Ultrafast Protein Response in Channelrhodopsin-2 Studied by Time

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Biophysical Chemistry, Biomolecules, and Biomaterials; Surfactants and Membranes

Ultrafast Protein Response in Channelrhodopsin-2 Studied by Time-Resolved IR Spectroscopy Elena Bühl, Peter Eberhardt, Christian Bamann, Ernst Bamberg, Markus Johann Braun, and Josef Wachtveitl J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.8b03382 • Publication Date (Web): 11 Dec 2018 Downloaded from http://pubs.acs.org on December 15, 2018

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The Journal of Physical Chemistry Letters

Ultrafast Protein Response in Channelrhodopsin-2 Studied by Time-resolved IR Spectroscopy Elena Bühl†, Peter Eberhardt†, Christian Bamann‡, Ernst Bamberg‡, Markus Braun†, Josef Wachtveitl†* †Institute

of Physical and Theoretical Chemistry, Goethe University, Max von Laue-Straße 7, 60438 Frankfurt am Main, Germany

‡Max

Planck Institute of Biophysics, Max von Laue-Straße 3, 60438 Frankfurt am Main, Germany

*E-mail: [email protected]

ACS Paragon Plus Environment

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ABSTRACT

Ultrafast IR transient absorption in the carbonyl vibrational region of protonated aspartate and glutamate residues in Channelrhodopsin-2 from Chlamydomonas reinhardtii shows immediate protein response to retinal excitation. The observed difference bands are formed directly after the excitation on the sub-ps time scale and were assigned to side chains in the retinal vicinity, such as D156 and E90. This finding implies an ultrafast and effective energy transfer from the retinal to its environment via hydrogen-bonded networks and reveals extraordinary strong chromophore-protein coupling and intense interaction within the protein. Relevance on the protein function as optically gated ion channel are discussed.

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Channelrhodopsin-2 (ChR-2) expressed in the eyespot of the green algae Chlamydomonas reinhardtii serves as sensory photoreceptor controlling its phototaxis.1,2 This membrane protein is known as a light-gated ion channel permeable for protons, potassium, sodium and calcium cations3 and is frequently used in optogenetics.4–7 Similar to other microbial rhodopsins, ChR-2 comprises a seven trans-membrane helix motif with a retinal chromophore covalently linked to a lysine via a Schiff base.8,9 In the dark-adapted state, the retinal is present in 100% all-trans conformation10 and isomerizes to 13-cis upon blue light excitation as the primary photo-product. Directly after retinal excitation a vibrational relaxation on the S1 potential surface within 150 fs takes place, followed by excited state decay and a simultaneous population of the 13-cis photo-product and the all-trans ground state within 400 fs. After cooling processes proceeding with a time component of 2.7 ps the K-like P1 intermediate is obtained.11,12 Retinal isomerization leads to a photo-cycle reaction similar to other type I retinal proteins like bacteriorhodopsin (BR) or proteorhodopsin (PR), where different ground state intermediates are formed before relaxation to the initial state occurs. In the course of this photo-cycle, a proton transport takes place including deprotonation (P2 intermediate) and reprotonation (P3 intermediate) of the retinal Schiff base (“leaky proton pump”).13–16 The aspartates in position 253 (D253) and 156 (D156) act as primary proton acceptor and proton donor, respectivaly.17,18 Additionally, the ion channel opening (t1/2 ~ 200 µs) and closing (t1/2 ~ 10 ms) are observed during the lifetime of the conducting P3 state. This channel activity is accompanied by alterations of the retinal environment such as protonation states of specific side chains as well as conformational changes of the protein backbone manifesting in the IR difference band at 1665 cm-1.8,17 Surprisingly this negative amide I band with an unusually large amplitude was already


