3A7K77), and N. marinus (D: NmHR, PDB 5B2N66). The structures of the four proteins were aligned and equal sections were chosen for each of them. The figures display the same line of sight.

4.2. Retinal Binding Pocket and Active Site

The polyene chain of retinal is sandwiched between aromatic side chains for both HsHR and NpHR in an identical topology.74,77 This positioning is almost identical to that of other microbial rhodopsins. According to the crystal structures, retinal assumes an all-trans configuration, which has also been confirmed by resonance-Raman for HsHR81 and NpHR.82 Like in BR a 15-anti configuration of the Schiff base of the retinal chromophore of HsHR was independently determined by crystallography and resonance-Raman spectroscopy.74,81 The architecture of the active sites of HsHR and NpHR facing the extracellular side are quite similar. The hydrogenbond pattern includes water molecules as well as charged and polar amino acids. The water clusters of NpHR and HsHR display a slightly different arrangement, although the chloride ions are placed in identical positions. In the original crystal structure of HsHR published by Kolbe et al.,74 the methyl group of T111 faces the chloride ion because the hydroxyl group interacts with the carboxyl group of a structural palmitate. Interestingly, crystallization of HsHR in a new crystal form with little constraints for the E-F loop65 reveals T111 in the same orientation as that found for NpHR77 with the hydroxyl group interacting with chloride. In Figure 3 the counterion complexes of the protonated Schiff base are depicted for BR (A), HsHR (B), NpHR (C), and NmHR (D). It is evident that the topologies are quite similar, although the functions as proton or chloride pumps are different. In each case, the topology encompasses water molecules and canonical amino acid side chains at defined sites. For HsHR (NpHR) the latter ones include T111(126) and D238(252). The corresponding amino acids in BR are D85 and D212, respectively. For NmHR, Cl−1 forms a hydrogen bond with the protonated Schiff base, which faces the extracellular side. Amino acids involved in Cl−1 binding in archaeal HRs (T and S residues) are replaced by N98 and

T102. However, the charge distribution of the counterion complex is generally conserved for the four proteins. 4.3. Anion Binding Sites

The crystal structures of HsHR65,74,84 and NpHR77,85,86 revealed, as discussed above, one high affinity-binding site close to the Schiff base. The topologies between the two homologues are almost identical (Figures 3 and 4). On the

Figure 4. Br binding sites in N. pharaonis HR.86 The structures shown are from subunit B (resting state and N state) and subunit C (O state; PDB 4QRY). The O state was superimposed by aligning the resting state structure of subunit C with that of subunit B. Color coding: resting state (yellow), N state (light green), and O state (dark green). In the resting state, Br− (orange) is bound at site I close to T126. In the N state (light green), a rotation of T126 and I134 causes the ion to move from a transiently generated binding site in L2 to site i134 still in close contact with the Schiff base. The O state (dark green) is structurally very similar to the N state but has a Br− ion situated at site II. E


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5. MECHANISM OF PHOTOCYCLE AND ANION TRANSPORT

other hand, low-affinity sites, which have been identified in various crystal structures are not congruent between HsHR and NpHR. Following previous structural studies, T. Kouyama and his co-workers described three anion binding sites in total.85 Besides the canonical site INpHR, the anion can bind to residues of neighboring subunits and the N-terminal end of bacterioruberin that is sandwiched between protomers (site IINpHR). The third site (site IIINpHR), only weekly populated, was found between the B−C loop and the F-G loop. A second site has also crystallographically been determined in the HsHR mutant T203 V84 but not in the crystal structure of the wild type. This site (IIHsHR) close to the extracellular surface is located at the B−C loop close to the start of helix C and is probably part of the chloride uptake pathway. It does not resemble the structure of site IIINpHR. Multiconformation continuum electro-statics calculations87 revealed further chloride-binding sites differing from those identified in structural analysis. Whether or not these additional sites are of functional importance is not clarified. It should be noted that in titration experiments only one binding site each for both HsHR and NpHR were identified. However, from functional investigations, e.g., photocycle measurements, at least an additional low affinity site was deduced (see below). As mentioned above for NmHR two Cl−1 binding sites have been identified.78 This second binding site is close to the A-B loop on the cytoplasmic site, which is different to those identified for HsHR and NpHR.

5.1. General Remarks on Photocycle Measurements and Evaluation

Early studies on the kinetics of light-activated microbial rhodopsins and other chromophore containing proteins like rhodopsins, phytochromes, or cryptochromes have been an important factor to elucidate the function of these proteins. There are three main aspects related to obtaining kinetic data, data evaluation, and interpretation. It is obvious that the database has to be as complete as possible. For example, the spectrum of an intermediate can only be obtained if a complete set of different wavelength is used for the kinetic experiments. To obtain information about the thermodynamics of the process, temperature dependencies have to be measured. The influence of external conditions, like, e.g., pH, salt concentration, or in the case of HR the type of anions, have also to be analyzed. It is possible to include other time-resolved techniques like vibrational spectroscopy or photocurrent measurements as well. An example of such a study is described in ref 90 where visible absorption, infrared, and photocurrent techniques were combined to study the BR reaction cycle. The data evaluation must cope with a large amount of information and different errors associated with each measurement. The presence of multiple kinetics in a given reaction requires methods for the exact and unbiased extraction of an unknown number of exponents from the data. There are a number of methods available, which deal with this problem. Gerwert et al.91 applied factor analysis and decomposition, which is in principle an extension of singular value decomposition (SVD). Another approach employed a global multiexponential nonlinear least-squares fitting program.90,92 These approaches assume that the reactions occur between well-defined intermediates and not between populations of closely related species, which would give rise to distributed kinetics. For this latter case, the corresponding fitting procedure is based on power law techniques. It has already been applied for the photocycle of BR 30 years ago but has not been followed up.93 Having determined a set of exponents, there is only a single case for which the problem of deriving the spectra of intermediates has an exact solution. This is the sequence of irreversible transitions between intermediates.94

