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Light and pH-induced Changes in Structure and Accessibility of Transmembrane Helix B and Its Immediate Environment in Channelrhodopsin-2*

Open AccessPublished:June 06, 2016DOI:https://doi.org/10.1074/jbc.M115.711200
      A variant of the cation channel channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) was selectively labeled at position Cys-79 at the end of the first cytoplasmic loop and the beginning of transmembrane helix B with the fluorescent dye fluorescein (acetamidofluorescein). We utilized (i) time-resolved fluorescence anisotropy experiments to monitor the structural dynamics at the cytoplasmic surface close to the inner gate in the dark and after illumination in the open channel state and (ii) time-resolved fluorescence quenching experiments to observe the solvent accessibility of helix B at pH 6.0 and 7.4. The light-induced increase in final anisotropy for acetamidofluorescein bound to the channel variant with a prolonged conducting state clearly shows that the formation of the open channel state is associated with a large conformational change at the cytoplasmic surface, consistent with an outward tilt of helix B. Furthermore, results from solute accessibility studies of the cytoplasmic end of helix B suggest a pH-dependent structural heterogeneity that appears below pH 7. At pH 7.4 conformational homogeneity was observed, whereas at pH 6.0 two protein fractions exist, including one in which residue 79 is buried. This inaccessible fraction amounts to 66% in nanodiscs and 82% in micelles. Knowledge about pH-dependent structural heterogeneity may be important for CrChR2 applications in optogenetics.

      Introduction

      Channelrhodopsins are involved in phototaxis and photophobia of unicellular green algae (
      • Sineshchekov O.A.
      • Govorunova E.G.
      • Spudich J.L.
      Photosensory functions of channelrhodopsins in native algal cells.
      ). They constitute a new class of light-gated ion channels containing the seven-transmembrane helix motif and the chromophore retinal as the light-sensitive cofactor, which are both common to the other retinal-containing proteins such as bacteriorhodopsin (bR)
      The abbreviations used are: bR
      bacteriorhodopsin
      AF
      acetamidofluorescein
      CrChR2
      Chlamydomonas reinhardtii channelrhodopsin-2
      DM
      n-decyl-β-d-maltopyranoside
      Fm
      proteolytic fragments containing transmembrane parts of CrChR2
      IAF
      5-iodoacetamidofluorescein
      SV
      Stern-Volmer.
      or visual rhodopsin (
      • Nagel G.
      • Ollig D.
      • Fuhrmann M.
      • Kateriya S.
      • Musti A.M.
      • Bamberg E.
      • Hegemann P.
      Channelrhodopsin-1: a light-gated proton channel in green algae.
      ). In CrChR2 the chromophore retinal is bound to Lys-257 (
      • Kato H.E.
      • Zhang F.
      • Yizhar O.
      • Ramakrishnan C.
      • Nishizawa T.
      • Hirata K.
      • Ito J.
      • Aita Y.
      • Tsukazaki T.
      • Hayashi S.
      • Hegemann P.
      • Maturana A.D.
      • Ishitani R.
      • Deisseroth K.
      • Nureki O.
      Crystal structure of the channelrhodopsin light-gated cation channel.
      ). Channelrhodopsin activation via light-induced isomerization of retinal from all-trans to 13-cis is coupled to a transient cofactor deprotonation and a functional protein structural change. In channelrhodopsin, as well as in other retinal-containing photoreceptors, subtle changes in the chromophore vicinity trigger large scale protein conformational changes remote from the chromophore binding pocket. Rearrangement of helix B is hypothesized to constitute a key element in channel opening by allowing the entry of water molecules (
      • Lórenz-Fonfría V.A.
      • Bamann C.
      • Resler T.
      • Schlesinger R.
      • Bamberg E.
      • Heberle J.
      Temporal evolution of helix hydration in a light-gated ion channel correlates with ion conductance.
      ,
      • Schneider F.
      • Grimm C.
      • Hegemann P.
      Biophysics of channelrhodopsin.
      ,
      • Watanabe H.C.
      • Welke K.
      • Sindhikara D.J.
      • Hegemann P.
      • Elstner M.
      Towards an understanding of channelrhodopsin function: simulations lead to novel insights of the channel mechanism.
      ). A continuous water wire is a prerequisite for the formation of the ion-conducting pathway. A hydrophilic pore between helices A, B, C, and G was suggested to serve as the ion permeation pathway based on the high resolution crystal structure of the C1C2 chimera (
      • Kato H.E.
      • Zhang F.
      • Yizhar O.
