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The Activation Pathway of Human Rhodopsin in Comparison to Bovine Rhodopsin*

  • Roman Kazmin
    Affiliations
    Institut für Medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany

    Institut für Biologie, Experimentelle Biophysik, Humboldt-Universität zu Berlin, 10115 Berlin, Germany
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  • Alexander Rose
    Footnotes
    Affiliations
    Institut für Medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany

    AG ProteInformatics, Charitéplatz 1, 10117 Berlin, Germany
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  • Michal Szczepek
    Footnotes
    Affiliations
    Institut für Medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany

    AG Protein X-ray Crystallography and Signal Transduction, Charitéplatz 1, 10117 Berlin, Germany
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  • Matthias Elgeti
    Footnotes
    Affiliations
    Institut für Medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany
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  • Eglof Ritter
    Correspondence
    To whom correspondence may be addressed.
    Affiliations
    Institut für Biologie, Experimentelle Biophysik, Humboldt-Universität zu Berlin, 10115 Berlin, Germany
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  • Ronny Piechnick
    Affiliations
    Institut für Medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany
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  • Klaus Peter Hofmann
    Affiliations
    Institut für Medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany

    Zentrum für Biophysik und Bioinformatik (BPI), Humboldt-Universität zu Berlin, 10115 Berlin, Germany
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  • Patrick Scheerer
    Correspondence
    To whom correspondence may be addressed.
    Affiliations
    Institut für Medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany

    AG Protein X-ray Crystallography and Signal Transduction, Charitéplatz 1, 10117 Berlin, Germany
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  • Peter W. Hildebrand
    Affiliations
    Institut für Medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany

    AG ProteInformatics, Charitéplatz 1, 10117 Berlin, Germany
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  • Franz J. Bartl
    Correspondence
    To whom correspondence may be addressed.
    Affiliations
    Institut für Medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany
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  • Author Footnotes
    * This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG, SFB740; to K. P. H., P. S., and P. W. H.; SFB1078 to P. S. and F. J. B.; HI1502 and BI893/8 to P. W. H.), DFG Cluster of Excellence “Unifying Concepts in Catalysis” (Research Field D3/E3-1; to P. S.) and Research Fellowship (EL 779-1; to M. E.), the German Federal Ministry of Education and Research (BMBF Verbundforschung, Grant 05K13KH1), and the European Research Council (Advanced Investigator Grant (ERC-2009/249910-TUDOR; to K. P. H.)
    This article contains supplemental Fig. 1.
    1 Both authors contributed equally to this work.
    2 Present address: Jules Stein Eye Institute and Dept. of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095-7008.
Open AccessPublished:June 23, 2015DOI:https://doi.org/10.1074/jbc.M115.652172
      Rhodopsin, the photoreceptor of rod cells, absorbs light to mediate the first step of vision by activating the G protein transducin (Gt). Several human diseases, such as retinitis pigmentosa or congenital night blindness, are linked to rhodopsin malfunctions. Most of the corresponding in vivo studies and structure-function analyses (e.g. based on protein x-ray crystallography or spectroscopy) have been carried out on murine or bovine rhodopsin. Because these rhodopsins differ at several amino acid positions from human rhodopsin, we conducted a comprehensive spectroscopic characterization of human rhodopsin in combination with molecular dynamics simulations. We show by FTIR and UV-visible difference spectroscopy that the light-induced transformations of the early photointermediates are very similar. Significant differences between the pigments appear with formation of the still inactive Meta I state and the transition to active Meta II. However, the conformation of Meta II and its activity toward the G protein are essentially the same, presumably reflecting the evolutionary pressure under which the active state has developed. Altogether, our results show that although the basic activation pathways of human and bovine rhodopsin are similar, structural deviations exist in the inactive conformation and during receptor activation, even between closely related rhodopsins. These differences between the well studied bovine or murine rhodopsins and human rhodopsin have to be taken into account when the influence of point mutations on the activation pathway of human rhodopsin are investigated using the bovine or murine rhodopsin template sequences.