4.4. Chloride Uptake and Release Channels

The architecture of chloride uptake channels facing the extracellular side in HsHR and NpHR are slightly different.74,77 The loop between helices B and C is much larger in NpHR than in HsHR. It covers part of the extracellular surface while forming a hydrophobic cap close to the chloride uptake zone. Between this natronobacterial cap and the active site, the uptake pathway is characterized by seven water molecules, which form hydrogen bonds with each other and with R123, R176, E234, and H100. H100 is located at the entrance of the channel. Its mutation to alanine caused a decrease in transport activity.88 Another amino acid, E234 (E219 in HsHR) becomes deprotonated during the early part (L2) of the photocycle.89 Although the extended BC loop is missing in HsHR, it also possesses a hydrophobic patch at the entrance of the chloride uptake channel comprising five hydrophobic residues.74 These residues are highly conserved in halorhodopsins. Kolbe et al.74 argue that this “greasy walling” might assist desolvation of chloride. The hydrophilic chloride uptake channel comprises , in addition to the chloride ion, two arginine residues and E105, which are involved in the coordination of this second chloride as well.65,84 Contrary to the uptake channel, a release channel for chloride is not discernible in the ground state structures of either HsHR or NpHR because of the close packing of the protein. To open a channel, light-activated conformational changes have to occur in order to enable the chloride to get from the Schiff base to the cytoplasm. Possible mechanism will be discussed below. The Cl1− transduction pathway of NmHR66,78 is quite similar to that of NpHR77 and HsHR74 but quite different to that of the Na+ pump KR2.79,80 The extracellular channel is open to the solvent, whereas the cytoplasmic channel is sealed from the cytoplasm blocking the backflow of Cl1− in the dark.

5.2. Photocycle of Halorhodopsin

The photocycles of NpHR and HsHR and mutants thereof have been extensively studied by various methods, including UV−vis and vibrational spectroscopy. The kinetics of charge transfer has also been investigated. Finally, the structures of low temperature trapped intermediates have been determined using X-ray crystallography. Generally, all archetypical intermediates, which have been defined in the analysis of the photocycle of BR have also been identified in HR. The M-intermediate, not detected in the native photocycle, can be generated artificially under special conditions, e.g. the addition of azide.60,95 Kinetic studies of NpHR and HsHR led to photocycle schemes84,96−101 that includes a spectrally silent transition between L1 and L2, which are only distinguished by protein and water FTIR bands.100,102 Váró et al. proposed a linear scheme for NpHR, which only includes reversible transitions with the hγ

exception of the last irreversible step.98 (→ K ↔ L1 ↔ L2 ↔ N F


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↔ O ↔ HR′ → HR). Contrary to the NpHR photocycle, for HsHR an O-intermediate has not been observed. Another difference concerns light-dark adaptation, which does not happen in NpHR.82,103 The all-trans/13-cis isomeric compositions of retinal for NpHR have been determined to be 84%/ 16% (trans/cis) under white light illumination and 86%/14% in the dark. The corresponding values for HsHR are 75%/16% and 45%/55%.103 The observation of a considerable contribution of 13-cis retinal in HsHR complicated the analysis of its photocycle. This is one of the reasons why most kinetic data has been gathered for NpHR. A later comprehensive study on the photocycle of NpHR, which also analyzed the effect of temperature, ionic strength, and type of anion, has put forward a novel scheme of the NpHR photocycle that is based on six apparent rate constants, which are assigned to irreversible transitions of a single relaxation chain (Figure 5).99 The data could be modeled over the whole

ignated A1 and A299). A1 can be assigned to ground state site I, for which a dissociation constant for chloride of 3 mM60 was determined. A2 has not been identified in titration experiments indicating that it is not closely located in the vicinity of the Schiff base. From the dependency of P4 on temperature but not on chloride concentration or type of anion it was deduced that in P4 A2 accepts the anion from A1, which implies that A2 is located at the halide release site but is excluded from the bulk. Both sites have sufficiently low affinities to be insensitive toward external chloride concentrations. In P5 the affinity of A1 decreases to about 1 M enabling a transfer of chloride from the external medium. A2 also has a low affinity toward chloride (estimated to be 5.7 M). One would expect that at chloride concentrations higher than 5.7 M the transport activity of HR should be inhibited. Indeed, this effect was observed by Bamberg et al.104 and more recently by Okuno et al.105 It should be noted that the dissociation constant for chloride binding to site 1 located close to the protonated Schiff base of the TSD- and the NTQ-motif rhodopsins are 1 order of magnitude smaller (SyHR = 0.1 mM30) or larger (FpHR (also called FR) = 84 mM28). The Kd for sulfate binding to SyHR is with 5.8 mM much larger than that for Cl−. The turnover of the photocycle of NpHRs is in the range of about 20 ms,99 which would generate around 10 Cl− pumped per second. However, Kleinlogel et al. determined under stationary light conditions a turnover of 1.5 ms, which lead to much higher currents.26 Under these conditions (stationary light excitation) the last step (NpHR′ → NpHR) becomes unimportant because already NpHR′ can be excited by orange light leading to an apparent acceleration of the photocycle time. This last slow photocycle step has not only been observed in NpHR but also for BR,94 MrHR,31 SyHR,30 but not in HsHR.97 For optogenetic applications, where steady state illumination conditions are applied, the existing of a ground state like intermediate can considerably enhance the turnover rate. One would expect for SyHR an acceleration by a factor of 2 to 17.5 ms30 and for MrHR by a factor 11 to 24 ms.31 Obviously, these two HRs are less suited as neural silencer than NpHR. Photocycles of the newly discovered HRs have already been studied, although not as extensive as those of NpHR and HsHR. Generally, the photocycles display a similar sequence of intermediates with K-, L-, N-, and O-like species.27,30,31,106 Unlike HsHR,97 all newly discovered HRs display an Ointermediate during the course of the photocycle. The photocycle of the TSD motif protein SyHR has a turnover of about 40 ms in the presence of NaCl whereas in the presence of sulfate a turnover of about 650 ms.30 Interestingly, in the salt free photocycle also L- and N-like intermediates are observed, contrary to the situation in NpHR.60 As shown in Figure 5 a characteristic of the photocycle of NpHR is the existence of two O-intermediates. Interestingly Tsukamoto et al.106 observe for NmHR also two red-shifted intermediates, which they assign to O1 and O2. Apparently, these two microbial rhodopsins follow, after light excitation, similar photocycle kinetics. If in other HRs this step is hidden because of kinetic reasons it has to be elucidated. Another interesting observation was made very recently by Harris et al.,31 who studied extensively MrHR, a cyanobacterial rhodopsin. In their vis- and FTIR-kinetic paper, they could convincingly show that light-driven chloride ion transfer is coupled with the translocation of protons and water.

