Letter pubs.acs.org/JPCL
Ultrafast Photoinduced Deactivation Dynamics of Proteorhodopsin C. Elias Eckert,† Jagdeep Kaur,‡ Clemens Glaubitz,‡ and Josef Wachtveitl*,† †
Institute of Physical and Theoretical Chemistry, Goethe-University Frankfurt am Main, Max-von-Laue-Str. 7, 60438 Frankfurt am Main, Germany ‡ Institute of Biophysical Chemistry, Goethe-University Frankfurt am Main, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany S Supporting Information *
ABSTRACT: We report femtosecond time-resolved absorption change measurements of the photoinduced deactivation dynamics of a microbial rhodopsin in the ultraviolet− visible and mid-infrared range. The blue light quenching process is recorded in green proteorhodopsin’s (GPR) primary proton donor mutant E108Q from the deprotonated 13-cis photointermediate. The return of GPR to the dark state occurs in two steps, starting with the photoinduced 13-cis to all-trans reisomerization of the retinal. The subsequent Schiff base reprotonation via the primary proton acceptor (D97) occurs on a nanosecond time scale. This step is two orders of magnitude faster than that in bacteriorhodopsin, potentially because of the very high pKA of the GPR primary proton acceptor.
R
SB reprotonation.31−34 It has been reported that the latter process occurs via the deprotonation of the primary proton acceptor Asp-8535−37 on a time scale of 100−200 ns.27,34,36,38 However, the ultrafast kinetics of the photoisomerization process has not been investigated to date. Hence, a complete mechanistic description of this process is still missing. This study reports the ultrafast photodynamics of the photoinduced deactivation process of a microbial rhodopsin. The study is based on the light-driven proton pump green proteorhodopsin (GPR).39−42 This well-investigated retinal protein has a high functional and structural similarity to BR and to other microbial rhodopsins. The BLQ effect is initiated by blue photoexcitation of the 13cis retinal in the M state with a deprotonated SB (dSB). Recent measurements for all-trans retinal have shown that the dynamics of dSB drastically differs from that of the protonated (pSB) species.43 Ultrafast experiments on pSB 13-cis retinal have not been performed to date. The detection of ultrafast BLQ photodynamics entails additional experimental effort. First, the initial M state population must be sufficiently large in order to achieve detectable absorption changes. Second, a mixture of different intermediates present in the sample may lead to the detection of different kinetic processes simultaneously occurring after photoexcitation. For these reasons the experiments were performed with GPR primary proton donor mutant E108Q. In this mutant the reprotonation kinetics of the SB is significantly delayed, leading to an increased M state lifetime
esearch on the functionality of microbial rhodopsins has become an important field in molecular life sciences. The investigation of these systems expedites the understanding of membrane proteins in structure and function and provides concepts and approaches for the continuously proceeding development of specifically designed optogenetic tools.1 Microbial rhodopsins act as light-triggered ion pumps, channels, or sensors. The initial step is the photoisomerization of the all-trans retinal, which is covalently bound to the protein via a Schiff base (SB). This primary reaction of retinal protein activation has been investigated in depth for numerous rhodopsins and is well-understood.2−13 In contrast to this, the effect of photoinduced deactivation is known for several retinal proteins like proteorhodopsin,14 halorhodopsin,15 sensory rhodopsin II,16 channelrhodopsin-2,17 and bacteriorhodopsin (BR).18−23 However, a time-resolved description of this process on a molecular level is available only for BR. The blue light quenching (BLQ) effect was discovered in the light-triggered proton pump BR: After the initial all-trans to 13cis isomerization (dark to K state transition of the retinal), a proton transfer from the SB to the primary proton acceptor occurs. Subsequently, a proton is released to the extracellular side. In a next step, the system decays thermally to form the N state, in which the SB is reprotonated by the primary proton donor. Afterwards, the retinal isomerizes back to all-trans configuration (O state). In the last step of the photocycle, the deprotonation of the primary proton acceptor and the recovery of the dark state takes place. However, blue light illumination of the M state leads to the deactivation of the proton-transfer process to the extracellular surface.18−20,24−26 The photocycle is interrupted and the protein returns to the ground state in at least two steps: First, the photoisomerization of the retinal in the M state from 13-cis to all-trans21,27−30 and subsequently the © XXXX American Chemical Society