      • Ramakrishnan C.
      • Nishizawa T.
      • Hirata K.
      • Ito J.
      • Aita Y.
      • Tsukazaki T.
      • Hayashi S.
      • Hegemann P.
      • Maturana A.D.
      • Ishitani R.
      • Deisseroth K.
      • Nureki O.
      Crystal structure of the channelrhodopsin light-gated cation channel.
      ). Polar and charged residues of transmembrane helix B line this pore. Important constituents of the pathway are the inner gate (intracellular (cytoplasmic) side) and the access channel (extracellular side), as well as the central gate with critical determinants to regulate cation selectivity in CrChR2 (for a review see Ref.
      • Schneider F.
      • Grimm C.
      • Hegemann P.
      Biophysics of channelrhodopsin.
      ). Residue Cys-128, close to the retinal binding pocket, constitutes the so-called DC gate together with Asp-156 (
      • Nack M.
      • Radu I.
      • Gossing M.
      • Bamann C.
      • Bamberg E.
      • von Mollard G.F.
      • Heberle J.
      The DC gate in channelrhodopsin-2: crucial hydrogen bonding interaction between C128 and D156.
      ). Mutants of Cys-128, e.g. C128T, show a prolonged lifetime of the conducting state (
      • Berndt A.
      • Yizhar O.
      • Gunaydin L.A.
      • Hegemann P.
      • Deisseroth K.
      Bi-stable neural state switches.
      ). These mutants are thus very well suited for trapping the P3520 state, the conducting open channel state (also named P520), in spectroscopic measurements, because no other effect except a prolonged open state was found upon mutation of Cys-128 (
      • Stehfest K.
      • Ritter E.
      • Berndt A.
      • Bartl F.
      • Hegemann P.
      The branched photocycle of the slow-cycling channelrhodopsin-2 mutant C128T.
      ,
      • Bamann C.
      • Gueta R.
      • Kleinlogel S.
      • Nagel G.
      • Bamberg E.
      Structural guidance of the photocycle of channelrhodopsin-2 by an interhelical hydrogen bond.
      ).
      The inner gate was hypothesized to be involved in CrChR2 activation (formation of the open conducting channel state) together with the tilt of helix B (
      • Schneider F.
      • Grimm C.
      • Hegemann P.
      Biophysics of channelrhodopsin.
      ). Evidence for light-induced movements of helix B comes from structural studies using electron crystallography and EPR spectroscopy (double electron-electron resonance) (
      • Krause N.
      • Engelhard C.
      • Heberle J.
      • Schlesinger R.
      • Bittl R.
      Structural differences between the closed and open states of channelrhodopsin-2 as observed by EPR spectroscopy.
      ,
      • Müller M.
      • Bamann C.
      • Bamberg E.
      • Kühlbrandt W.
      Light-induced helix movements in channelrhodopsin-2.
      ,
      • Sattig T.
      • Rickert C.
      • Bamberg E.
      • Steinhoff H.J.
      • Bamann C.
      Light-induced movement of the transmembrane helix B in channelrhodopsin-2.
      ). Together these data suggest that in contrast to bR and sensory rhodopsin, where large helix movements were observed for helix F upon reprotonation of the retinal Schiff base (
      • Sass H.J.
      • Büldt G.
      • Gessenich R.
      • Hehn D.
      • Neff D.
      • Schlesinger R.
      • Berendzen J.
      • Ormos P.
      Structural alterations for proton translocation in the M state of wild-type bacteriorhodopsin.
      ,
      • Radzwill N.
      • Gerwert K.
      • Steinhoff H.J.
      Time-resolved detection of transient movement of helices F and G in doubly spin-labeled bacteriorhodopsin.
      ,
      • Alexiev U.
      • Rimke I.
      • Pöhlmann T.
      Elucidation of the nature of the conformational changes of the EF-interhelical loop in bacteriorhodopsin and of the helix VIII on the cytoplasmic surface of bovine rhodopsin: a time-resolved fluorescence depolarization study.
      ,
      • Moukhametzianov R.
      • Klare J.P.
      • Efremov R.
      • Baeken C.
      • Göppner A.
      • Labahn J.
      • Engelhard M.
      • Büldt G.
      • Gordeliy V.I.
      Development of the signal in sensory rhodopsin and its transfer to the cognate transducer.
      ,
      • Wegener A.A.
      • Chizhov I.
      • Engelhard M.
      • Steinhoff H.J.
      Time-resolved detection of transient movement of helix F in spin-labelled pharaonis sensory rhodopsin II.