Figure 5. Kinetic model of the photocycle of NpHR. P1−6 denote the kinetic states, which are characterized by archetypical spectral states (K, L, O, N, and the precursor of the ground state NpHR′). The half times refer to 20 °C. Aadapted with permission from ref 99. Copyright 2001 Elsevier.

range of parameters. The spectra of kinetic states were calculated by choosing solely the fraction of cycling as free parameter. The resulting kinetic states (P1−6) are characterized by their composition of the archetypical spectral states (S1−4) K, L, O, N, and NpHR′. The spectra of P4 and P5 represent fast equilibria between spectral states S2 (L) and S3 (O) as well as between S3 (O) and S4 (N), respectively. The equilibrium between L3520 and O1600 (P4) is temperature dependent whereas that of P5 (O2600 and N520) is sensitive toward the chloride concentration and the type of anion. These results have been interpreted in analogy to the M1 → M2 transition in BR as switch of accessibility. During the first L3520↔O1600 equilibrium chloride is released to the cytoplasmic surface, whereas during the O2600↔N520 state chloride is picked up from the extracellular side. O1600 and O2600 must be differentiated from each other by their conformation and the isomerization of retinal. Indeed, in static and time-resolved FTIR experiments, an all-trans conformation of retinal was determined.102 Ergo, the reaction from P4 to P5 resembles the switch changing the ion accessibility from the cytoplasmic channel to the extracellular channel as proposed for the M1-M2 transition in BR. An important aspect of these photocycle experiments is the information gained about two chloride binding sites (desG


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5.3. Mechanism of Anion Transport

changes of the protein. The FTIR spectra of later intermediates have only been published for NpHR.102 For the later intermediates of HsHR, no FTIR data have been published to date. The results for NpHR show that the O-intermediate is characterized by an intense chromophore as well as protein bands in the amide I region. Furthermore, it was shown that retinal has assumed an all-trans configuration, a result that was also seen in the crystal structure of NpHR.86 Summarizing, the FTIR data provided evidence that major structural changes occur during the L1 → L2 transition and during the lifetime of O. After light excitation all-trans → 13-cis isomerization of retinal is triggered, which is manifest in the early Kintermediate. The back reaction occurs in the time domain of the O-intermediate. The structural and photocycle data can provide the basis for a molecular mechanism of Cl−1 transport across the membrane (Figure 6). In this review, we are using a slightly different

In recent years multiple structures of HR intermediates have been published,84,86 including those of the L1,84,86 L2,86 N,86 and O86 states. Additionally, a homologous structure to the Ointermediate was obtained in the blue form of NpHR85 by the removal of Cl1− from site I60. Furthermore, the addition of azide to NpHR converts the chloride pump107 into a proton pump with a photocycle comparable to that of BR. This observation made it possible to determine the structure of an M-like intermediate.108 It should be noted that adding azide to HsHR converts the chloride pump in a two-photon reaction into an inverted proton pump.109 It was somewhat surprising that the first structural investigation of HsHR intermediates only led to an L1 structure.84 In more recent work, Schreiner et al.65 provided a reason for this observation. These authors crystallized HsHR in a different crystal form than that which was originally obtained. Under these conditions, crystal contacts involving the loop between helices E and F were not made. It is expected that a movement of helix F like in BR110,111 or NpSRII112 is of functional importance because it provides the means for opening a channel connecting the active side with the cytoplasm. The L1 structure, verified by light-induced FTIR difference spectra, revealed that Cl−1 is still located at the same position as in the wild type structure.84 Similarly, low temperature crystallographic studies on NpHR disclosed that the anion has not yet moved from its binding site.86 Resonance Raman data showed that the halide ion forms a stronger hydrogen bond with the Schiff base,82 which might indicate that Cl−1 has moved closer to the cytoplasmic channel. Similar results were obtained for HsHR with the difference that the frequency of νCN is lower than in NpHR113,114 pointing again to different properties of the two HR homologues. In their structural investigations on NpHR Kouyama and coworkers86 were able to determine structures of intermediates which they assigned to L, N, X(N′), and O species. In the following, their structural data will be discussed with regard to the photocycle data and other results from the literature. In their structural investigation of NpHR intermediates,86 the authors isolated a structure in which the anion has moved across the barrier of retinal, which the authors designated as L2. The next intermediate in their reaction scheme assigned to N contains a halide ion located not very far from that in L2 (site i134 in Figure 4). According to their scheme Cl1− is released to the cytoplasm in X(N′). The reaction between X(N′) and O is characterized by the isomerization of retinal, the resulting structure is, with the exception of an anion situated at site II (Figure 4) similar to the anion-depleted blue membrane.85 The conformational changes accompanied by these transitions are of particular interest because they provide information about possible transfer pathways for Cl1−. Major remodelling of the protein backbone are observed for helices C and F and are connected to the formation of the N state. Reestablishing the ground state structure occurs in two steps. In O, helix F returns to its original state whereas helix C establishes its final conformation only during the last reaction. These structural data and their assignment to specific intermediates by the authors have to be reconciled by spectroscopic data. FTIR results on HsHR revealed large changes in the amide I region during the L1 → L2 transition84 indicating considerable conformational changes. Likewise, timeresolved and static FTIR experiments on NpHR100,102 demonstrated that the L1 → L2 transition solely involves

Figure 6. Reaction scheme based on the structures intermediates published by Kouyama et al.86 Adapted with permission from Figure 8 in ref 86. (Copyright 2015 Elsevier) by using PDB 4QRY.

