Article pubs.acs.org/JPCB
Transient Conformational Changes of Sensory Rhodopsin II Investigated by Vibrational Stark Effect Probes Hendrik Mohrmann,† Ines Kube,‡ Víctor A. Lórenz-Fonfría,†,§ Martin Engelhard,‡ and Joachim Heberle*,† †
Department of Physics, Experimental Molecular Biophysics, Freie Universität Berlin, 14195 Berlin, Germany Max Planck Institute of Molecular Physiology, Otto-Hahn-Str. 11, 44227 Dortmund, Germany
‡
S Supporting Information *
ABSTRACT: Sensory rhodopsin II (SRII) is the primary light sensor in the photophobic reaction of the halobacterium Natronomonas pharaonis. Photoactivation of SRII results in a movement of helices F and G of this seven-helical transmembrane protein. This conformational change is conveyed to the transducer protein (HtrII). Global changes in the protein backbone have been monitored by IR difference spectroscopy by recording frequency shifts in the amide bands. Here we investigate local structural changes by judiciously inserting thiocyanides at different locations of SRII. These vibrational Stark probes absorb in a frequency range devoid of any protein vibrations and respond to local changes in the dielectric, electrostatics, and hydrogen bonding. As a proof of principle, we demonstrate the use of Stark probes to test the conformational changes occurring in SRII 12 ms after photoexcitation and later. Thus, a methodology is provided to trace local conformational changes in membrane proteins by a minimal invasive probe at the high temporal resolution inherent to IR spectroscopy.
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INTRODUCTION Infrared difference spectroscopy is a powerful tool to analyze protein function by detection of changes in their vibrational modes. For instance, CO stretching vibrations appear/ disappear transiently upon protonation/deprotonation of carboxylic amino acid side chains. The assignment of the vibrations to the corresponding residues is facilitated by sitedirected mutagenesis. Minute catalytic changes like, for example, proton translocation and hydrogen-bonding changes involving the S−H group of cysteines and the O−H group of threonine, serine, and water,1−3 have been traced with highest spatial sensitivity and temporal resolution as provided by IR spectroscopy.4−7 Absorption changes of the amide I band, the carbonyl (CO) vibration of the peptide bond, report on conformational and H-bonding changes of the protein backbone.8 Because of vibrational coupling, spectral differences in the amide I region do not allow for localizing the conformational changes. Isotope labeling of particular CO oscillators would be an ideal tool for this purpose as the electronic structure of the peptide bond is not affected by the replacement to 13C or 18O;9 however, sitedirected isotope labeling in complex membrane proteins is notoriously difficult and has hardly been achieved so far.10,11 An alternative means to identify local environmental changes is the site-directed incorporation of a probe with customized vibrational properties.12−14 Molecular vibrations respond to electric fields through dipole interaction by a shift in vibrational frequency. This so-called © 2016 American Chemical Society
vibrational Stark effect (VSE) has been successfully applied to determine the strength of electrostatic fields in proteins15−18 or to measure the transmembrane potential in vesicles.17 In another study, the detected electrostatic changes were used to gain local sensitivity for conformational changes in proteins, for example, after urea-induced unfolding19 or in freeze-trapped intermediate states of bovine visual rhodopsin.12 Here, we aim at resolving structural changes of a membrane protein both in time and space. Sensory rhodopsin II (SRII) is the primary photoreceptor in the photophobic response of Natronomonas pharaonis. Akin to bacteriorhodopsin, the polypeptide folds in the form of seven transmembrane helices into the membrane with the chromophore retinal bound via a Schiff base linkage to K205. Upon illumination retinal isomerizes from all-trans to 13-cis configuration, which initiates a reaction cycle that comprises conformational changes conveyed to the halobacterial transducer protein (HtrII in Figure 1). Photoisomerization of retinal induces local changes in the hydrogen-bonding network in its close vicinity. Such structural and electrostatic perturbations lead to the dissociation of the proton from the retinal Schiff base and transfer to D75 (formation of the M1 state, τ = 30 μs). The functionally important conformational changes, the target of our invesReceived: February 24, 2016 Revised: April 22, 2016 Published: April 25, 2016 4383