Received: December 19, 2016 Accepted: January 10, 2017 Published: January 10, 2017 512
DOI: 10.1021/acs.jpclett.6b02975 J. Phys. Chem. Lett. 2017, 8, 512−517
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The Journal of Physical Chemistry Letters by several orders of magnitude compared to that of wild-type GPR.44 Therefore, the E108Q mutation induces a dramatically slower photocycle.42 As a consequence, electrophysiological experiments on GPR primary proton donor mutants reveal weak photocurrents compared to those of the wild type.45 By continuous intense green light illumination, the ensemble of GPR E108Q proteins reaches a photostationary equilibrium leading to a high accumulation of proteins in the M state. Early intermediates do not accumulate to a detectable level because of their fast kinetics. Moreover, flash photolysis experiments reveal that the population of late intermediates in the GPR E108Q photocycle is very small.42 Although in the photostationary state a small fraction of ground-state GPR E108Q proteins is present, its low extinction coefficient in the blue spectral range renders it unlikely that signals following a λ = 390 nm excitation originate from this species. Hence, the kinetics observed after blue light excitation of GPR E108Q in a photostationary equilibrium arises almost exclusively from the BLQ dynamics. In BR it could be shown that the M state of D96N mutant, which is equivalent to the E108Q mutant in GPR, undergoes a back photoreaction, which is kinetically and spectrally very similar to the corresponding dynamics of the wild type.34 Consequently, the results presented here should reflect the BLQ photodynamics of wild-type GPR. The time-resolved absorption measurement of the BLQ process in the ultraviolet−visible (UV−vis) spectral range is depicted in Figure 1a. The photoexcitation with blue light introduces a negative signature around 400 nm and a spectrally very broad positive absorption from 460 to 650 nm, which almost entirely disappears within picoseconds. At long delay times (see Figure 1b), a remaining absorption band centered at 460 nm decays on a nanosecond time scale, while an absorption band at 525 nm builds up. Additionally, a small positive signal around 630 nm is detected, which disappears on a subnanosecond time scale. The transient data are fitted with a global lifetime analysis (GLA) routine using six time constants: 0.2 ps, 2 ps, 13 ps, 200 ps, 1 ns, and ∞. The decay-associated spectra (DAS) are depicted in Figure 1e. The DAS for the infinity lifetime is not shown, because it solely reflects the absorption change at the latest delay time. The spectral shapes for the first four lifetimes highly resemble each other. All of them describe a broad absorption decay from 460 to 650 nm, while the absorption in the blue region increases. The 1 ns lifetime reflects the late photodynamics in the transient data: the decay in absorption at 450 nm and simultaneously the formation of a species at 525 nm. The negative signature in the transient data at 400 nm is attributed to the bleaching signal of the M state. The broad absorption band which builds up directly after excitation is interpreted as excited-state absorption (ESA). Its decay predominantly consists of three fast lifetimes (0.2, 2, and 13 ps) and to a minor extent of a fourth 0.2 ns lifetime. The DAS of these rate constants reveal an absorption increase in the blue spectral region (Figure 1e) indicating fast back conversion to the initial M state. However, the spectral position of the DAS minima are red-shifted with respect to the M state absorption maximum. Therefore, this signature most likely also describes the formation of a spectrally red-shifted photoproduct located at 460 nm in the transient data (here called M′ state). The origin of the small signature around 630 nm could not be assigned yet.
Figure 1. Transient absorbance changes of GPR E108Q in D2O at pD 8.5 after photoexcitation of the retinal in the M state at 390 nm obtained in the visible spectral region from (a) −1 to 1900 ps (lin/log) and (b) 500 and 1900 ps (lin) delay time. (c) Solid curve, normalized absorbance change spectrum at 1.9 ns delay time; dotted curve, normalized steady-state absorption of dark state GPR E108Q. (d) Transient absorption changes (black) with fit (red) at selected wavelengths. (e) Decay-associated spectra obtained from global lifetime analysis of transient data. The DAS of the 0.2 ps lifetime is scaled with a factor of 0.25 for better comparability.