interpretation and assignment of the structures of the intermediates than presented by Kouyama et al.86 The authors described a first intermediate L2 in which the Cl−1 has crossed the Schiff base barrier to the cytoplasmic side. In the next structure, designated N Cl−1 moves a little further away into the vicinity of an Ile (i134). Concomitantly, the cytoplasmic channel opens and water molecules can enter providing a direct pathway for Cl−1 to reach site A2 and subsequently be released into the cytoplasm. Considering the photocycle data, this intermediate could be assigned to L3520 that undergoes a fast equilibrium with O1600 in the P4 state. The next intermediate, designated N(X′), lacks a Cl−1 close to the Schiff base. The expected absorption maximum for such a motif would be at 600 nm, which would define this intermediate as O and therefore will here be assigned to O1600. The switch, reopening the extracellular channel, would occur in the next equilibrium between O2600 and N. This last intermediate has not been observed in the crystallographic study but might have a structure more similar to the ground state. As depicted in Figure 6, conformational changes mainly involve helices C and F, which alter the ion accessibility from the cytoplasmic channel to the extracellular channel. The switch between the two Ointermediates involves the reorientation of helix F, thereby closing the cytoplasmic channel and removing water molecules. Presumably, this transition would also see retinal isomerizing back to all-trans.102 It is important to note that this model of the mechanism of anion transfer of NpHR might not be applicable to HsHR. Both proteins display distinct differences in light-dark adaptation, in the spectral properties of anion-depleted protein, and in the photocycle characteristics, e.g., the missing O-intermediate. H


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Table 2. Optogenetic Silencer action spectra silencer

function

origin

Arch ArchT

proton pump proton pump

Mac NpHR

proton pump chloride pump chloride pump anion channel

Halorubrum sodomense Halorubrum strain TP009 Leptospaeria maculans Natronomonas pharaonis Haloarcula salinarum

Jaws GtACR1

Guillardia theta

photocycle

λmax (nm)

halfwidth (nm)

566a121 570125

500−632a121 480−630125

550a121 586121

484−616a121 534−638121

600120

490−640120

515127

460−560127

turnover (ms)

sow component (ms)

30−40b123

100b123

1.599

2099

current halfdecay photocurrent density (pA/pF)

(ms) 5.1c124 4.2c124

6.426 2.2120

40c124 6.9126 4.2c124

1.826 1.9d120 5.7120

200 (3s)e128

128,4129 3.2f129

a

Taken from the supplement of ref 121. bTaken from the supplement of ref 123. cData estimated from supplemental Figure 16d of ref 124. dData for eNpHR3.0. eFull recovery to the ground state takes 3 s. fHalf-decay for ZipACR.

Clearly, additional structural and kinetic information on HsHR is needed to understand its mechanism of chloride transfer. Furthermore, crystal structures of intermediates of the novel HRs would enable more educated conclusions to be drawn about the molecular mechanism of light-activated chloride transport.

following years, two main foci of research were followed. On the one hand, the optogenetic toolkit was used to delineate neuronal circuitries or animal behavior. On the other hand, extensive research has been undertaken to widen the scope of optogenetic tools in order to meet essential biophysical and physiological demands. 6.1. Properties of Optogenetic Tools: General Aspects

6. PHOTORECEPTORS FOR OPTOGENETIC SILENCING OF NEURONAL ACTIVITY Since the early 2000s optogenetics has experienced an exponential growth concerning its development of new tools and its usage in in vivo and ex vivo applications.115,116 Although the term “optogenetic” was not yet coined a first optogenetic experiment was already published in 1994. Büldt and colleagues were able to express BR into yeast mitochondria and proved its proper function as energy converter, demonstrating that a light driven proton pump can be transferred into an organelle of a nonphotoactive eukaryotic host.117 A couple of years later Crick made a farsighted proposal to turn on and off the firing of neurons.118 He stated that “The ideal signal would be light, probably at an infrared wavelength to allow light to penetrate far enough. This seems rather farfetched but it is conceivable that molecular biologists could engineer a particular cell type to be sensitive to light in this way”. Shortly before he died in 2004, his vision started to become true. His suggestion to develop light-sensitive tools in the infrared region is one important aspect of developing new optogenetic tools that is still the focus of intensive research. Elucidating static and dynamic neural circuitries is considerably facilitated by methods that allow targeted perturbation of neuronal function. Microbial rhodopsins have been proven as versatile tools to control activation and silencing of neuronal tissues and cells. In 2005, it was demonstrated that ChR2 can be expressed stably in mammalian neurons.119 When activated with a series of brief pulses of light, defined trains of spikes with a millisecond temporal resolution were generated. In search for an optimized silencing tool Han and Boyden expressed a mammalian codon-optimized NpHR gene in neurons.25 With this construct, they could demonstrate that alternating short irradiation of hippocampal neurons, coexpressing ChR2 and NpHR, with blue and yellow light can drive trains of neural hyperpolarizations and depolarizations. This repertoire of activating and silencing techniques conveys optical and temporal control with cellular precision and enables the elucidation and manipulation of neuronal circuitry. In the

The efficacy of tools for optogenetic applications depends chiefly on two general characteristics. First, the biochemical and biophysical properties of the photoreceptors have to match the experimental requirements. Second, the proteins have to be expressed efficiently in a target organism or target cells, which is not generally guaranteed. In the following, some important points for efficient applications in neurophysiological research will be outlined. 6.1.1. Biophysical and Biochemical Properties. The biophysical properties of microbial rhodopsins in general and those of the chromophore in particular are of decisive importance for optogenetic applications. The optical properties of the chromophore are determined by the absorption maxima and their absorption coefficients, the quantum yields, the photocycle characteristics, and the all-trans 13-cis isomerization ratio of the retinylidene Schiff base. As already noted by Crick probes absorbing in the long wavelength region would allow higher tissue penetration depth of light. Chuong et al. measured the relative fluence of green (532 nm) and red light (635 nm) in mouse brains depending on the distance from the optical fiber (ref 120, supplement Figure 1). At 1 mm distance green light intensity declined to about 25%, whereas the red light only weakened to about 65%. Absorption maxima around 600 nm are still rare with solely the proton pump Jaws absorbing at 600 nm,120 the chloride pump NpHR,121 and the cation channel Chrimson with an absorption maximum at 585 nm.122 The position of λmax is also important in experiments that afford bidirectional control of membrane potential, which afford spectrally orthogonal absorption maxima (see Table 2 for a list of absorption maxima of selected proteins). For example, Mac and NpHR (excitation wavelengths: 470 and 630 nm, respectively) can serve in multicolour silencing experiments.121 In another approach, using a gene-fusion strategy permitted stoichiometric expression of neuronal silencers and activators like, e.g., ChR2 and NpHR.26 A thorough analysis of photocycles is still lacking for most of the optogenetic tools. For BR and NpHR as archetypical representatives of proton and chloride pumps, the photocycles I