      ), helix B in CrChR2 undergoes prominent light-induced rearrangements. However, the EPR distance measurements are not able to discriminate between conformational changes of the spin label and structural changes of the protein moiety. Therefore, it is not entirely clear whether the light-induced changes (
      • Krause N.
      • Engelhard C.
      • Heberle J.
      • Schlesinger R.
      • Bittl R.
      Structural differences between the closed and open states of channelrhodopsin-2 as observed by EPR spectroscopy.
      ,
      • Sattig T.
      • Rickert C.
      • Bamberg E.
      • Steinhoff H.J.
      • Bamann C.
      Light-induced movement of the transmembrane helix B in channelrhodopsin-2.
      ) originate from helix B displacements or local structural changes of the loop or label conformation.
      To investigate the conformational dynamics of the cytoplasmic end of helix B in real time, i.e. on the picosecond and nanosecond time scale, we selectively labeled position Cys-79 in a C128T variant of CrChR2 (
      • Krause N.
      • Engelhard C.
      • Heberle J.
      • Schlesinger R.
      • Bittl R.
      Structural differences between the closed and open states of channelrhodopsin-2 as observed by EPR spectroscopy.
      ) with a fluorescent dye. Then we followed the conformational changes of the channel in detergent (CrChR2 micelles) and lipid environment (CrChR2 nanodiscs) by time-resolved fluorescence depolarization. This technique is very well suited to obtain information on local and global protein dynamics on the nanosecond time scale, because the dynamics of the covalently bound dye is affected by the motion of the protein segment to which it is covalently attached. The different modes of motion can be separated in the time domain (
      • Alexiev U.
      • Rimke I.
      • Pöhlmann T.
      Elucidation of the nature of the conformational changes of the EF-interhelical loop in bacteriorhodopsin and of the helix VIII on the cytoplasmic surface of bovine rhodopsin: a time-resolved fluorescence depolarization study.
      ,
      • Alexiev U.
      • Farrens D.L.
      Fluorescence spectroscopy of rhodopsins: insights and approaches.
      ,
      • Kirchberg K.
      • Kim T.Y.
      • Möller M.
      • Skegro D.
      • Dasara Raju G.
      • Granzin J.
      • Büldt G.
      • Schlesinger R.
      • Alexiev U.
      Conformational dynamics of helix 8 in the GPCR rhodopsin controls arrestin activation in the desensitization process.
      ,
      • Schröder G.F.
      • Alexiev U.
      • Grubmüller H.
      Simulation of fluorescence anisotropy experiments: probing protein dynamics.
      ). We measured the time-resolved anisotropy curves in the dark state of the channel and after light activation at pH 6.0 and pH 7.4. The increase in final anisotropy at both pH values in nanodiscs clearly showed that the light-induced formation of the open channel state is associated with a strong increase in sterical hindrance of helix B motion, suggesting a large conformational change at the cytoplasmic surface. Accessibility studies by means of fluorescence quenching with iodide, a polar quencher, support these interpretations. Moreover, the latter experiments revealed a pH-dependent structural heterogeneity of helix B in close vicinity to the inner gate.

      Discussion

      Our goal was to gain new insights from structural dynamics into the mechanism underlying the formation of the open channel state in CrChR2. In particular, we focused on the cytoplasmic part of helix B. A key role for helix B in light-induced channel opening and closing has been suggested. Evidence for light-induced movements of helix B is based on structural studies using electron crystallography and EPR spectroscopy (double electron-electron resonance) (
      • Krause N.
      • Engelhard C.
      • Heberle J.
      • Schlesinger R.
      • Bittl R.
      Structural differences between the closed and open states of channelrhodopsin-2 as observed by EPR spectroscopy.
      ,
      • Müller M.
      • Bamann C.
      • Bamberg E.
      • Kühlbrandt W.
      Light-induced helix movements in channelrhodopsin-2.
      ,
      • Sattig T.
      • Rickert C.
      • Bamberg E.
      • Steinhoff H.J.
      • Bamann C.
      Light-induced movement of the transmembrane helix B in channelrhodopsin-2.
      ). Although double electron-electron resonance measurements suggest a light-induced displacement/outward tilt of helix B (
      • Krause N.
      • Engelhard C.
      • Heberle J.
      • Schlesinger R.
      • Bittl R.
      Structural differences between the closed and open states of channelrhodopsin-2 as observed by EPR spectroscopy.
      ,
      • Sattig T.
      • Rickert C.
      • Bamberg E.