It is worth noting that this early photokinetics of the M state strongly resembles the primary photoreactions of the GPR dark state: for both events, the ESA decay and the formation of a photoproduct (M′ state and K state, respectively) are described by three highly similar rate constants.8,9 Figure 1c depicts the difference absorption spectrum at maximum delay time (solid line) and the steady-state absorption of GPR E108Q in the dark state (dashed line). The spectral shape of the species at 525 nm at long delay times is remarkably similar to the dark state absorption of GPR E108Q (Figure 1c). This result leads to the assumption that the dark state recovery via BLQ starts on a nanosecond regime. Following this interpretation, the photoproduct at 460 nm is assigned as an intermediate in the BLQ dynamics. The BLQ retinal dynamics requires at least two steps: reisomerization from 13-cis to all-trans and SB reprotonation. The reisomerization step occurs after the initial photoexcitation. The photoproduct at 460 nm (M′ state) most likely describes the absorption of an all-trans retinal with a dSB. The bathochromic shift with respect to the M state absorption may arise from the fact that the π-electron system of the all-trans retinal is elongated compared to a 13-cis retinal, with the consequence of a red-shifted absorption maximum. Consequently, the decay of the M′ state and the formation of the 513
DOI: 10.1021/acs.jpclett.6b02975 J. Phys. Chem. Lett. 2017, 8, 512−517
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The Journal of Physical Chemistry Letters 525 nm photoproduct describe the reprotonation step of the SB and by that the ultrafast return to the retinal configuration of the GPR dark state. To substantiate the results of the UV−vis transient data, the time-resolved femtosecond pump−probe experiments were expanded to the mid-infrared (MIR) spectral region between 1500 and 1700 cm−1, where the absorption bands of the retinal CC and CN stretch modes appear (Figure 2a).42,46,47 For the assignment of the signatures in the time-resolved MIR data, a Fourier-transform infrared (FTIR) difference absorption measurement was conducted. The dotted curve in Figure 2a depicts the difference absorption spectrum between
the dark state and the M state. The solid curve describes the absorption change 1.8 ns after the photoexcitation of the retinal in the M state. This spectrum consists of three measurements in different spectral regions. All of them are scaled for a better comparability with the FTIR data. Negative signatures in the FTIR difference spectrum and in the femtosecond data describe the M state absorption. Hence, positive amplitudes in the FTIR data arise from dark state absorption as well as late, positive absorption changes in the time-resolved data. The comparison of these measurements reveals a high similarity in their spectral shape, indicating fast dark state recovery via BLQ on a nanosecond time scale. Indeed, the transients at the spectral positions where the dark state CC (1537 cm−1, Figure 2b) and dark state CN (1628 cm−1, Figure 2d) retinal stretch modes appear42,46,47 both reveal a positive absorption change with a nanosecond rising time. The corresponding DAS are depicted in panels c and e of Figure 2, respectively. Synchronously with the dark state CC recovery, a 1 ns absorption decay at 1554 cm−1 occurs (Figure 2b). This observation becomes even more obvious from the decayassociated spectra of the 1 ns lifetime (Figure 2c). The DAS of the 40 ps lifetime reflects the formation of the signature at 1554 cm−1. The kinetics for the absorption increase and decay at 1554 cm−1 is almost identical to the kinetics of the M′ state from the transient UV−vis data (Figure 1). Hence, the photoproduct at 1554 cm−1 can be assigned to the M′ state dynamics. It is well-known that the spectral position of the retinal C C stretch mode is inversely correlated to the retinal absorption maximum for the electronic transition.48,49 Based on this corresponding pair of values for the M state and dark state, the interpolated wavenumber for the CC stretch mode of the chromophore in the M′ state is 1553 cm−1, perfectly matching with the signature at 1554 cm−1. Therefore, this observation is assigned to the retinal CC stretch mode in the M′ state. The previous results are related to the photodynamics of the retinal. Between 1640 and 1700 cm−1, mainly amide-I resonances of the protein (CO stretching vibrations) are observed6,14,42,50 and to a lower extent CN stretch modes. At 1650 cm−1, a strong negative bleaching signature appears (Figure 2f) and is thus