      Introduction

      Rhodopsin (Rho),
      The abbreviations used are: Rho
      rhodopsin
      hRho
      human Rho
      bRho
      bovine Rho
      GPCR
      G protein-coupled receptor
      TM
      transmembrane
      EL2
      extracellular loop 2
      Gt
      G protein transducin
      bis-tris-propane
      1,3-bis[tris(hydroxymethyl)methylamino]propane
      GTPγS
      guanosine 5′-O-(thiotriphosphate)
      Batho
      bathorhodopsin
      Lumi
      lumirhodopsin
      TEC
      T5E2C region
      SB
      Schiff base.
      the rod cell photoreceptor that mediates dim light vision in the vertebrate eye, is the archetype of class A G protein-coupled receptors (GPCR) and consists of 348 amino acids. It shares with other GPCRs a seven-transmembrane (TM) α helical structure. Rho is unique among GPCRs as its light-sensitive ligand 11-cis-retinal is covalently bound to the apoprotein via a protonated Schiff base. Light absorption induces 11-cis to all-trans isomerization thus replacing the inverse agonist by a strong agonist in situ. Subsequently the receptor proceeds through a number of intermediates, eventually leading to the late metarhodopsin states, which include the active form Meta II that binds and activates the G protein (
      • Hofmann K.P.
      • Scheerer P.
      • Hildebrand P.W.
      • Choe H.-W.
      • Park J.H.
      • Heck M.
      • Ernst O.P.
      A G protein-coupled receptor at work: the rhodopsin model.
      ). Rho activation is the first step of a highly optimized catalytic system allowing single photon response due to high amplification on the background of virtually no dark noise.
      The majority of biochemical and biophysical investigations on Rho were undertaken with the easily accessible bovine rhodopsin (bRho) (
      • Rakoczy E.P.
      • Kiel C.
      • McKeone R.
      • Stricher F.
      • Serrano L.
      Analysis of disease-linked rhodopsin mutations based on structure, function, and protein stability calculations.
      ). Bovine and human rhodopsin (hRho) show a sequence identity of 93.4% (
      • Nathans J.
      • Hogness D.S.
      Isolation and nucleotide sequence of the gene encoding human rhodopsin.
      ), with different amino acids at 23 positions (Fig. 1). Remarkably some sequence differences occur in regions that may play a crucial role during receptor activation. This applies to the connection from TM5 to the extracellular loop 2 (EL2, amino acids 175–198), hereafter defined as the T5E2C region, and amino acids 297–300 in the direct proximity of the Schiff base, which deprotonates during the transition from inactive Meta I to the active Meta II state (Fig. 2). Because both regions are connected to activating conformational changes, it is possible that these sequence alterations significantly affect the conformations of the dark state and light-activated photointermediates and thereby change the functional properties of the photoreceptor.
      Figure thumbnail gr1
      FIGURE 1.Sequence alignment of human and bovine rhodopsin. Green, differences between both sequences; orange, conserved D(E)RY motif; blue, TM3-TM5 microdomain (Glu-122, Trp-126, and His-211) near the retinal β-ionone ring; magenta, conserved NPXXY(X)5,6F motif; red, Schiff base lysine (Lys-296); the bold lettering denotes the known retinitis pigmentosa positions.
      Figure thumbnail gr2
      FIGURE 2.Homology model and areas of interest. a, homology model of dark state human rhodopsin based on bovine rhodopsin structure (PDB entry 1U19) containing 11-cis-retinal bound via a protonated Schiff base to Lys-296 (red). Black boxes highlight the regions (amino acids 297–300) and (amino acids 194–198) that are in the focus of this study. Differences between human (green) and bovine (gray) amino acids are indicated. b and d, enlarged side view of the two regions shows the details of the structural differences. c and e, sequence alignment segments of bovine and human rhodopsin for residues 294–304 and 190–200 as well WebLogo diagram of all available mammalian rhodopsin sequences. Dashed arrows below e indicate the known secondary structure elements, namely EL2, TM5, and the T5E2C region (residues 194–198; TEC).