      • Steinhoff H.J.
      • Bamann C.
      Light-induced movement of the transmembrane helix B in channelrhodopsin-2.
      ), electron crystallographic studies found additional evidence for a loss of order in that helix (
      • Müller M.
      • Bamann C.
      • Bamberg E.
      • Kühlbrandt W.
      Light-induced helix movements in channelrhodopsin-2.
      ).
      The prominent light-induced movement/structural changes of helix B in CrChR2 are unique, because for other microbial rhodopsin, such as sensory rhodopsin and bR, major helix displacements occur upon light activation in helix F (
      • Sass H.J.
      • Büldt G.
      • Gessenich R.
      • Hehn D.
      • Neff D.
      • Schlesinger R.
      • Berendzen J.
      • Ormos P.
      Structural alterations for proton translocation in the M state of wild-type bacteriorhodopsin.
      ,
      • Radzwill N.
      • Gerwert K.
      • Steinhoff H.J.
      Time-resolved detection of transient movement of helices F and G in doubly spin-labeled bacteriorhodopsin.
      ,
      • Alexiev U.
      • Rimke I.
      • Pöhlmann T.
      Elucidation of the nature of the conformational changes of the EF-interhelical loop in bacteriorhodopsin and of the helix VIII on the cytoplasmic surface of bovine rhodopsin: a time-resolved fluorescence depolarization study.
      ,
      • Moukhametzianov R.
      • Klare J.P.
      • Efremov R.
      • Baeken C.
      • Göppner A.
      • Labahn J.
      • Engelhard M.
      • Büldt G.
      • Gordeliy V.I.
      Development of the signal in sensory rhodopsin and its transfer to the cognate transducer.
      ,
      • Wegener A.A.
      • Chizhov I.
      • Engelhard M.
      • Steinhoff H.J.
      Time-resolved detection of transient movement of helix F in spin-labelled pharaonis sensory rhodopsin II.
      ). In the latter case, the light-induced helix F movements are connected to the reprotonation of the retinal Schiff base via the internal proton donor Asp-96 in the proton pump cycle (
      • Dickopf S.
      • Alexiev U.
      • Krebs M.P.
      • Otto H.
      • Mollaaghababa R.
      • Khorana H.G.
      • Heyn M.P.
      Proton transport by a bacteriorhodopsin mutant aspartic acid-85 → asparagine, initiated in the unprotonated Schiff-base state.
      ,
      • Alexiev U.
      • Mollaaghababa R.
      • Scherrer P.
      • Khorana H.G.
      • Heyn M.P.
      Rapid long-range proton diffusion along the surface of the purple membrane and delayed proton-transfer into the bulk.
      ). Because CrChR2 is a light-triggered cation channel, it was suggested that the changes around helix B seem to be involved in cation permeation by creating a water-filled pore (
      • Müller M.
      • Bamann C.
      • Bamberg E.
      • Kühlbrandt W.
      Light-induced helix movements in channelrhodopsin-2.
      ).
      To detect dynamic structures and conformational changes of helix B via time-resolved fluorescence depolarization (
      • Alexiev U.
      • Rimke I.
      • Pöhlmann T.
      Elucidation of the nature of the conformational changes of the EF-interhelical loop in bacteriorhodopsin and of the helix VIII on the cytoplasmic surface of bovine rhodopsin: a time-resolved fluorescence depolarization study.
      ,
      • Schröder G.F.
      • Alexiev U.
      • Grubmüller H.
      Simulation of fluorescence anisotropy experiments: probing protein dynamics.
      ), we selectively labeled position Cys-79 in a CrChR2 variant, in which the native cysteines, except for Cys-79 and Cys-208, were exchanged to alanine (
      • Krause N.
      • Engelhard C.
      • Heberle J.
      • Schlesinger R.
      • Bittl R.
      Structural differences between the closed and open states of channelrhodopsin-2 as observed by EPR spectroscopy.
      ). Because the two remaining cysteines exhibit differential reactivity toward IAF, we were able to exclusively label position 79 at helix B (Fig. 3). The cysteine mutations were generated in the slowly cycling variant C128T (
      • Berndt A.
      • Yizhar O.
      • Gunaydin L.A.
      • Hegemann P.
      • Deisseroth K.
      Bi-stable neural state switches.
      ), which accumulates the conducting state by slowing down the respective time constants (Fig. 1). This allowed us to specifically address the conducting state after blue light illumination. In addition, these so-called step function mutants of CrChR2 are particularly interesting in optogenetics because they offer the opportunity to generate a more permanent cell depolarization (
      • Berndt A.