downshifted with respect to the corresponding minimum at 1658 cm−1 in the FTIR data. The spectral mismatch probably arises from the presence of a positive absorption band at 1662 cm−1 (Figure 2f). This signature overlaps with the bleaching band and spectrally downshifts its minimum. This observation can be interpreted as an amide-I shift, suggesting that the BLQ dynamics of the protein (in contrast to the retinal) is still ongoing after 1.8 ns. The transients at 1654 and 1666 cm−1 reveal a synchronous, counteracting behavior (Figure 2d). Initially, the signal at 1654 cm−1 (1666 cm−1) increases (decreases), and after 100−200 ps it decays (increases). The global lifetime analysis (Figure 2e) shows that the later process occurs with a 1 ns lifetime just like the M′ state decay observed in the UV−vis and CC stretch mode spectral region. Therefore, this dynamics can be interpreted as the protein response to conformational changes which are coupled to the formation and decay of the M′ state. The second step in the BLQ process is the reprotonation of the SB. The CN stretch mode of the retinal is expected to be highly influenced by the protonation state of the SB. Hence, the detection of the CN stretch mode appearing at the spectral position of the dark state suggests that the SB reprotonation happens on a nanosecond time scale. The counterion of the SB
Figure 2. (a) Solid curve: absorbance changes of GPR E108Q in D2O (at pD 8.5) 1.8 ns after photoexcitation of the retinal in the M state at 390 nm in the infrared spectral region. These data are obtained from three independent measurements. The three curves are scaled with respect to the dotted curve for better comparability. Dotted curve: FTIR difference spectrum of dark state minus M state absorption. (b, d) Transient absorption changes at selected probe wavenumbers (gray) with fit (black). (c, e) Decay-associated spectra obtained from global lifetime analysis of transient data. (f) Transient absorbance changes in the amide-I region. 514
DOI: 10.1021/acs.jpclett.6b02975 J. Phys. Chem. Lett. 2017, 8, 512−517
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The Journal of Physical Chemistry Letters
second SB reprotonation (M′ to dark state transition). For BR, it is reported that the primary proton acceptor D85 is in close proximity to the SB in the ground state as well as in the M state. In both states, the distance does not exceed 5 Å.54,55 Consequently, the SB reprotonation step via the primary proton acceptor is expected to happen on an ultrafast time scale. Interestingly, in GPR the SB reprotonates 2 orders of magnitude faster than in BR. Additionally, the spectral shift between the M′ and M state in GPR (about 50 nm) is much higher than in BR (about 15 nm). We believe that in line with the drastically faster formation dynamics this provides additional evidence that the BLQ effect in GPR is quite different with respect to BR. These observations may be explained by the very high pKA of the GPR primary proton acceptor D97.14 During the photocycle, the retinal SB in the M state is reprotonated from the cytoplasmic side of the protein by the primary proton donor E108 (Figure 4). It could be shown that
in the dark state is the deprotonated primary proton acceptor D9751 under alkaline conditions, which is protonated in the M state.52 Hence, D97 can potentially act as the proton donor for the retinal SB in the BLQ process. To investigate this, the FTIR and femtosecond pump−probe measurements were expanded to higher wavenumbers in order to detect the CO stretch mode of D97. This stretching vibration appears at 1743 cm−1.14,42,47 With the deprotonation of D97, this vibrational band is supposed to vanish and the symmetric COO− stretch mode is expected to appear simultaneously. However, the spectral position of the COO− mode is strongly downshifted to under 1400 cm−153 and is therefore beyond the spectral range in the experiment. The main feature in the transient data is the distinct bleaching band at 1743 cm−1 (Figure 3a−c), describing the
Figure 3. Transient absorbance changes of D97 CO stretch mode at pD 8.5 in D2O after photoexcitation of GPR E108Q M state at 390 nm. (a) 2D contour map of femtosecond pump−probe measurement. (b) Solid curve, normalized absorption change spectrum from panel a for 1.8 ns delay time; dotted curve, normalized FTIR difference spectrum of dark state minus M state absorption. (c) Spectra from panel a for the following delay times (from gray to black): 0.1, 0.5, 0.6, 1, 1.2, 1.5, and 1.8 ns. (d) Decay-associated spectra obtained from global lifetime analysis of transient data.