      For our studies, bRho and hRho were expressed and purified in COS-1 cells, which yields sufficient quantities and purity to conduct spectroscopic investigations. Subsequent incorporation into lipid vesicles allows their investigation in a native-like membrane environment. The main technique of our investigation is FTIR difference spectroscopy, which has high structural sensitivity and allows investigation of Rho samples at physiologically relevant millimolar concentrations. Furthermore, the FTIR peptide binding assay applied here is capable of monitoring the conformational changes induced by binding of the C terminus of the Gt α-subunit to the active receptor (
      • Bartl F.
      • Ritter E.
      • Hofmann K.P.
      FTIR spectroscopy of complexes formed between metarhodopsin II and C- terminal peptides from the G-protein α- and γ-subunits.
      ,
      • Nishimura S.
      • Kandori H.
      • Maeda A.
      Interaction between photoactivated rhodopsin and the C-terminal peptide of transducin α-subunit studied by FTIR spectroscopy.
      • Szczepek M.
      • Beyrière F.
      • Hofmann K.P.
      • Elgeti M.
      • Kazmin R.
      • Rose A.
      • Bartl F.J.
      • von Stetten D.
      • Heck M.
      • Sommer M.E.
      • Hildebrand P.W.
      • Scheerer P.
      Crystal structure of a common GPCR-binding interface for G protein and arrestin.
      ). The spectroscopic data are complemented by classical molecular dynamics (MD) simulations of the dark state and active conformations of hRho and bRho, and the G protein activation capability was tested using a catalytic fluorescence assay.
      At least 100 mutations at 68 positions (see Fig. 1) in Rho genes are known to cause retinitis pigmentosa, which is linked to a gradual loss of peripheral and night vision (see Ref.
      • Rakoczy E.P.
      • Kiel C.
      • McKeone R.
      • Stricher F.
      • Serrano L.
      Analysis of disease-linked rhodopsin mutations based on structure, function, and protein stability calculations.
      for examples). Some of these mutations have an effect on the structural integrity of the protein like impairment of protein folding and transport (
      • Singhal A.
      • Ostermaier M.K.
      • Vishnivetskiy S.A.
      • Panneels V.
      • Homan K.T.
      • Tesmer J.J.
      • Veprintsev D.
      • Deupi X.
      • Gurevich V.V.
      • Schertler G.F.
      • Standfuss J.
      Insights into congenital stationary night blindness based on the structure of G90D rhodopsin.
      ). Other mutations primarily have an impact on the conformational equilibria of the agonist-bound receptor (Meta-equilibria) (
      • Bosch L.
      • Ramon E.
      • Del Valle L.J.
      • Garriga P.
      Structural and functional role of helices I and II in rhodopsin. A novel interplay evidenced by mutations at Gly-51 and Gly-89 in the transmembrane domain.
      ), whereas congenital night blindness is predominantly caused by an increase in constitutive activity (
      • Rao V.R.
      • Cohen G.B.
      • Oprian D.D.
      Rhodopsin mutation G90D and a molecular mechanism for congenital night blindness.
      ). Many of these diseases are caused by single amino acid mutations. The structural and mechanistic differences between bRho and hRho found in this study should thus be considered when the activation pathway of malfunctioning human receptor mutants is investigated.