DOI: 10.1021/acs.jpcb.6b01900 J. Phys. Chem. B 2016, 120, 4383−4387
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EXPERIMENTAL METHODS
Expression, Solubilization, Purification, and Stark Probe Labeling. Wild-type SRII and the variants S158C, L159C, Y160C, and F210C were heterologuously expressed in E. coli and purified as previously described.31 The proteins were solubilized in 0.05% n-dodecyl-β-D-maltoside, 50 mM sodium phosphate (pH 8), and 300 mM NaCl. The introduced cysteine residues were modified by 2-nitro-5-thiobenzoate-catalyzed addition of cyanide.32 Labeling efficiency was determined by calculating the ratio of the amide II band (at 1550 cm−1) and the cyanide absorbance (CN stretching vibration at ∼2160 cm−1), taking into account the different absorption coefficients.33 The absence of the cysteine S−H stretching vibration (at around 2560 cm−1) was used as an additional control (not shown). All variants show a labeling efficiency of >70%. FTIR Experiments. SRII samples were washed and concentrated by centrifugation using pore filters with a cutoff size of 50 kDa (Merck Millipore, Germany), dried onto BaF2 windows, and rehydrated at ∼93% relative humidity with the saturated vapor phase from a glycerol/water mixture (3:7 wt/ wt).34 Light-induced steady-state FTIR difference spectra were recorded at room temperature and under continuous illumination with green light (505 nm) with a spectral resolution of 4 cm−1. For time-resolved experiments, the output of an optical parametric oscillator (OPTA, Germany), driven by the third harmonic of a Nd:YAG Laser (Quanta-Ray, Spectra Physics, USA), was used to initiate the photoreaction. The laser emission was tuned to a wavelength of 500 nm with a 10 ns pulse width and an energy density of 3 mJ/cm2 at the sample. Experiments were performed on a Bruker Vertex 80v spectrometer with a spectral resolution of 8 cm−1 and a mirror velocity of 7.6 cm/s (HeNe frequency of 240 kHz). The acquisition of the first interferogram was completed after 12 ms. 800 spectra were recorded at room temperature for a satisfactory S/N ratio.
Figure 1. Extracellular view on SRII in complex with HtrII including the different single-site cysteine mutants used in the current work to incorporate cyanide labels. The movement of helix F and rotation of helix TM2 upon photoactivation are indicated by arrows (PDB entry 1H2S20 modified by backbone-aligned and cyanide-labeled cysteines). It is noted that SRII was used in all of the presented experiments in the absence of HtrII. The box shows the cyanylation reaction of cysteine catalyzed by Ellman’s reagent.
tigation, occur with τ = 2 ms, while the retinal Schiff base remains deprotonated. This transition (M1 ⇒ M2) is spectroscopically silent in UV−vis21 but observable by FTIR spectroscopy.22−25 The gained flexibility in combination with a redistribution of charges through proton transfer causes changes in the tertiary structure near the interface with its transducer, formed by helices F and G.26−28 Specifically, a translation of helix G toward the extracellular side has been observed along with a screw-like motion plus outward tilt of helix F.28 Both movements cause a screw-like motion of TM2 of HtrII, the latter interacting via a cytoplasmic hydrogen bond with helix G.20 We have previously shown by surface-enhanced IR absorption spectroscopy that specific steps in the reaction mechanism of SRII are sensitive to the applied transmembrane potential.29,30 Most of the previously described studies were performed under steady-state conditions. Here we present a time-resolved IR spectroscopic study on cyanide-labeled SRII, resolving the dynamics of physiologically important structural changes of the signaling state of SRII. Time-resolved FTIR techniques on such modified samples facilitate the observation of local structural changes during the onset of signal transfer from the photoreceptor to the transducer. We could verify that the observed changes in the electrostatic environment of our local probes correlate temporally with vibrational changes elsewhere in the protein. Furthermore, we were able to distinguish between conformational and electrostatic changes inside the protein. To the best of our knowledge, this is the first time-resolved application of vibrational Stark probes to study functional changes of proteins.