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can be affected or transgene-specific immune responses can be triggered. In the case of neural tissues, opsins do not seem toxic to human neurons in the brain or retina.142,143 In order to reach the target site functionally intact microbial rhodopsins have to be folded correctly and have to be transported from the ribosome to their and only to their final location. Indeed, this is not always the case. NpHR provides an illustrative example. First-generation of NpHR was found to be impaired in trafficking to the plasma membrane.144 Changing two motifs, an N-terminal signal peptide and a C-terminal ER export sequence led to a considerably increased membrane location with an absence of aggregation and toxicity.145 6.1.3. General Requirements for Optogenetic Applications. It is important to note that a successful use of optogenetic tools especially for applications in vertebrates also depends on their effects on biophysical consequences and unwanted cellular answers. For example, steady state irradiation might lead to phototoxic effects like tissue heating and/or tissue damage. It is also important to ensure stable and constant function; however, prolonged activation of the photoreceptors might cause decline in photocurrent amplitudes. This inactivation can give rise to an inhibition of 50−90%,124 which might be inter alia due to a branching of the photocycle.124,135 Another reason might be that the depletion of H+ in the case of proton pumps might alter the efficiency of continued transport.146 Inward pumping eNpHR3.0 alters the intracellular Cl− concentration,147 which can influence GABAA reversal potential.148 Using optogenetic chloride pumps or channels one has also to address the question about the intracellular chloride concentration. In neurons, for example, the Cl− concentration depends on the compartment with a decreasing gradient from axons to dendrites.149

have been studied in extenso (see above). Early photocycle models for channelrhodopsins described linear models.130,131 However, later work on the ChR2 Cys128Thr mutant indicated a branched model.132 Recently, the photocycle of GtACR1 has been studied in more detail.128 A comparison of spectral intermediates and channel states of this anion channel with those of ChR2 displays quite distinct differences indicating that they are members of different families of microbial rhodopsins. Archaerhodopsins, originally discovered by Mukohata and colleagues and in 1994 display photocycles quite similar to that of BR.133 This similarity is also seen in the crystal structures of Arch-1 and Arch-2.134 The demands on photocycle properties are different for ion pumps and channels. In order to become efficient activators the latter rhodopsins should have a decelerated photocycle kinetics, which allows for a prolonged open state. Ideally, they are activated by a short laser pulse at, e.g., blue, and deactivated by a second pulse of red or UV light (for a recent review on optogenetic tools see ref 116). On the other hand, the turnover of microbial rhodopsin pumps are in the range of 20−30 ms, which limits the temporal resolution of ion transport. However, a close analysis of the photocycle kinetics reveals that in some cases the photocycle can be short-circuited in a two-photon process. In the NpHR photocycle the rate constant k6 = 20 ms describes a transition between a state (NpHR′) and the ground state (NpHR).99 Both NpHR′ and NpHR possess identical spectral properties. Because in silencing experiments irradiation of neurons are performed under stationary light conditions already NpHR′ is excited, which accelerates the ion-turnover cosiderably.26 The existence of intermediates similar to the ground state is not unique for NpHR′. For example, a similar state has also been described for the BR photocycle.94 These examples demonstrate the importance for determining detailed photocycle data of optogenetic tools for their deployment in neurophysiological experiments. Unfortunately, detailed photocycle data for most ion pumps is still missing. There is another aspect concerning the need to determine reliable photocycle data. Geibel et al. reported that pumping efficiency of BR is dependent on the membrane potential ΔΨ.135 Strong cell hyperpolarization causes a splitting of the photocycle at the M1 intermediate (where the conformational switch has not yet occurred) into a nonpumping branch. This phenomenon might also be true for Arch26 although exact data has not been published so far. Quantum yields are also important factors influencing the efficacy of optogenetic tools. Unfortunately, these values were only determined for BR (Φ = 0.6136), NpHR (Φ = 0.5264), and channelrhodopsin (C1C2, Φ = 0.3137). There is certainly an urgent need for further biophysical studies on newly discovered light-gated membrane proteins. 6.1.2. Expression and Targeting. The efficacy of optogenetic tools depends on their expression level in the target cell(s) and on the presence of retinal. Whereas retinal is synthesized in most vertebrate tissues138 this is not true, e.g., for C. elegans139 or Drosophila (e.g., ref 140) where it has to be provided through food. The expression of optogenetic tools in target cells or their subcellular location depends on a couple of factors. The gene of interest can be delivered by various techniques like electroporation, microinjection, transfection, or viral delivery. If a targeting on a subcellular level is desired, one has to rely on existing subcellular trafficking signals (for reviews, see refs 116 and 141). Expression into subcellular compartments has some limitations because the cellular health

6.2. New Silencing Optogenetic Tools

In recent years, the toolbox of available proteins for optogenetic applications has been stocked up continuously not only for neural activators but also for neural silencers (for reviews refer to refs 116 and 141). At the present time, there are three major families of genetically encoded silencing proteins: chloride pumps, proton pumps, and anion channels (Figure 7). Table 2 tabulates some of the biophysical properties of silencers (see ref 141 for a detailed list silencing tools). NpHR has been the first chloride pump introduced as a neural silencer and has been used in experiments ranging from mammalian cells to behaving mammals and freely moving

Figure 7. Tools for silencing and activating neurons. Halorhodopsins, archaerhodopsins, and anion channelrhodopsins enable hyperpolarization of cells, whereas channelrhodopsins as cation-permeable channels support the depolarization of cells. J