      • Yizhar O.
      • Gunaydin L.A.
      • Hegemann P.
      • Deisseroth K.
      Bi-stable neural state switches.
      ).
      Time-resolved fluorescence depolarization experiments allowed us to monitor the conformational dynamics of the cytoplasmic end of helix B in the dark state and the changes upon blue light illumination via the analysis of the rotational correlation time and the amplitude assigned to the anisotropy decay component (second decay component in Table 1), which reflects the motion of helix B. Moreover, the final anisotropy is a measure for the steric hindrance of helix B motion. The increase in final anisotropy r at both pH values in nanodiscs upon light activation (Fig. 5) clearly shows that the light-induced formation of the open channel state is associated with an increased steric hindrance of helix B motion at the inner gate because of a large conformational change at the cytoplasmic surface. These changes are schematically indicated in Fig. 3C, which shows the structural model of dark-adapted CrChR2 and thus a closed channel conformation. The red-colored area around position Cys-79 indicates the reduction of conformational space of helix B/dye in the open channel conformation compared with dark CrChR2. In addition to changes in conformational space for helix B motion, the rotational correlation times of the cytoplasmic end of helix B in the dark and in the open conducting P3520 state indicate changes in helix B flexibility. In CrChR2 micelles the cytoplasmic end of helix B is immobile in the dark state and becomes flexible in the P3520 state with a rotational correlation time ϕ2 of 1.3 ns. Similarly, a faster motion of helix B was observed in nanodiscs at pH 6.0 in the P3520 state with a rotational correlation time φ2 of 1.8 ns compared with 3.3 ns in the dark state. Because only in CrChR2 micelles was immobility of the cytoplasmic end of helix B in the dark state observed, we speculate that the detergent molecules restrict the mobility of the first intracellular loop. It is known from the literature (
      • Böckmann R.A.
      • Caflisch A.
      Spontaneous formation of detergent micelles around the outer membrane protein OmpX.
      ) that detergent molecules may interact with solvent-exposed surface regions of membrane proteins.
      As in channelrhodopsin, light-induced conformational changes were observed at the cytoplasmic surface of the retinal proteins bR and visual rhodopsin (
      • Kim T.Y.
      • Schlieter T.
      • Haase S.
      • Alexiev U.
      Activation and molecular recognition of the GPCR rhodopsin: insights from time-resolved fluorescence depolarisation and single molecule experiments.
      ,
      • Kim T.Y.
      • Möller M.
      • Winkler K.
      • Kirchberg K.
      • Alexiev U.
      Dissection of environmental changes at the cytoplasmic surface of light-activated bacteriorhodopsin and visual rhodopsin: sequence of spectrally silent steps.
      ). A general increase in protein flexibility/protein softening was assumed to be a prerequisite to overcome potential barriers in the large scale structural changes during the M-intermediate of bR (
      • Pieper J.
      • Buchsteiner A.
      • Dencher N.A.
      • Lechner R.E.
      • Hauss T.
      Transient protein softening during the working cycle of a molecular machine.
      ). The latter were correlated with anisotropy changes in the M-intermediate and the Meta-II state of bR and visual rhodopsin, respectively (
      • Kim T.Y.
      • Schlieter T.
      • Haase S.
      • Alexiev U.
      Activation and molecular recognition of the GPCR rhodopsin: insights from time-resolved fluorescence depolarisation and single molecule experiments.
      ,
      • Kim T.Y.
      • Möller M.
      • Winkler K.
      • Kirchberg K.
      • Alexiev U.
      Dissection of environmental changes at the cytoplasmic surface of light-activated bacteriorhodopsin and visual rhodopsin: sequence of spectrally silent steps.
      ). The clear reduction in conformational space of helix B movement in nanodiscs upon light activation together with a shortening of the rotational correlation time at pH 6 indicates that changes in the dynamics of the cytoplasmic part of helix B are involved in the formation of the open conducting state. Moreover, a higher steric restriction of the fluorescent label in position 79 pointing toward the putative dimer interface (Fig. 3C) would agree with the structural model of an open conductive state with changes at the dimer interface (
      • Krause N.
      • Engelhard C.
      • Heberle J.
      • Schlesinger R.
      • Bittl R.
      Structural differences between the closed and open states of channelrhodopsin-2 as observed by EPR spectroscopy.
      ) and the tilt of helix B observed in MD simulations (
      • Watanabe H.C.
      • Welke K.
      • Sindhikara D.J.
      • Hegemann P.