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observed in the P1 difference spectrum at 80 K well before the channel opening.19–21 The fspump/probe experiments provided for the 1665 cm-1 band a formation time of 500 fs. Additionally, many side chain bands were detected. Thus, an ultrafast energy transfer from the retinal to the protein backbone during the isomerization was observed, implying the formation of a pre-gating state on the sub-ps time scale.22 In the present work, we investigate the effect of retinal excitation on its environment employing ultrafast vis-pump/IR-probe spectroscopy in the region of the protonated aspartate and glutamate C=O vibrational bands. Based on the established band assignment in this region, we determine the formation dynamics of the difference signals caused by environmental alterations of special amino acid residues important for protein functionality, such as E90 or D156.8 Especially, we focus on the ultrafast protein response induced by chromophore excitation as crucial precursor of the channel opening. We investigate the effect of the retinal isomerization and its fast energy transfer on the protein environment employing ultrafast vis-pump/IR-probe spectroscopy in the region of the protonated aspartate and glutamate C=O vibrational bands (Figure 1 and Figure 2 B). Data analysis via global fitting procedure starts at a delay time of 0.4 ps, signals at early delay times are dominated by cross-phase modulation. The contour plot and transient spectra at selected delay times clearly show that all spectral signatures detected at 1.5 ns can be observed already 400 fs after excitation. From UV/vis transient absorption data two ultrafast time constants of 150 and 400 fs were derived to describe initial dynamics and the isomerization process.12 Thus, at a delay time of 400 fs isomerization is mainly completed. Global fit analysis requires two decay times of τ1 = (1.5 ± 1.0) ps and τ2 = (30 ± 5) ps to account for the observed dynamics and an additional τ3 time constant (set to infinity) to


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describe the final product spectrum at 1.5 ns (for the respective decay associated spectra, DAS, see Figure 2 A). The shortest time constant τ1 describes a weak signal decrease and narrowing of the difference bands, while the time constant τ2 shows a signal decay especially below 1700 cm-1. These signatures can be explained by cooling processes or vibrational relaxation on the ground state potential surface. During these processes the excess energy is distributed to the retinal low frequency modes and the chromophore environment including solvent molecules.23 It has to be stressed that the spectral position of all prominent maxima and minima (1692, 1709, 1718, 1738, and 1746 cm-1, marked by dashed lines in Figure 1 A-B) remain unchanged from 400 fs to 1.5 ns (complete experimental time window). At 1692 cm-1 a dominant positive signal can be observed. This band has a small positive shoulder at 1700 cm-1 at short delay times, which decays with τ1. Similar to other signals within the investigated spectral range, this band is formed on the sub-ps time range and can already be detected at 400 fs. After a small decay with τ2 = 30 ps caused by cooling processes the (+)1692 cm-1 difference band shows a constant offset at the end of the measurement. Next to this band a negative signal at 1709 cm-1 as well as a positive band at 1718 cm-1 can be observed. Compared to other signals this positive band shows only a small amplitude after 100 ps. Furthermore, its spectral shape implies an overlap of different positive and negative signals. Such an overlap of different signals is also observed at the high energy end of the spectrum. In this range the infinite spectrum shows a broad, weak positive band with a maximum at about 1766 cm-1. In contrast, a clear spectral signature can be detected at (+)1746/(-)1738 cm-1. Similar to the difference band at ()1709 cm-1 these two bands are present at a delay time of 400 fs and decay with two time


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constants τ1 = 1.5 ps and τ2 = 30 ps, resulting in a considerable remaining signal amplitude at 1.5 ns.

Figure 1. Results of the IR-pump-probe experiments on ChR-2 A) Contour plot of fitted data of ChR-2 after 480 nm laser pulse excitation presented from 0.4 ps to 1.5 ns. The time scale is linear up to 1 ps and logarithmic afterwards. The positive and negative absorbance changes are colored red and blue, respectively. B) Transient absorbance changes of ChR-2 at selected delay times.

In order to assess the detected vibrational bands, the final transient spectrum for a delay time of 1.5 ns was parametrized using 11 Gaussians to determine the accurate band positions (see Figure 2 C). At this delay time the P1 intermediate state of ChR-2 is populated, as mentioned above. Thus the respective difference spectrum consists of initial ground state (negative) and P1 state (positive) vibrational bands.


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Figure 2. Data analysis of the time-resolved measurements on ChR-2. A) Decay associated spectra of three time constants determined by global lifetime analysis of ChR-2 data. B) Transient absorbance changes of ChR-2 at 1662 cm-1,22 1692 cm-1, 1709 cm-1, 1718 cm-1, 1739 cm-1, 1746 cm-1. C) The infinite spectrum (DAS of τ3) of ChR-2 (red line) fitted using 11 Gaussians (grey dotted lines).