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worms. To improve its properties, NpHR was modified in such a way to efficiently direct it to the plasma membrane and to optimize its neurophysiological properties.145 The resulting photoreceptor eNpHR3.0 displayed an increased photocurrent and was less prone to aggregation or toxicity. It also showed superior light-induced inhibition, more than 20-fold stronger than that of wild-type NpHR.145 The peak current between 560 and 590 nm allows far-red optogenetic inhibition.144 A new light-driven chloride pump identified in Haloarcula salinarum (strain Shark), subsequently named Jaws, was successfully introduced as an optogenetic inhibitor.120 At 635 nm illumination, photocurrents, hyperpolarization, and neural inhibition were still constant, and the neural activity showed only a reduction to 95% after prolonged irradiation. Compared to eNpHR3.0, Jaws displays an even stronger red-shifted action spectrum (Table 2), making it an interesting tool for dual-color experiments and for applications in mammalian brains.120 The family of proton pumps has also grown in recent years. Early on, archaerhodopsin was determined to be a proton pump.150 This functional property led to genomic screens that revealed other pigments with the potential to serve as multiplecolor silencers of neural activity. One candidate, archaerhodopsin-31 (Arch), enables an almost 100% silencing of neurons.121 Another proton pump, Mac, derived from the eukaryote Leptospaeria maculans151 inhibited neural activity under illumination with blue light. These experiments were pursued by Mattis et al.,124 who streamlined the proteins in such a manner that they became suitable for a varied set of optogenetic applications. An interesting property of certain microbial rhodopsins can be exploited such that they can serve as pH and voltage indicators. In the 1980s, it was shown for the BR mutant D85N that electrical fields can shift the pK of the Schiff base, thus shifting the absorption maximum hypsochromically to 400 nm.152 This observation indicated that microbial rhodopsins might also serve as voltage sensors. Indeed, this has been proven for Arch, which was utilized as a genetically encoded voltage sensor in neurons.153 The mechanism of voltagesensitive fluorescence was further examined by Maclaurin et al.123 that was followed by optimization studies. In a comprehensive mutational study of Gloeobacter violaceus rhodopsin (GR), 70 GR analogues with absorption maxima covering a range from about 450 to 620 nm were analyzed.154 The information about indicator mutations found in “bright” GR were used to mutate corresponding sites in Arch (named Arch(DETC). Directed evolution of Arch(DETC) further improved quantum efficiency and brightness.155 As a proof of concept, Arch-based sensors have been introduced into neurons of Caenorhabditis elegans, showing fluorescence-voltage sensing in behaving worms.156 This work nicely shows that the membrane potential can be determined in parallel with the optogenetic experiment. It could also be a valuable tool to study functional properties of membrane proteins in general at defined membrane potentials. Since 2015 members of a new family of anion channels were described, which turned out to be efficient neuronal silencers. One subgroup was derived from ChR2157 and the channelrhodopsin chimera C1C2158 by mutational alterations of functional amino acids. The other subgroup was discovered in the cryptophyte alga Guillardia theta.159 This latter light-gated anion channel is of particular interest because it has almost perfect anion selectivity and high single-channel conductance. One important advantage of anion channels over ion pumps is

that the former shunt the membrane potential whereas the hyperpolarization caused by the latter is a subtractive mode of inhibition. Shunting processes minimize ionic flux, possibly preventing nonphysiological changes in intracellular chloride concentrations. These new microbial rhodopsins were applied in various model systems. For example, GtACR1 has been introduced in cultured neurons146 or in fruit flies,140 while the anion pump Jaws has been used in primates160 and the proton pump Mac in C. elegans.161

7. CONCLUDING REMARKS The discovery of HR being an inward directed chloride pump and the observation that the BR mutant D85T can serve as a chloride pump as well led quite early to the development of general concepts for ion translocation.162,163 The discovery of new microbial rhodopsins showing a wide range of diversity of their functional properties5 emphasizes the need to consider if general reaction schemes are applicable. Oesterhelt and colleagues have put forward a general model of light activated ion pumps and sensors, which might be also applicable for channels.163 The basic idea of the isomerization/switch/transfer (IST) model is combining the three functional elements isomerization of retinal (I), protein switch altering the extracellular and cytoplasmic accessibilities (S), and ion transfer across the membrane (T). Light excitation triggers the three processes after which the protein returns back to its original ground state. In order to meet this essential requirement the three processes (I/S/T) have to be reversed in order to establish the properties of the ground state. Another essential feature of the model concerns the kinetic independence of ion transfer and switch, which governs the accessibility of the Schiff base. The relative rates of these two processes defines the vectoriality of the transport. In their original paper, Haupts et al. were able to use this model to explain some experimental results like the difference of vectorial proton transfer in BR observed for one and two photon processes. In their analysis, the authors also proposed chloride transport in HsHR and BRD85T. Considering this general mechanism, one could state that the pump mechanisms of BR and NpHR or NmHR are quite similar, with the switch occurring between M1 → M2 and O1 → O2, respectively. In light of recent results, there are two aspects, which corroborate the IST model. First, the proposal that the transfer of ions and the accessibility switch are kinetically independent are substantiated by FTIR and photocycle measurements. Proteorhodopsin alters the vectoriality of the proton transfer as a function of pH, although the photocycle kinetics do not change between the acidic and alkaline state.164 As supported by FTIR, the only difference between high and low pH samples is the protonation status of D97, while other vibrational changes are essentially pH-independent. Another example stems from NpSRII, whose D75N mutant, which lacks an Mlike intermediate, has a much faster photocycle than the wild type, while transitions involving structural changes remain similar.165 Comparable observations have been made for NpHR in comparison with anion-depleted blue NpHR. The FTIR difference spectrum of L2 of the anion-transporting photocycle and the spectrum of the second intermediate of the photoreaction of blue NpHR are qualitatively similar.166 Comparing the charge distribution in the central cluster of HR and BR87 derived from electrostatic calculations shows that the mobile chloride anion in HR substitutes for the fixed K


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existence of a general underlying mechanism. For example, a light-driven proton pump can be converted into a proton channel.172 BR was changed into a sensor by strategically placed mutations including on helix F, which allowed the binding of a cognate transducer.173 Mutating D85 into T or S174 turns BR into a chloride pump, while adding azide to HR converts a chloride pump into a proton pump.107 The question has to be raised, if these different functions imprinted on an archetypical scaffold are a general phenomenon in Nature. It is obvious that the number of secondary structural elements are limited. From the point of view of designing enzymes with new functions and properties, it would be advantageous if an existing protein scaffold can be manipulated in such a way that new catalytic activities can be generated. This strategy has been successfully applied by introducing beta-lactamase activity into the alphabeta/betaalpha metallohydrolase scaffold of glyoxalase II.175 The seven transmembrane helices scaffold of microbial rhodopsins with thousands of different genes identified7 seems to be an ideal structural frame for the development and design of new light-sensitive proteins with tailor-made functions.