      Discussion

      The aim of this study was to compare the activation pathways of bRho and hRho with respect to structure, function, and kinetics of photointermediate formation. For this purpose we first analyzed the sequences of both pigments and modeled and simulated the structures of dark and active state of hRho on the basis of the available crystal structures of bRho. The combination of these modeling techniques with FTIR difference spectroscopy of the photointermediates provides us with a detailed picture of conformational changes involved in activation. Thus we can describe the similarities and differences between these two photoreceptors.

      Structural Differences between Bovine and Human Rhodopsin

      In the majority of the 23-amino acid alterations, the chemical and physical side-chain properties are preserved (i.e. F88L, T93S, V173A, V218I, L266V, L321I), and no major influence on receptor function is expected. However, significant differences occur in two distinct regions with potential influence on receptor structure (Fig. 1) and may thus have functional consequences.
      The first region comprises the residues 297–300 in the direct vicinity of the retinal Schiff base with the sequence 297SAAI300 in hRho and 297TSAV300 in bRho (Fig. 2, b and c). The serine to alanine exchange may contribute to two distinct effects on the retinal Schiff base environment. First, the electrostatic properties of the retinal Schiff base environment will be altered when the hydroxyl group is removed. Second, our simulations reveal additional space in hRho (Fig. 3), potentially occupied by an additional water molecule, which influences the vicinity of the Schiff base (
      • Sekharan S.
      • Mooney V.L.
      • Rivalta I.
      • Kazmi M.A.
      • Neitz M.
      • Neitz J.
      • Sakmar T.P.
      • Yan E.C.
      • Batista V.S.
      Spectral tuning of ultraviolet cone pigments: an interhelical lock mechanism.
      ) and thus the UV-visible absorption maximum (
      • Sullivan J.M.
      • Shukla P.
      Time-resolved rhodopsin activation currents in a unicellular expression system.
      ) and the typical infrared absorptions of the dark state. In agreement, the absorption maximum of the SB chimera, in which the bovine 297TSAV300 sequence was introduced, was shifted back toward that typical of bRho, whereas the kinetics of Meta II formation remain the same as seen by hRho, suggesting only a local influence of the TSAV region on the retinal Schiff base.
      The second sequence deviation comprises the T5E2C region (residues 194–198) at the connection from TM5 to EL2 (Fig. 2, d and e). The human T5E2C sequence is conserved in many mammalian rhodopsins, except in Rho of sea mammals or nocturnal animals including bRho (see the Weblogo plot in Fig. 2, c and e). The differences in the T5E2C region alter its hydrogen bond network with the end of TM5 and leads, according to our MD simulations, to a considerable difference in conformational flexibility of the T5E2C region. In bRho this region exhibits a flapping motion during the MD simulations that still allows coupling of EL2 motion to TM5 movement, whereas in hRho the whole region shows an additional sliding motion that loosens the tight EL2/TM5 coupling (see Fig. 4). Consequently, in bRho this region functions as a rigid lever (
      • Ahuja S.
      • Smith S.O.
      Multiple switches in G protein-coupled receptor activation.
      ), whereas in hRho it is a rather flexible linker with limited ability to trigger TM5 movement connected with Meta I formation (
      • Ye S.
      • Zaitseva E.
      • Caltabiano G.
      • Schertler G.F.
      • Sakmar T.P.
      • Deupi X.
      • Vogel R.
      Tracking G-protein-coupled receptor activation using genetically encoded infrared probes.
      ). Together with the faster formation of the Schiff base deprotonated species, this implies that necessary conformational changes must already have taken place before the formation of Meta I or do not have to take place in hRho. Our simulations suggest that the loosening of the EL2/TM5 coupling present in the active conformation of bRho is anticipated in the dark state of hRho. The dynamic properties of the EL2 have previously been connected with the constitutive activity of other GPCRs (
      • Wheatley M.
      • Wootten D.
      • Conner M.T.
      • Simms J.
      • Kendrick R.
      • Logan R.T.
      • Poyner D.R.
      • Barwell J.
      Lifting the lid on GPCRs: the role of extracellular loops.
      ,
      • Wifling D.
      • Bernhardt G.
      • Dove S.
      • Buschauer A.
      The extracellular loop 2 (ECL2) of the human histamine H4 receptor substantially contributes to ligand binding and constitutive activity.
      ). Note that sequence differences in the 297–300 region around the Schiff base could also contribute to constrictions in EL2 motion and consequently be responsible for the limitation of TM5 movement in Meta I. However, replacing amino acids 194–196 of the human receptor with the bovine sequence, as in the TEC chimera, does partially restore the bovine Meta I difference spectrum (especially the band at 1661 cm−1) (Fig. 6c). Consequently, we favor the view that the alteration in the T5E2C region influences TM5/EL2 coupling linked to Meta I formation. Interestingly the absorption maximum of the TEC chimera is shifted to 492 nm. This indicates a long range influence of this region on the electrostatic properties of the Schiff base and/or the retinal environment, which is most likely caused by slight rearrangements of the helical bundle in the inactive conformations including the dark state.