      • Elstner M.
      Towards an understanding of channelrhodopsin function: simulations lead to novel insights of the channel mechanism.
      ). It seems that these conformational changes on the cytoplasmic surface induced by retinal isomerization led to the required structural rearrangements allowing transient water influx necessary for either proton uptake (e.g. in bR) or cation permeation in CrChR2.
      As indicated in Fig. 3C, position 79 at the cytoplasmic end of helix B is located at the entrance of the conducting pore (for a review see Ref.
      • Schneider F.
      • Grimm C.
      • Hegemann P.
      Biophysics of channelrhodopsin.
      ) in close proximity to the inner gate, a hydrophilic amino acid cluster that blocks the cation permeation pathway at the cytoplasmic (intracellular) side. The amino acids belonging to the inner gate are Glu-82, Glu-83, Tyr-70, His-134, His-265, and Arg-268 (Fig. 3C). It was hypothesized that a reorientation of this cluster might be the final step in CrChR2 gating (
      • Schneider F.
      • Grimm C.
      • Hegemann P.
      Biophysics of channelrhodopsin.
      ). To detect possible changes at the inner gate, we investigated the accessibility of the bound fluorophore in position 79 to the polar quencher I in the dark and after light illumination and in dependence of pH. When bound to the surface of the channel, fluorescein exhibits a fluorescence lifetime of ∼3 ns, clearly smaller than for free fluorescein (∼4 ns). This faster fluorescence lifetime of fluorescein when bound to the channel surface can be explained mainly by quenching effects from the protein surface. A similar effect on the fluorescence lifetime was found when fluorescein was covalently bound to the surface of bR (
      • Kim T.Y.
      • Winkler K.
      • Alexiev U.
      Picosecond multidimensional fluorescence spectroscopy: a tool to measure real-time protein dynamics during function.
      ) and visual rhodopsin (
      • Alexiev U.
      • Rimke I.
      • Pöhlmann T.
      Elucidation of the nature of the conformational changes of the EF-interhelical loop in bacteriorhodopsin and of the helix VIII on the cytoplasmic surface of bovine rhodopsin: a time-resolved fluorescence depolarization study.
      ,
      • Boreham A.
      • Kim T.Y.
      • Spahn V.
      • Stein C.
      • Mundhenk L.
      • Gruber A.D.
      • Haag R.
      • Welker P.
      • Licha K.
      • Alexiev U.
      Exploiting fluorescence lifetime plasticity in FLIM: target molecule localization in cells and tissues.
      ). At pH 7.4 we observed reduced quenching rates for the collisional polar quencher iodide in both micelles and nanodiscs compared with free fluorescein in solution. No changes were observed after blue light illumination. Linear SV plots, both in the dark state and after illumination (Fig. 9), suggest the presence of a single population of fluorophores, i.e. a homogeneous protein conformation. In contrast, at pH 6.0 structural heterogeneity with two different protein conformations was observed as deduced from the analysis of the modified SV plots (Fig. 10). Thus, a pH-dependent conformational change takes place at the cytoplasmic surface of the channel, close to the inner gate. Analysis of this structural heterogeneity (
      • Eftink M.R.
      • Ghiron C.A.
      Fluorescence quenching studies with proteins.
      ,
      • Lehrer S.S.
      Solute perturbation of protein fluorescence: the quenching of the tryptophyl fluorescence of model compounds and of lysozyme by iodide ion.
      ) reveals a protein population in which position 79, i.e. the side of helix B pointing toward the putative dimer interface (Fig. 3C), is not solute accessible from the aqueous solution. This protein population constitutes the majority of the sample with ∼82% (micelles) and 66% (nanodiscs). The higher final anisotropy r observed in nanodiscs at pH 6.0 compared with pH 7.4 in the dark state (Figs. 4 and 6C) can be explained by the large buried fraction of bound fluorescein at pH 6.0 that is expected to experience a higher degree of steric restriction. Note that this structural heterogeneity does not affect the overall change observed in the time-resolved anisotropy signal after illumination. However, only minor changes were detected in the fractions after illumination, indicating that the conformational heterogeneity is rather pH-dependent than light activation-dependent. The protein fraction that was accessible, however, showed an unusual high accessibility for a fluorescein bound to a protein surface, because quenching values on the order of free fluorescein were observed. We speculate that in this fraction fluorescein bound in position 79 is oriented toward the hydrophilic cluster of the inner gate attracting water molecules to the cytoplasmic surface (
      • Watanabe H.C.
      • Welke K.