The ultrafast vis-pump/IR-probe measurements on ChR-2 in the carbonyl stretching vibration range of protonated aspartate and glutamate residues show many difference bands already 400 fs after the retinal excitation. The spectral signature detected at this short delay time and also at 1.5 ns mirrors the 80 K difference spectrum of the freezetrapped P1 intermediate in H2O19–22 as well as D2O22. Based on the above shown Gaussian fit


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of the transient spectrum at 1.5 ns we will assign the observed difference signals to specific residues, as already described in the literature. The spectral signature at (+)1746 cm-1 and (-)1738 cm-1 was observed after illumination of ChR-2 at 80 K and assigned to the carbonyl stretching vibration of residue D156 caused by hydrogen-bonded network alterations.19–22 Also time-resolved measurements with ns time resolution exhibited a similar difference band for the P1 state.24 Based on point mutation experiments these signals were also assigned to a shift of the carbonyl stretching vibration of D156 in the P1 intermediate caused by alterations of the hydrogen-bonded network of this side chain.17,18 The negative band at 1709 cm-1 in D2O (1718 cm-1 in H2O) is consistently assigned in the literature.18,21,24 This signal is attributed to the protonated amino acid residue E90, which is supposed to be responsible for ion selectivity of ChR-2.25 This difference band is also present in the 80 K FTIR spectrum of the freeze-trapped P1 intermediate21,22 as well as in time-resolved measurements with ns time resolution already 200 ns after excitation.25,26 The strongest IR signal for the P1 intermediate is observed at 1692 cm-1 (or 1690 cm-1 according to the Gaussian fit) with a small shoulder at 1700 cm-1. The low-temperature FTIR spectrum of P1 in H2O also shows a 5 cm-1 upshifted positive band representing the result of overlapping vibrational bands.19,21. However, there is no conclusive band assignment in the literature yet. Based on the intermediate lifetimes investigations combined with studies on point mutation part of the positive band at 1695 cm-1 was assigned to the protonation of D253 during P2 formation.17 However, P2 formation is accompanied by retinal deprotonation and takes place on a µs time scale, 13,17 it is thus not expected to be observed in our ultrafast pump-probe experiments.9,27


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The formation of the detected carbonyl vibrational bands in ChR-2 induced after the chromophore excitation is unusually fast. This indicates a strong retinal interaction with its direct environment leading to an ultrafast energy transfer to the protein. This strong coupling was observed in earlier time-resolved IR studies in the amide I region.22 In addition to an intense amide I band at 1665 cm-1 several difference bands originating from protein side chains were detected directly after the decay of the cross-phase modulation signal. Unfortunately, these signals could not be assigned to specific amino acids. However, in the present study, using the existing band assignment in the C=O vibrational region, we can now attribute the detected signals to specific side chains. Compared to the amide I band, which is formed with a time constant of 500 fs, the signal formation in the C=O region is already finished at 400 fs after excitation (see transients in Figure 2 B). Our previous ultrafast experiments in the UV/vis spectral region12 provide two sub-ps time constants for the primary photoreaction dynamics of ChR-2. The shortest time constant τ1 = 150 fs was associated with wave packet motions on the S1 surface out of the Franck-Condon region along the C=C stretching coordinate. The longer decay time τ2 = 400 fs was assigned to the S1 depopulation and the 13-cis photoproduct formation. Therefore, the amide I band formation takes place on a similar time scale as the retinal isomerization. Earlier processes like the retinal stretching in the excited state may also affect the side chain vibrations in the carbonyl region. The IR difference spectra for the P1 state of ChR-2 in the carboxylic vibrational range significantly differ from those of other microbial rhodopsins. While the 77K difference spectrum of Anabena Sensory Rhodopsin (ASR) does not show any signals in the C=O vibrational range,28 the K intermediate spectrum in bacteriorhodopsin (BR) shows a


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difference signal at (-)1740/(+)1733 cm-1 assigned to D115 (homologue of D156 for ChR2).8,29 Also for the chloride pump halorhodopsin (HR) a similar band originating from the homologue D141 vibration could be detected.30 Compared to the signals of D156 in ChR-2 this band has a very small amplitude and opposite signs. Furthermore, in time-resolved IR measurements on BR with ps time resolution no absorbance changes of carboxylic vibrations were observed between 100 ps and 14 ns, which is due to the fact that the D115 signal formation occurs on later ns time scale.31 The residue D156 in ChR-2 is located in helix D 9 and forms together with C128 the so called “DC gate” 19. The replacement of one of these amino acids leads to a 102- to 105-fold deceleration of the channel closing.32,33 Also, D156 was identified as the proton donor during the photo-cycle.17,18 The IR band position at 1738 cm-1 corresponds to the vibrational frequency of a carboxylic group with one hydrogen-bond.34 Therefore, the spectral shift to 1746 cm-1 after retinal excitation represents a weakening of this hydrogen bond and, in consequence, alterations within the D156 hydrogen-bonded network during the primary photoreaction. The glutamate residue in position 90 (Helix B) is part of an inter-helical hydrogenbonded network coupled with N258 (Helix G) and S63 (Helix A) forming a constriction within the opsin framework.9 There is a dispute in literature about the E90 deprotonation time.17,18,24,25 In the investigated time range the E90 band does not show particularly pronounced dynamics. The E90 difference signal is already formed 400 fs after retinal excitation, which is very similar to the dynamics of the D156 C=O ground state vibration. Thus, within the investigated time range the hydrogen-bonded network formed by E90, N258 and S63 rather than the protonation state of E90is affected.