      Differences between the Activation Pathway of Bovine and Human Rhodopsin

      Rho activation is known to proceed gradually from the photochemical core to the cytoplasmic surface. This means that changes due to photon absorption are initially localized to the retinal itself leading to its ultrafast isomerization. Consequently, the first stable intermediate, Batho, shows virtually no structural changes of the ligand binding pocket. This results in a highly constrained all-trans-retinal configuration indicated by a red-shifted absorption maximum (
      • Nakamichi H.
      • Okada T.
      Crystallographic analysis of primary visual photochemistry.
      ). The first significant side chain movements take place during formation of the Lumi intermediate (
      • Nakamichi H.
      • Okada T.
      Local peptide movement in the photoreaction intermediate of rhodopsin.
      ). FTIR difference spectra can only reflect those parts of the protein that undergo changes during the reaction investigated. Consistently, bands in the Batho and Lumi FTIR difference spectra are confined to changes of the retinal geometry due to cis/trans isomerization and direct interaction with adjacent groups. The small differences between the Batho and Lumi FTIR difference spectra of hRho and bRho, albeit more pronounced in Lumi, are predominantly due to differences in the sequence around the Schiff base (297SAAI300 in hRho and 297TSAV300 in bRho), which are also indicated by different UV-visible absorption maxima in the dark state.
      Our FTIR results reveal the first significant differences between the two activation pathways in the Meta I difference spectra of both pigments. Especially, the positive band at 1661 cm−1, assigned to conformational changes occurring with Meta I formation, is not observed in human Meta I. In bovine Meta I the primary counterion of the Schiff base is known to switch from Glu-113, which serves as counterion in the dark state, to Glu-181 positioned in EL2 (
      • Sandberg M.N.
      • Greco J.A.
      • Wagner N.L.
      • Amora T.L.
      • Ramos L.A.
      • Chen M.H.
      • Knox B.E.
      • Birge R.R.
      Low temperature trapping of photointermediates of the rhodopsin E181Q mutant.
      ). This decisive reorganization of the Schiff base hydrogen-bond network leads to several conformational changes that unlock the photoreceptor irreversibly and facilitate its activation by thermal energy and proton uptake (
      • Mahalingam M.
      • Martínez-Mayorga K.
      • Brown M.F.
      • Vogel R.
      Two protonation switches control rhodopsin activation in membranes.
      ). This is achieved by a collective motion of EL2 and TM5, realized by a relatively rigid connection of both regions in bRho (
      • Ye S.
      • Huber T.
      • Vogel R.
      • Sakmar T.P.
      FTIR analysis of GPCR activation using azido probes.
      ). The stability of this rigid connection is, however, strongly affected in the simulations of hRho. Our simulations further revealed an increased lateral mobility of EL2 in the dark state of hRho, which enables Glu-181 to move closer to the retinal Schiff base and thereby at least partially anticipates the counterion shift (Fig. 4). This notion is also corroborated by a TEC chimera between hRho and bRho (Fig. 6c). In this hRho based chimera, the “flexible” sequence 194LKP196 of hRho is replaced with the more rigid bovine sequence 194PHE196, thus restoring the putative tight coupling of EL2 movement to TM5 motion. In this mutant the band intensity at +1661 cm−1 is almost restored to the level found in the Meta I difference spectra of bRho.
      Formation of the species with deprotonated Schiff base proceeds much faster in hRho as observed in the present time-resolved flash photolysis measurements and is in agreement with results of detergent-solubilized hRho (
      • Lewis J.W.
      • van Kuijk F.J.
      • Thorgeirsson T.E.
      • Kliger D.S.
      Photolysis intermediates of human rhodopsin.
      ) and in vivo experiments (
      • Pugh E.N.
      Rhodopsin flash photolysis in man.
      ). This finding supports the view that some rate-limiting structural changes going along with Meta I in bRho already have formed in hRho. The SB chimera exhibits no influence on the Meta II formation kinetics. It is formed with a comparable rate as hRho (Fig. 8a, Table 1), although the absorption maximum of the bRho dark state is restored. More intriguing is the behavior of the TEC chimera. The exchange of EL2/TM5 connection influences not only both rate constants of Meta II formation but also restores the structure of bovine Meta I at least partially and shows a hypochromic shift of the dark state absorption maximum. The pKa of the Meta I ↔ Meta II equilibrium is a very sensitive monitor of the conformational equilibrium. The two pKa values are virtually identical, which means that both pigments share the same conformational equilibria of Meta II states and that the mechanisms for TM6 outward movement and proton uptake at Glu-134 are conserved. Finally, binding of the GtαCT peptide surrogate and catalytic activation of the transducin reflect that the functional properties of the activated receptor conformations are similar.
      It is tempting to correlate our results with the existence of two different Meta I species that form on the ms and μs timescale, respectively, as it has been described for bRho (
      • Thorgeirsson T.E.
      • Lewis J.W.
      • Wallace-Williams S.E.
      • Kliger D.S.
      Effects of temperature on rhodopsin photointermediates from lumirhodopsin to metarhodopsin-II.
      ). The two Meta I states are formed in parallel reaction pathways accessed in a temperature-dependent manner. However, because the Meta equilibria are not significantly shifted in hRho, we suggest that the dark state conformation is such that the mutations in the Schiff base and T5E2C region facilitate faster activation by allowing conformational changes characteristic for bovine Meta I (e.g. Schiff base counterion shift).