residues D85 and E204. Apparently, the electrostatic changes in the two systems are comparable, pointing to a common mechanism for chloride pumping vs proton pumping. The required changes in proton and chloride affinities are mediated by protein conformational changes. All of these examples demonstrate that with the photoisomerization of the retinal chromophore conformational changes are triggered that are mostly independent of external conditions or mutations of functional groups that interfere with the transfer of ions like, e.g., the proton transfer from the Schiff base to its counterion. The second aspect is connected to the question of the nature of the switch. Certainly, the isomerization of retinal plays a crucial role; however, conformational changes are of particular importance as well. There is ever growing evidence obtained not only for microbial rhodopsins but also for animal rhodopsin that an outward movement of helix F, as demonstrated in crystallographic data gathered for NpHR85,86,108 (Figure 6), is crucial for their distinctive function. An essential property of this movement is the opening of the cytoplasmic channel, which allows the proton to reprotonate the Schiff base (BR167,168) or chloride to be released into the cytoplasm (NpHR). The previous discussion was based on the assumption that solely H+ or Cl− are transported across the membrane. However, the observation that chloride is pumped by halorhodopsins stimulated the idea that in BR not a proton but a hydroxyl ion is transported.169−171 This concept was refined with the assumption that the cytoplasmic half of HR functions as a proton/HCl antiporter whereas BR functions as a proton/water antiporter.77 Further experiments are needed to validate this model. However, one should keep in mind that a new member of microbial rhodopsins (K. eikastus KR2)57 pumps sodium or protons in the absence of sodium.80 In this case, an antiporter type mechanism is difficult to reconcile. It is also interesting to note that light-driven chloride ion transfer by the newly analyzed cyanobacterial rhodopsin MrHR is coupled with the translocation of protons and water.31 The recently discovered microbial rhodopsins provided considerable new information about the properties of HRs. The elucidation of their structure, in particular the molecular details of the anion transfer pathways, the structure of intermediates, and their spectroscopic analysis to obtain detailed kinetic data, will be necessary to achieve a comprehensible understanding of the anion transfer mechanism. It will be of great importance to establish if Nature has developed a general mechanism, on which light-activated ion transfer is based. With the advent of optogenetics as means to manipulate neuronal activity in a tailor-made manner the necessity arose for increasing the repertoire within the toolkit. Indeed, extensive genome mining of different organism ranging from bacteria to eukaryotes has revealed not only new members of light activated chloride pumps like Jaws but also proton pumps with novel properties and anion channels. These proteins have already been utilized in neurophysiological studies displaying their potential as efficient silencing tools. However, a thorough analysis of their biophysical properties like cis−trans ratio of the retinal chromophore or the mechanism and kinetics of the photocycle is still missing. With this data, it will become feasible to streamline the properties of these new silencers according to the needs of a particular optogenetic experiment. It is intriguing that functions of microbial rhodopsins can easily be altered into new functionalities, which indicates the

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. ORCID

Martin Engelhard: 0000-0003-1921-9830 Present Address ⊥

Huygens Laboratory, Niels Bohrweg 2, 2333 CA Leiden, The Netherlands.

Notes

The authors declare no competing financial interest. Biographies Christopher Engelhard was born in Essen, Germany, in 1984. He obtained his degree in Physics at the Freie Universität Berlin (Berlin, Germany) in 2010. He received a Ph.D. degree with highest honors from the Freie Universität Berlin in 2015, working under the supervision of Prof. Robert Bittl on channelrhodopsins as well as flavin-based photo receptor proteins. He is currently working as a PostDoc in the group of Prof. Michel Orrit at the Universiteit Leiden (Leiden, Netherlands), where his research is focused on the long-term tracking of protein conformational transitions using single-moleccule spectroscopy. Igor Chizhov obtained his degree in Physics from Moscow Physical Engineering Institute (USSR) in 1978 and his Ph.D. from General Physics Institute (Academy of Science, Moscow) in 1988. In 1992 he joined the group of F. Siebert at the Institute of Biophysics (AlbertLudwig-University, Freiburg, Germany). From 1993 to 2002 he worked as a research assistant Max-Planck Institute for Molecular Physiology, Dortmund. In 2002 he became group leader at the Institute for Biophysical Chemistry, Hannover Medical School, Hannover. Currently, he is leading the Institute for laboratory of time-resolved spectroscopy of biological molecules. He is an expert in flash photolysis studies and computational kinetics. Friedrich Siebert studied Physics at the Universities of Freiburg and Hamburg. He obtained the Diploma degree in Physics from the University of Freiburg and later the Ph.D. degree in Physics also from the University of Freiburg. He had already developed an interest in the application of vibrational spectroscopy, encompassing Brillouin scattering, far and mid-IR spectroscopy, and Raman spectroscopy, to L