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RESULTS AND DISCUSSION To resolve the structural changes at the interface between SRII and HtrII, we introduced single-cysteine residues that were modified with KCN under the catalytic reaction by Ellman’s reagent to form thiocyanides as VSE probes (Figure 1). As a structural guidance, cysteine variants were chosen that have been shown in previous EPR spectroscopic experiments sensitive to light-induced structural changes.23,35 Light-induced FTIR difference spectra recorded at room temperature and under continuous illumination with green light (505 nm) exhibited bands in the fingerprint region (Figure 2b) characteristic to the M2 state,36 the intermediate where functionally relevant conformational changes have occurred. The steady-state spectra are very similar for WT and all variants tested, indicating that observed vibrational structural changes are invariant to the amino acid replacements.5,21 Inspection of the frequency range where the CN stretching vibration absorbs shows very distinct spectral changes (Figure 2a) depending on the site where the VSE probe was introduced. The largest changes were observed for the S158C-CN variant (green trace in Figure 2b). Here the VSE probe is located on the outside of helix F, which, when SRII interacts with the transducer, is tilting outward (Figure 1). Contrary to our expectation, we found only minor VSE for the Y160C-CN and L159C-CN variants. These residues are also located in helix F, but their side chains are pointing into the interface with helices E and G, respectively. Apparently and unlike the larger MTSL (S-(1-oxyl-2,2,5,54384
DOI: 10.1021/acs.jpcb.6b01900 J. Phys. Chem. B 2016, 120, 4383−4387
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The Journal of Physical Chemistry B
include F2int in the parameters A, B, and C, respectively15 (see the Supporting Information for further details). For an anisotropic orientation of the VSE probe with respect to the electric field, B is larger than C, resulting in a (predominantly) first-order derivative band shape, while B is smaller than C for an isotropically distributed Stark probe, retaining a second-order derivative band shape.15 In the context of the highly anisotropic protein environment, the orientation of the VSE probe with respect to the electric field relates to the flexibility of the side chain where the probe was attached. The spectral derivatives have been calculated by two different but related approaches.37 The first method is the numerical differentiation of the absorbance spectrum with the benefit that a parametric band shape of A(v) does not need to be assumed but at the cost of noise degradation. The second approach is a parametrical fit to the absorbance band, which allows for noisefree analytical derivatives to be obtained. Both approaches provided derivative band shapes too broad to fit the experimental light-induced difference absorption of the CN vibrational band in the S158C-CN variant (Figure S1). An implicit assumption of both approaches is that all cyanide-labeled protein is functional and thus equally contributes to the absorbance and to the light-induced spectral changes. In our case, the absorbance spectrum arises mainly from functional protein, but contaminations from protein devoid of chromophore (opsin) and from denatured protein cannot be strictly excluded. The contaminant populations are labeled by cyanide but do not contribute to the light-induced difference signal. As an alternative approach, we fitted the absorption changes of the CN band to a weighted sum of a Gaussian and its first- and second-order derivatives (Figure 2a and Figure S2). The fitting model contains two nonlinear parameters, maximum and width of the band, and three linear parameters A, B, and C (see eq 1). The isotropic component C is a measure for the rotational freedom of the probe comprising rotational isomerization around internal bonds and the flexibility of the protein backbone. Differences in the kinetics of the first- and second-order derivatives thus allow us to
Figure 2. Steady-state differences of thiocyanate variants of SRII in the spectral region sensitive to the vibrational Stark probe (CN stretching vibration at around 2160 cm−1) (a) and protonation changes of carboxylic groups and conformational changes in the protein backbone and the retinal cofactor (b). Spectra have been scaled to the band at 1545 cm−1 (ethylenic mode of retinal overlapped by amide II contributions).22
tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate) spin label, the small cyanide group does not fully experience the movement of helix F.35 Fitting the cyanide difference band to the sum of zeroth-, first-, and second-order derivatives of the absorbance band provides information on the electric field strength, Fint, and the degree of orientation of the Stark probe.15,16,37 ⎡ ∂ 2 ⎛ A(ν) ⎞⎤ ∂ ⎛ A(ν) ⎞ 2 ⎜ ⎟ + C̃ν 2 ⎜ ⎟⎥ ΔA(F ) = Fint ⎢Ã ·A(ν) + Bν̃ ∂ν ⎝ ν ⎠ ⎣ ∂ν ⎝ ν ⎠⎦ (1)
à , B̃ , and C̃ are parameters that can be derived from fitting ΔA, the difference absorption spectrum, for a known external electric field, F. Because only relative changes have been recorded, we