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A straightforward explanation for an ultrafast protein response should address the immediate vicinity of the respective amino acids to the retinal chromophore. However, regarding E90 and D156 there is no direct contact to the excited retinal and the direct excess energy transfer seems not to be possible.9,35 On the other hand the crystal structure of ChR-2 reveals a large hydrogen-bonded network within the channel pore consisting of four cavities.35 Three gates with retinal at the center separate the cavities. Hence, E90 as a part of the inner gate forms a hydrogen bond to the residue D253 via a water molecule, which is in direct contact to the retinal chromophore (see Figure 3).35 Thus, retinal excitation leads to a decrease of the hydrogen bond strength resulting in a negative 1709 cm-1 difference band.

Figure 3. Illustration of the hydrogen-bonded network weakening of ChR-2 after retinal isomerization (PDB ID: 6EID).35 Black dotted lines represend the hydrogen-bonded network, black dashed lines the altered hydrogen-bonded network after excitation. Red color indicates the changes discussed in this paper.


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Concerning D156 the most probable conjunction of this side chain to the retinal is across C128 (“DC gate”). Residue C128 has van-der-Waals contact to the retinal and interacts with its π electron system. Consequently, it is feasible that the retinal excitation directly influences C128 and excess energy is rapidly transferred to D156 via a hyrdogen-bonded water molecule on the sub-ps time scale.9,35 Our study provides evidence that energy transfer from the chromophore to nearby amino acids without van-der-Waals contact to the retinal occurs on an ultrafast time-scale. This unexpectedly fast process within the protein implies strong interactions via the hydrogenbonded network that is already present in the initial ground state. The hydrogen-bonded network connects the optical excitation of the retinal to the amino acids at the three gates and induces signal changes, which are indicative for later intermediate steps. The efficient energy redistribution in the ChR-2 protein due to its unique structure induces this pregating state that is responsible for the subsequent channel opening on a much longer timescale36.

ASSOCIATED CONTENT Supporting Information Detailed description of the experimental procedures and data analysis


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AUTHOR INFORMATION Corresponding Author E-Mail: [email protected] Notes The authors declare no competing interests.

ACKNOWLEDGMENT This work has been funded by the DFG via SFB 807 “Transport and Communication across Biological Membranes”.

REFERENCES (1)

Foster, K. W.; Saranak, J.; Patel, N.; Zarilli, G.; Okabe, M.; Kline, T.; Nakanishi, K. A Rhodopsin Is the Functional Photoreceptor for Phototaxis in the Unicellular Eukaryote Chlamydomonas. Nature 1984, 311, 756–759.

(2)

Sineshchekov, O. A.; Jung, K.-H.; Spudich, J. L. Two Rhodopsins Mediate Phototaxis to Low- and High-Intensity Light in Chlamydomonas Reinhardtii. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 8689–8694.

(3)

Nagel, G.; Szellas, T.; Huhn, W.; Kateriya, S.; Adeishvili, N.; Berthold, P.; Ollig, D.; Hegemann, P.; Bamberg, E. Channelrhodopsin-2, a Directly Light-Gated Cation-Selective Membrane Channel. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 13940–13945.

(4)

Fenno, L.; Yizhar, O.; Deisseroth, K. The Development and Application of Optogenetics.


13


Page 14 of 19

Annu. Rev. Neurosci. 2011, 34, 389–412. (5)

Boyden, E. S.; Zhang, F.; Bamberg, E.; Nagel, G.; Deisseroth, K. Millisecond-Timescale, Genetically Targeted Optical Control of Neural Activity. Nat. Neurosci. 2005, 8, 1263– 1268.

(6)

Gunaydin, L. A.; Yizhar, O.; Berndt, A.; Sohal, V. S.; Deisseroth, K.; Hegemann, P. Ultrafast Optogenetic Control. Nat. Neurosci. 2010, 13, 387–392.