      • Sindhikara D.J.
      • Hegemann P.
      • Elstner M.
      Towards an understanding of channelrhodopsin function: simulations lead to novel insights of the channel mechanism.
      ) and that positively charged residues, such as Arg-268, locally increase the iodide concentration, leading to an apparent higher quenching compared with the lower bulk I concentrations. This effect may also be transient, because transient surface changes (in addition to the discussed helix tilt) are known to occur at the cytoplasmic surface of both bR (
      • Alexiev U.
      • Scherrer P.
      • Marti T.
      • Khorana H.G.
      • Heyn M.P.
      Time-resolved surface-charge change on the cytoplasmic side of bacteriorhodopsin.
      ) and visual rhodopsin (
      • Möller M.
      • Alexiev U.
      surface charge changes upon formation of the signaling state in visual rhodopsin.
      ) upon light activation. For instance, a conformational change of the EF-Loop at the cytoplasmic surface of bR was correlated with surface charge changes (
      • Alexiev U.
      • Rimke I.
      • Pöhlmann T.
      Elucidation of the nature of the conformational changes of the EF-interhelical loop in bacteriorhodopsin and of the helix VIII on the cytoplasmic surface of bovine rhodopsin: a time-resolved fluorescence depolarization study.
      ,
      • Alexiev U.
      • Scherrer P.
      • Marti T.
      • Khorana H.G.
      • Heyn M.P.
      Time-resolved surface-charge change on the cytoplasmic side of bacteriorhodopsin.
      ).
      Because conformational heterogeneity was not observed at pH 7.4, a structural pH-dependent rearrangement of helix B below pH 7 must occur that affects the region of the inner gate. Support for this conclusion comes from recent x-ray crystallographic studies of channelrhodopsin, because the available C1C2 x-ray crystal structures (
      • Kato H.E.
      • Zhang F.
      • Yizhar O.
      • Ramakrishnan C.
      • Nishizawa T.
      • Hirata K.
      • Ito J.
      • Aita Y.
      • Tsukazaki T.
      • Hayashi S.
      • Hegemann P.
      • Maturana A.D.
      • Ishitani R.
      • Deisseroth K.
      • Nureki O.
      Crystal structure of the channelrhodopsin light-gated cation channel.
      ,
      • Kato H.E.
      • Kamiya M.
      • Sugo S.
      • Ito J.
      • Taniguchi R.
      • Orito A.
      • Hirata K.
      • Inutsuka A.
      • Yamanaka A.
      • Maturana A.D.
      • Ishitani R.
      • Sudo Y.
      • Hayashi S.
      • Nureki O.
      Atomistic design of microbial opsin-based blue-shifted optogenetics tools.
      ) suggest pH-dependent changes at the cytosolic side of helix B. Although the crystal structure of a C1C2 chimera was solved at pH 6.0 (
      • Nack M.
      • Radu I.
      • Gossing M.
      • Bamann C.
      • Bamberg E.
      • von Mollard G.F.
      • Heberle J.
      The DC gate in channelrhodopsin-2: crucial hydrogen bonding interaction between C128 and D156.
      ), another C1C2 variant was crystallized at pH 7.0 (
      • Kato H.E.
      • Kamiya M.
      • Sugo S.
      • Ito J.
      • Taniguchi R.
      • Orito A.
      • Hirata K.
      • Inutsuka A.
      • Yamanaka A.
      • Maturana A.D.
      • Ishitani R.
      • Sudo Y.
      • Hayashi S.
      • Nureki O.
      Atomistic design of microbial opsin-based blue-shifted optogenetics tools.
      ). The latter C1C2 variant contains two amino acid substitutions (corresponding amino acids in CrChR2: T159G/G163A) in close proximity to the β-ionone ring of the retinal. When comparing these C1C2 structures at the two pH values of 6.0 and 7.0, different protonation states for four amino acids were resolved; three of them are located in the N-terminal region. The only structural difference observed in the transmembrane part (apart from the amino acid substitutions) is at position Glu-83 (Fig. 3, A and C), which is part of the inner gate and located in helix B close to residue Cys-79 (Fig. 3C). In its protonated state at pH 6.0, Glu-83 is hydrogen-bonded to His-134 (helix C), whereas at pH 7.0, it forms a salt bridge with Arg-268 (helix G). Thus, protonation of Glu-83 at pH 6.0 in CrChR2 and consequently hydrogen bonding to His-134 might lead to the observed structural pH-dependent rearrangement of the cytoplasmic end of helix B below pH 7 in our experiments. Because Glu-83 belongs to the 12 polar residues along the channel pore, forming a hydrophilic and strongly electronegative surface, structural changes close to the cytoplasmic end of this pore (i.e. close to the inner gate) may affect gating. This will be the subject of future studies.