Figure 3. (a) Time-resolved rapid-scan FTIR spectra of the SRII-S158C−CN variant. Inset: Zoom-in of the CN stretching region. (b) Difference spectra extracted from the time-resolved data set (a) at indicated times; (c) Time traces of the three fitting parameters A, B, and C derived from eq 1. Please note that A, B, and C are parameters with different units (see SI for details) and the amplitudes have been scaled to facilitate the comparison of the kinetics. (d) Time-resolved difference spectra of the CN stretching band fitted to eq 1. The blue dots and lines indicate the position and fwhm of the Gaussian. The spectra of all time points could be fitted with an R2 of >0.99. 4385
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The Journal of Physical Chemistry B discriminate between changes in the backbone flexibility and oriented conformational changes. Because the S158C-CN variant exhibited the strongest difference bands in the steady-state experiment, it was used for studying the kinetics by time-resolved rapid-scan FTIR spectroscopy. Figure 3a shows the transient absorption changes in the amide and CN regions. The spectral shape of the CN difference band corresponds well to the steady-state difference band (cf. Figures 2a and 3a). The difference spectrum reflects the presence of M-like intermediates in both the steady-state and the time-resolved data at 12 ms after light excitation.38 Because of the limited time resolution of the rapid-scan technique, only the decay of the M intermediate to the initial ground state was monitored.23 Kinetic traces of the CN band at 2158 cm−1, the protonation of Asp75 at 1764 cm−1, the ethylenic stretch vibration of the retinal at 1545 cm−1, and the structural changes of amide I at 1619 cm−1 are presented in the Supporting Information (Figure S3a). Maximum entropy inversion was used to transform the time-resolved spectra into lifetime-resolved spectra for these distinct bands.21,39,40 The lifetime distributions (Figure S3b) confirm that the electrostatic changes in helix F, as detected by the vibrational Stark band at 2158 cm−1, and the conformational changes of the amide I band at 1545 cm−1 are correlated. They happen simultaneously during the O ⇒ SRII transition within 3 s. The quantitative analysis of the CN band, as previously introduced, was used as a basis for the time-resolved analysis. The time traces of the fitting parameters A, B, and C reflect the decay of signal while the protein conformational changes are reversed (τA = 4.2 s, τB = 2.4 s, and τC = 2.8 s). The time constants are in agreement with those found by lifetime analysis of the same data (Figure S2). Interestingly, the parameter B that represents the orientational part of the cyanide moiety does not decay in a single exponential fashion, but an additional time constant of 0.5 s dominates the decay (cf. Figure 3c), which refers to the M2 ⇒ O transition.22 It is known that the transducer helix TM2 returns back to its original position during this decay;41 however, the observed VSE change is of intrinsic SRII offspring because the transducer is absent in our samples. This change does not perturb the flexibility of the local probe, as we determined by the anisotropic component C. Instead, it is a pure change in the electrostatic environment that precedes reprotonation of Asp75 with a time constant of 2.7 s. Our data generally agree with previous EPR experiments.23,28 Flash photolysis data showed that the incorporated MTSL spin label in SRII-Y160R1 results in altered reaction kinetics through steric hindrance.35 Our flash photolysis experiments on the cyanide-labeled variant SRII-Y160C-CN displayed identical kinetics of the cyanide-labeled variant and wild-type SRII (Figure S4), indicating that the smaller cyanide probe does not interfere with the native conformational changes of the protein. Thus, the presented method produces physiologically relevant data with no perturbation of the native protein structure and function. In conclusion, we present here a novel method to locally probe transient structural changes in a membrane protein. We derived information through the qualitative analysis of spectral data that separates electrostatic from conformational contributions. The millisecond time resolution of rapid-scan FTIR spectroscopy renders this approach complementary to EPR spectroscopy and constitutes a promising start for further studies at improved temporal resolution. Recent developments like high-power quantum cascade lasers that cover the spectral range of the
cyanide probe, overcome the limitations of step-scan FTIR spectroscopy.42 Temporal resolution as high as a few femtoseconds (10−15 s) is reached by applying pump−probe spectroscopy to thiocyanate-labeled proteins.43
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b01900. Details of the function used for the fit, lifetime analysis of kinetic data, UV−vis kinetic traces of wild-type SRII and SRII-Y160C-CN, and numerical differentiation of absorbance data fails to fit the difference data. (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Tel: +49 (0)30 838 56151. Present Address §
V.A.L.-F.: Department of Biochemistry and Molecular Biology, Universitat de València, 46100 Burjassot, Spain. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS H.M. thanks Till Stensitzki and Emanuel Pfitzner for fruitful discussions on data analysis. V.A.L.-F. is a Ramón y Cajal fellow. J.H. acknowledges the financial support by the Deutsche Forschungsgemeinschaft (SFB 1078, projects A1 and B3).
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