(7)

Zhang, F.; Wang, L.-P.; Brauner, M.; Liewald, J. F.; Kay, K.; Watzke, N.; Wood, P. G.; Bamberg, E.; Nagel, G.; Gottschalk, A.; et al. Multimodal Fast Optical Interrogation of Neural Circuitry. Nature 2007, 446, 633–639.

(8)

Lórenz-Fonfría, V. A.; Heberle, J. Channelrhodopsin Unchained: Structure and Mechanism of a Light-Gated Cation Channel. Biochim. Biophys. Acta 2014, 1837, 626–642.

(9)

Kato, H. E.; Zhang, F.; Yizhar, O.; Ramakrishnan, C.; Nishizawa, T.; Hirata, K.; Ito, J.; Aita, Y.; Tsukazaki, T.; Hayashi, S.; et al. Crystal Structure of the Channelrhodopsin Light-Gated Cation Channel. Nature 2012, 482, 369–374.

(10)

Becker-Baldus, J.; Bamann, C.; Saxena, K.; Gustmann, H.; Brown, L. J.; Brown, R. C. D.; Reiter, C.; Bamberg, E.; Wachtveitl, J.; Schwalbe, H.; et al. Enlightening the Photoactive Site of Channelrhodopsin-2 by DNP-Enhanced Solid-State NMR Spectroscopy. Proc. Natl. Acad. Sci. 2015, 112, 9896–9901.

(11)

Scholz, F.; Bamberg, E.; Bamann, C.; Wachtveitl, J. Tuning the Primary Reaction of


14

Page 15 of 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Channelrhodopsin-2 by Imidazole, PH, and Site-Specific Mutations. Biophys. J. 2012, 102, 2649–2657. (12)

Verhoefen, M.-K.; Bamann, C.; Blöcher, R.; Förster, U.; Bamberg, E.; Wachtveitl, J. The Photocycle of Channelrhodopsin-2: Ultrafast Reaction Dynamics and Subsequent Reaction Steps. ChemPhysChem 2010, 11, 3113–3122.

(13)

Bamann, C.; Kirsch, T.; Nagel, G.; Bamberg, E. Spectral Characteristics of the Photocycle of Channelrhodopsin-2 and Its Implication for Channel Function. J. Mol. Biol. 2008, 375, 686–694.

(14)

Feldbauer, K.; Zimmermann, D.; Pintschovius, V.; Spitz, J.; Bamann, C.; Bamberg, E. Channelrhodopsin-2 Is a Leaky Proton Pump. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 12317–12322.

(15)

Friedrich, T.; Geibel, S.; Kalmbach, R.; Chizhov, I.; Ataka, K.; Heberle, J.; Engelhard, M.; Bamberg, E. Proteorhodopsin Is a Light-Driven Proton Pump with Variable Vectoriality. J. Mol. Biol. 2002, 321, 821–838.

(16)

Lozier, R. H.; Bogomolni, R. a; Stoeckenius, W. Bacteriorhodopsin: A Light-Driven Proton Pump in Halobacterium Halobium. Biophys. J. 1975, 15, 955–962.

(17)

Lórenz-Fonfría, V. a; Resler, T.; Krause, N.; Nack, M.; Gossing, M.; Fischer von Mollard, G.; Bamann, C.; Bamberg, E.; Schlesinger, R.; Heberle, J. Transient Protonation Changes in Channelrhodopsin-2 and Their Relevance to Channel Gating. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, E1273-81.


15


(18)

Page 16 of 19

Resler, T.; Schultz, B.-J.; Lórenz-Fonfría, V. A.; Schlesinger, R.; Heberle, J. Kinetic and Vibrational Isotope Effects of Proton Transfer Reactions in Channelrhodopsin-2. Biophys. J. 2015, 109, 287–297.

(19)

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. Photochem. Photobiol. Sci. 2010, 9, 194–198.

(20)

Radu, I.; Bamann, C.; Nack, M.; Nagel, G.; Bamberg, E.; Heberle, J. Conformational Changes of Channelrhodopsin-2. J. Am. Chem. Soc. 2009, 131, 7313–7319.

(21)

Ritter, E.; Stehfest, K.; Berndt, A.; Hegemann, P.; Bartl, F. J. Monitoring Light-Induced Structural Changes of Channelrhodopsin-2 by UV-Visible and Fourier Transform Infrared Spectroscopy. J. Biol. Chem. 2008, 283, 35033–35041.