Figure 4. Scheme of the GPR photocycle (green arrows). Blue arrows indicate the BLQ deactivation pathway. A, primary proton acceptor; D, primary proton donor.
in case of the BLQ process, this SB reprotonation occurs via the primary proton acceptor D97 and thus from the extracellular side, as reported for BR. Hence, the photoisomerization of the 13-cis retinal with a dSB (M state) to all-trans leads to a switch45,56 in the orientation of the SB resulting in a reversal of direction in the proton transfer. With this approach, the ultrafast BLQ dynamics in similar systems could also be addressed. Especially for channelrhodopsins, this effect is of high interest for optogenetic applications, because the simultaneous control of ion channel opening and closing by light would improve the temporal precision of ion permeation significantly.
disappearance of the CO stretch mode after photoexcitation of the retinal in the M state. Its spectral shape perfectly matches with the corresponding absorption band of the M state in the FTIR measurement (Figure 3b). Close to the bleaching band, the sharp positive absorption at 1738 cm−1 is interpreted as the slightly downshifted absorption band of the CO stretch mode. This signature decays with a lifetime of 0.8 ns, as it is described by the DAS (Figure 3d) at 1740 cm−1. Therefore, this lifetime is assigned to the decay of the CO stretch of D97 and consequently to the proton-transfer process from the amino acid residue of D97 to the retinal SB. With a M-accumulating GPR mutant, the ultrafast photodynamics of the photoinduced deactivation process of a retinal protein could be measured. This study reveals that the interruption of the proton pump process of proteorhodopsin and the return to the dark state of the chromophore occur in a subnanosecond regime. This reaction consists of two steps: reisomerization of the retinal from 13-cis to all-trans (M to M′ state transition) in picoseconds and subsequently the nano-
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EXPERIMENTAL METHODS The preparation of the GPR mutant E108Q samples in a D2O buffer is described in detail in the Supporting Information. For the measurements in the photostationary equilibrium, the samples were continuously illuminated with intense green light (central wavelength, 532 nm; power, 350 mW; Verdi-5W laser, Coherent). The femtosecond pump−probe setup used for the MIR and the UV−vis transient absorption measurements is explained in detail in the Supporting Information. 515
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(8) Lenz, M. O.; Huber, R.; Schmidt, B.; Gilch, P.; Kalmbach, R.; Engelhard, M.; Wachtveitl, J. First Steps of Retinal Photoisomerization in Proteorhodopsin. Biophys. J. 2006, 91, 255−262. (9) Huber, R.; Köhler, T.; Lenz, M. O.; Bamberg, E.; Kalmbach, R.; Engelhard, M.; Wachtveitl, J. pH-Dependent Photoisomerization of Retinal in Proteorhodopsin. Biochemistry 2005, 44, 1800−1806. (10) Imasheva, E. S.; Balashov, S. P.; Wang, J. M.; Dioumaev, A. K.; Lanyi, J. K. Selectivity of Retinal Photoisomerization in Proteorhodopsin Is Controlled by Aspartic Acid 227. Biochemistry 2004, 43, 1648− 1655. (11) Hamm, P.; Zurek, M.; Röschinger, T.; Patzelt, H.; Oesterhelt, D.; Zinth, W. Subpicosecond Infrared Spectroscopy on the Photoisomerisation of the Protonated Schiff Base of All-Trans Retinal. Chem. Phys. Lett. 1997, 268, 180−186. (12) Lenz, M.; Woerner, A.; Glaubitz, C.; Wachtveitl, J. Photoisomerization in Proteorhodopsin Mutant D97N. Photochem. Photobiol. 2007, 83, 226−231. (13) Lutz, I. Primary Reactions of Sensory Rhodopsins. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 962−967. (14) Friedrich, T.; Geibel, S.; Kalmbach, R.; Chizhov, I.; Ataka, K.; Heberle, J.; Engelhard, M.; Bamberg, E. Proteorhodopsin Is a LightDriven Proton Pump with Variable Vectoriality. J. Mol. Biol. 2002, 321, 821−838. (15) Hegemann, P.; Oesterhelt, D.; Bamberg, E. The Transport Activity of the Light-Driven Chloride Pump Halorhodopsin Is Regulated by Green and Blue Light. Biochim. Biophys. Acta, Biomembr. 