      Conclusion

      Despite their close evolutionary relationship, hRho and bRho display differences in their primary structure that we connect to specific impacts on structural elements during receptor function. On the one hand the electrostatic environment of the retinal Schiff base linkage is altered. On the other hand we have identified an increase of conformational flexibility in the linker region between extracellular loop 2 and transmembrane helix 5 of hRho, which is a feature of the active conformation and has been reported to increase the constitutive activity in other GPCRs. Flexibility in this region seems to be anticipated in hRho leading to a shifted absorption maximum in the dark state and faster formation of active Meta II. Although the structural properties of the inactive receptor seem to have adapted to the specific environment of the host species (e.g. diurnality or nocturnality), the active conformation itself is surprisingly similar. Apparently, the architecture of the receptor is such that host specific activation pathways evolve; however, the active conformation appears highly optimized for G protein catalysis and is thus conserved.
      Our findings highlight that although bRho provides a valuable template for hRho, the differences between the inactive conformations have to be considered carefully, especially when the activation mechanism of disease-associated mutations of hRho are to be examined using the bovine template.

      Author Contributions

      F. J. B. coordinated the study and wrote the paper. R. K. prepared the samples, performed FTIR UV-visible and photolysis measurements, analyzed the spectroscopic data, wrote the paper, and prepared Figs. 1, 5, 6, 7, and 8. M. S. and P. S. prepared all expression vectors, Fig. 2, and wrote the paper. A. R. performed MD simulations, prepared Figs. 3, 4, and supplemental Fig. 1, and wrote the paper. P. W. H. supervised the theoretical part of this study and wrote the paper. M. E. analyzed and interpreted the spectroscopic data, provided data analysis tools, and wrote the paper. E. R. analyzed and interpreted the spectroscopic data and wrote the manuscript. R. P. performed Gt activation measurements. K. P. H. wrote the paper. R. K., E. R., M. S., and P. S. has the initial idea and initiated the project.

      Acknowledgments

      We thank Martha E. Sommer for critical reading of the manuscript, Anja Koch, Jana Engelmann, and Brian Bauer for technical assistance and Rho purification, and Martin Heck for fruitful discussions. The computer time necessary was provided by the “Norddeutscher Verbund für Hoch-und Höchstleistungsrechner” (HLRN) project bec00085.

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