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(3) Grote, M.; Engelhard, M.; Hegemann, P. Of Ion Pumps, Sensors and Channels - Perspectives on Microbial Rhodopsins between Science and History. Biochim. Biophys. Acta, Bioenerg. 2014, 1837, 533−545. (4) Zhang, F.; Vierock, J.; Yizhar, O.; Fenno, L. E.; Tsunoda, S.; Kianianmomeni, A.; Prigge, M.; Berndt, A.; Cushman, J.; Polle, J. The Microbial Opsin Family of Optogenetic Tools. Cell 2011, 147, 1446− 1457. (5) Govorunova, E. G.; Sineshchekov, O. A.; Li, H.; Spudich, J. L. Microbial Rhodopsins: Diversity, Mechanisms, and Optogenetic Applications. Annu. Rev. Biochem. 2017, 86, 845−872. (6) Kandori, H. Ion-Pumping Microbial Rhodopsins. Front. Mol. Biosci. 2015, 2, 1−11. (7) Kurihara, M.; Sudo, Y. Microbial Rhodopsins: Wide Distribution, Rich Diversity and Great Potential. Biophys. Physicobiol. 2015, 12, 121−129. (8) Inoue, K.; Kato, Y.; Kandori, H. Light-Driven Ion-Translocating Rhodopsins in Marine Bacteria. Trends Microbiol. 2015, 23, 91−98. (9) Ernst, O. P.; Lodowski, D. T.; Elstner, M.; Hegemann, P.; Brown, L. S.; Kandori, H. Microbial and Animal Rhodopsins: Structures, Functions, and Molecular Mechanisms. Chem. Rev. 2014, 114, 126− 163. (10) Oesterhelt, D.; Stoeckenius, W. Rhodopsin-Like Protein from the Purple Membrane of Halobacterium Halobium. Nature - New Biology 1971, 233, 149−152. (11) Oesterhelt, D.; Stoeckenius, W. Functions of a New Photoreceptor Membrane. Proc. Natl. Acad. Sci. U. S. A. 1973, 70, 2853−2857. (12) Mitchell, P. Keilin’s Respiratory Chain Concept and Its Chemiosmotic Consequences. Science 1979, 206, 1148−1159. (13) Racker, E.; Stoeckenius, W. Reconstitution of Purple Membrane Vesicles Catalyzing Light- Driven Proton Uptake and Adenosine Triphosphate Formation. J. Biol. Chem. 1974, 249, 662−663. (14) Matsuno-Yagi, A.; Mukohata, Y. Two Possible Roles of Bacteriorhodopsin; a Comparative Study of Strains of Halobacterium Halobium Differing in Pigmentation. Biochem. Biophys. Res. Commun. 1977, 78, 237−243. (15) Mukohata, Y.; Kaji, Y. Light-Induced Membrane-Potential Increase, ATP Synthesis, and Proton Uptake in Halobacterium Halobium, R1mr Catalyzed by Halorhodopsin: Effects of N,N′Dicyclohexylcarbodiimide, Triphenyltin Chloride, and 3,5-Di-TertButyl-4-Hydroxybenzylidenemalononitrile (Sf6847). Arch. Biochem. Biophys. 1981, 206, 72−76. (16) Hildebrand, E.; Dencher, N. Two Photosystems Controlling Behavioural Responses of Halobacterium Halobium. Nature 1975, 257, 46−48. (17) Bogomolni, R. A.; Spudich, J. L. Identification of a Third Rhodopsin-Like Pigment in Phototactic Halobacterium Halobium. Proc. Natl. Acad. Sci. U. S. A. 1982, 79, 6250−6254. (18) Spudich, J. L.; Bogomolni, R. A. Mechanism of Colour Discrimination by a Bacterial Sensory Rhodopsin. Nature 1984, 312, 509−513. (19) Takahashi, T.; Tomioka, H.; Kamo, N.; Kobatake, Y. A Photosystem Other Than Ps370 Also Mediates the Negative Phototaxis of Halobacterium Halobium. FEMS Microbiol. Lett. 1985, 28, 161−164. (20) Tomioka, H.; Takahashi, T.; Kamo, N.; Kobatake, Y. Flash Spectrometric Identification of a Fourth Rhodopsin-Like Pigment in Halobacterium Halobium. Biochem. Biophys. Res. Commun. 1986, 139, 389−395. (21) Wolff, E. K.; Bogomolni, R. A.; Scherrer, P.; Hess, B.; Stoeckenius, W. Color Discrimination in Halobacteria: Spectroscopic Characterization of a Second Sensory Receptor Covering the BlueGreen Region of the Spectrum. Proc. Natl. Acad. Sci. U. S. A. 1986, 83, 7272−7276. (22) Spudich, E. N.; Sundberg, S. A.; Manor, D.; Spudich, J. L. Properties of a Second Sensory Receptor Protein in Halobacterium Halobium Phototaxis. Proteins: Struct., Funct., Genet. 1986, 1, 239−246.

the study of complex systems. After completing his Ph.D., he switched to Biophysics by joining the newly established Institute of Biophysics and Radiation Biology. He established new methods for the application of IR and Raman spectroscopy for the study of function and structure of biological systems. A special flavour was the inclusion of modern techniques of Biochemistry and Molecular Biology by collaboration with corresponding experts. Among many research papers he published several review papers with special emphasis on chromophoric systems such as retinal proteins and tetrapyrrolic proteins. He coauthored a book together with Peter Hildebrandt on the application of vibrational spectroscopy in Life Science. Martin Engelhard, retired Principal Investigator at the Max Planck Institute for Molecular Physiology in Dortmund (Germany), has been carrying out research on archaeal microbial rhodopsins with the focus on transmembrane signal transduction. His other main interest has been questions on the chemical synthesis of proteins with the aim to generate samples with tailor-made properties. He obtained his Ph.D. from the Georg August University Gö ttingen (Germany) in Chemistry. After his postdoc training at the Rockefeller University (New York) in the lab of Bruce Merrifield he joined the Max Planck Institute in Dortmund. In 1996 he became an external member of the Faculty of Chemistry at the Technical University of Dortmund and in 2004 Adjunct Professor in Biochemistry. In 2005 he became until his retirement Speaker of the International Max Planck Research School for Chemical Biology (IMPRS-CB).

ACKNOWLEDGMENTS We would like to thank the Max Planck Gesellschaft and the Deutsche Forschungsgemeinschaft for continuous support. ABBREVIATIONS Arch archaerhodopsin-31 ArchT proton pump from Halorubrum strain TP009 BR bacteriorhodopsin ChR-1 channelrhodopsin-1 ChR-2 channelrhodopsin-2 FpHR halorhodopsin from Fulvimarina pelagi (also named FR) GR Gloeobacter violaceus rhodopsin GtACR1 anion channel from Guillardia theta HR halorhodopsin HsSR sensory rhodpsin from Halobacterium salinarum Jaws chloride pump from Haloarcula salinarum KR2 Na+-pumping rhodopsin Mac proton pump from Leptospaeria maculans MrHR halorhodopsin from Mastigocladopsis repens NmHR halorhodopsin from Nonlabens marinus (also named ClR or NM-R3) NpHR halorhodopsin from Natronobacterium pharaonis SRI sensory rhodopsin I SRII sensory rhodopsin II SyHR halorhodopsin from a Synechocystis species REFERENCES (1) Adamantidis, A.; Arber, S.; Bains, J. S.; Bamberg, E.; Bonci, A.; Buzsaki, G.; Cardin, J. A.; Costa, R. M.; Dan, Y.; Goda, Y.; et al. Optogenetics: 10 Years after ChR2 in Neurons–Views from the Community. Nat. Neurosci. 2015, 18, 1202−1212. (2) Mühlhäuser, W. W.; Fischer, A.; Weber, W.; Radziwill, G. Optogenetics - Bringing Light into the Darkness of Mammalian Signal Transduction. Biochim. Biophys. Acta, Mol. Cell Res. 2017, 1864, 280− 292. M


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