1985, 819, 195−205. (16) Balashov, S. P.; Sumi, M.; Kamo, N. The M Intermediate of Pharaonis Phoborhodopsin Is Photoactive. Biophys. J. 2000, 78, 3150− 3159. (17) 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. (18) Dancsházy, Z.; Drachev, L. A.; Ormos, P.; Nagy, K.; Skulachev, V. P. Kinetics of the Blue Light-Induced Inhibition of Photoelectric Activity of Bacteriorhodopsin. FEBS Lett. 1978, 96, 59−63. (19) Ormos, P.; Dancsházy, Z.; Karvaly, B. Mechanism of Generation and Regulation of Photopotential by Bacteriorhodopsin in Bimolecular Lipid Membrane. The Quenching Effect of Blue Light. Biochim. Biophys. Acta, Bioenerg. 1978, 503, 304−315. (20) Karvaly, B.; Dancsházy, Z. Bacteriorhodopsin: A Molecular Photoelectric Regulator Quenching of Photovoltaic Effect of Bimolecular Lipid Membranes Containing Bacteriorhodopsin by Blue Light. FEBS Lett. 1977, 76, 36−40. (21) Hess, B.; Kuschmitz, D. The Photochemical Reaction of the 412 Nm Chromophore of Bacteriorhodopsin. FEBS Lett. 1977, 74, 20−24. (22) Balashov, S. P. Photochemical Processes of Light Energy Transformation in Bacteriorhodopsin. Sov. Sci. Rev., Sect. D: Physiochem. Biol. Rev. 1988, 8, 1−61. (23) Ohno, K.; Govindjee, R.; Ebrey, T. G. Blue Light Effect on Proton Pumping by Bacteriorhodopsin. Biophys. J. 1983, 43, 251−254. (24) Ormos, P.; Dancsházy, Z.; Keszthelyi, L. Electric Response of a Back Photoreaction in the Bacteriorhodopsin Photocycle. Biophys. J. 1980, 31, 207−213. (25) Hwang, S. B.; Korenbrot, J. I.; Stoeckenius, W. Transient Photovoltages in Purple Membrane Multilayers. Charge Displacement in Bacteriorhodopsin and Its Photointermediates. Biochim. Biophys. Acta, Biomembr. 1978, 509, 300−317. (26) Keszthelyi, L.; Ormos, P. Electric Signals Associated with the Photocycle of Bacteriorhodopsin. FEBS Lett. 1980, 109, 189−193. (27) Kalisky, O.; Ottolenghi, M.; Honig, B.; Korenstein, R. Environmental Effects on Formation and Photoreaction of the M412 Photoproduct of Bacteriorhodopsin: Implications for the Mechanism of Proton Pumping. Biochemistry 1981, 20, 649−655. (28) Balashov, S. P. Phototransformation of Metabacteriorhodopsin. Photobiochem. Photobiophys. 1981, 2, 111−117. (29) Grieger, I.; Atkinson, G. H. Photolytic Interruptions of the Bacteriorhodopsin Photocycle Examined by Time-Resolved Resonance Raman Spectroscopy. Biochemistry 1985, 24, 5660−5665.
The time-resolved data are analyzed with the global lifetime analysis (GLA) method. In this routine, all spectrally resolved transients are analyzed with a fixed set of exponential functions delivering decay-associated spectra for every exponential term. The analysis is conducted with the program “OPTIMUS”.57,58 The Fourier transform infrared spectra were recorded with a Vertex 80 (Bruker, Ettlingen) spectrometer. For the measurement in the photostationary equilibrium, the sample was continuously illuminated with green light under the same conditions as in the transient measurements.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpclett.6b02975. Sample preparation, femtosecond Vis-pump MIR-probe spectroscopy, femtosecond Vis-pump UV−vis-probe spectroscopy (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Josef Wachtveitl: 0000-0002-8496-8240 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are thankful to Ingrid Weber for providing excellent technical assistance. The work was funded by Cluster of Excellence Frankfurt: Macromolecular Complexes Frankfurt and Sonderforschungsbereich 807 Transport and Communication across Biological Membranes.
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REFERENCES
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DOI: 10.1021/acs.jpclett.6b02975 J. Phys. Chem. Lett. 2017, 8, 512−517
Letter
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DOI: 10.1021/acs.jpclett.6b02975 J. Phys. Chem. Lett. 2017, 8, 512−517