(22)

Neumann-Verhoefen, M.-K.; Neumann, K.; Bamann, C.; Radu, I.; Heberle, J.; Bamberg, E.; Wachtveitl, J. Ultrafast Infrared Spectroscopy on Channelrhodopsin - 2 Reveals Efficient Energy Transfer from the Retinal Chromophore to the Protein. J. Am. Chem. Soc. 2013, 135, 6968–6976.

(23)

Hamm, P.; Ohline, S. M.; Zinth, W. Vibrational Cooling after Ultrafast Photoisomerization of Azobenzene Measured by Femtosecond Infrared Spectroscopy. J. Chem. Phys. 1997, 106, 519–529.

(24)

Kuhne, J.; Eisenhauer, K.; Ritter, E.; Hegemann, P.; Gerwert, K.; Bartl, F. Early Formation of the Ion-Conducting Pore in Channelrhodopsin-2. Angew. Chemie (International ed.)


16

Page 17 of 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2015, 54, 4953–4957. (25)

Eisenhauer, K.; Kuhne, J.; Ritter, E.; Berndt, A.; Wolf, S.; Freier, E.; Bartl, F.; Hegemann, P.; Gerwert, K. In Channelrhodopsin-2 Glu-90 Is Crucial for Ion Selectivity and Is Deprotonated during the Photocycle. J. Biol. Chem. 2012, 287, 6904–6911.

(26)

Wietek, J.; Wiegert, J. S.; Adeishvili, N.; Schneider, F.; Watanabe, H.; Tsunoda, S. P.; Vogt, A.; Elstner, M.; Oertner, T. G.; Hegemann, P. Conversion of Channelrhodopsin into a Light-Gated Chloride Channel. Science 2014, 344, 409–412.

(27)

Watanabe, H. C.; Welke, K.; Sindhikara, D. J.; Hegemann, P.; Elstner, M. SI: Towards an Understanding of Channelrhodopsin Function: Simulations Lead to Novel Insights of the Channel Mechanism. J. Mol. Biol. 2013, 425, 1795–1814.

(28)

Furutani, Y.; Kawanabe, A.; Jung, K.-H.; Kandori, H. FTIR Spectroscopy of the All-Trans Form of Anabaena Sensory Rhodopsin at 77 K: Hydrogen Bond of a Water between the Schiff Base and Asp75. Biochemistry 2005, 44, 12287–12296.

(29)

Braiman, M. S.; Mogi, T.; Marti, T.; Stern, L. J.; Khorana, H. G.; Rothschild, K. J. Vibrational Spectroscopy of Bacteriorhodopsin Mutants: Light-Driven Proton Transport Involves Protonation Changes of Aspartic Acid Residues 85, 96, and 212. Biochemistry 1988, 27, 8516–8520.

(30)

Rothschild, K. J.; Bousché, O.; Braiman, M. S.; Hasselbacher, C. A.; Spudich, J. L. Fourier Transform Infrared Study of the Halorhodopsin Chloride Pump. Biochemistry 1988, 27, 2420–2424.


17


(31)

Page 18 of 19

Diller, R.; Iannone, M.; Cowen, B. R.; Maiti, S.; Bogomolni, R. A.; Hochstrasser, R. M. Picosecond Dynamics of Bacteriorhodopsin, Probed by Time-Resolved Infrared Spectroscopy. Biochemistry 1992, 31, 5567–5572.

(32)

Bamann, C.; Gueta, R.; Kleinlogel, S.; Nagel, G.; Bamberg, E. Structural Guidance of the Photocycle of Channelrhodopsin-2 by an Interhelical Hydrogen Bond. Biochemistry 2010, 49, 267–278.

(33)

Berndt, A.; Yizhar, O.; Gunaydin, L. A.; Hegemann, P.; Deisseroth, K. Bi-Stable Neural State Switches. Nat. Neurosci. 2009, 12, 229–234.

(34)

Nie, B.; Stutzman, J.; Xie, A. A Vibrational Spectral Maker for Probing the HydrogenBonding Status of Protonated Asp and Glu Residues. Biophys. J. 2005, 88, 2833–2847.

(35)

Volkov, O.; Kovalev, K.; Polovinkin, V.; Borshchevskiy, V.; Bamann, C.; Astashkin, R.; Marin, E.; Popov, A.; Balandin, T.; Willbold, D.; et al. Structural Insights into Ion Conduction by Channelrhodopsin 2. Science 2017, 358, eaan8862.

(36)

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. Proc. Natl. Acad. Sci. 2015, 112, E5796--E5804.


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