      Moreover, pH-dependent structural heterogeneity of helix B in the C128T variant would add a new perspective to the finding of multiple dark-adapted states in C128T (
      • Ritter E.
      • Piwowarski P.
      • Hegemann P.
      • Bartl F.J.
      Light-dark adaptation of channelrhodopsin C128T mutant.
      ). Hence, our data not only show the nature of the conformational change of the cytoplasmic end of helix B in channel opening but also highlight the pH-dependent conformational heterogeneity of a CrChR2 step function mutant that belongs to the neurophysiological optogenetics tool kit.
      By a combination of different time-resolved fluorescence techniques, we have presented results that lend support to the helix tilt model (
      • Schneider F.
      • Grimm C.
      • Hegemann P.
      Biophysics of channelrhodopsin.
      ,
      • Krause N.
      • Engelhard C.
      • Heberle J.
      • Schlesinger R.
      • Bittl R.
      Structural differences between the closed and open states of channelrhodopsin-2 as observed by EPR spectroscopy.
      ,
      • Müller M.
      • Bamann C.
      • Bamberg E.
      • Kühlbrandt W.
      Light-induced helix movements in channelrhodopsin-2.
      ,
      • Sattig T.
      • Rickert C.
      • Bamberg E.
      • Steinhoff H.J.
      • Bamann C.
      Light-induced movement of the transmembrane helix B in channelrhodopsin-2.
      ) of helix B upon formation of the open channel state and gave insight in the possible mechanism by comparing the CrChR2 data with time-resolved anisotropy data from other retinal proteins. Accessibility studies revealed a pH-dependent structural heterogeneity close to the inner gate. This kind of information is important when comparing and interpreting crystallographic and spectroscopic results for CrChR2 photocycle or electrophysiological measurements, because crystal structures were obtained at pH 6 and 7 (discussed above), and spectroscopic as well as electrophysiology data were measured at a variety of pH values (
      • Stehfest K.
      • Ritter E.
      • Berndt A.
      • Bartl F.
      • Hegemann P.
      The branched photocycle of the slow-cycling channelrhodopsin-2 mutant C128T.
      ,
      • Kato H.E.
      • Kamiya M.
      • Sugo S.
      • Ito J.
      • Taniguchi R.
      • Orito A.
      • Hirata K.
      • Inutsuka A.
      • Yamanaka A.
      • Maturana A.D.
      • Ishitani R.
      • Sudo Y.
      • Hayashi S.
      • Nureki O.
      Atomistic design of microbial opsin-based blue-shifted optogenetics tools.
      ,
      • Ritter E.
      • Piwowarski P.
      • Hegemann P.
      • Bartl F.J.
      Light-dark adaptation of channelrhodopsin C128T mutant.
      ,
      • Lórenz-Fonfría V.A.
      • Heberle J.
      Channelrhodopsin unchained: structure and mechanism of a light-gated cation channel.
      ). Structural heterogeneity might also contribute to the observed multiple dark states of the ChR2-C128T variant (
      • Ritter E.
      • Piwowarski P.
      • Hegemann P.
      • Bartl F.J.
      Light-dark adaptation of channelrhodopsin C128T mutant.
      ). Moreover, structural heterogeneity may affect the photocurrent, open state time of the channel, and degree of inactivation, important parameters when employing step function mutants of CrChR2 in optogenetics experiments.

      Author Contributions

      P. V. performed time-resolved fluorescence experiments, analyzed and interpreted the data, and contributed to the writing of the manuscript; N. K. generated the mutant protein, developed the preparation in nanodiscs, and contributed to the writing of the manuscript; J. B. contributed to the time-resolved fluorescence experiments and sample preparation; C. S. characterized the fluorescently labeled sample and performed experiments shown in Figs. 1 and 2; M. W. prepared the protein sample; F. S. performed the electrophysiology experiments and wrote the corresponding parts of the manuscript; R. S. conceived and coordinated the study, contributed to the design of the experiments, and contributed to the writing of the manuscript; U. A. conceived and coordinated the study, designed the experiments, analyzed and interpreted the data, and wrote the manuscript. All authors reviewed the results and approved the final version of the manuscript.

      Acknowledgment

      We thank Peter Hegemann (Humboldt-Universität zu Berlin) for support.

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