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Reply to ”Comment on ‘Transient Conformational Changes of Sensory Rhodopsin II Investigated by Vibrational Stark Effect Probes’” Hendrik Mohrmann, and Joachim Heberle J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b03453 • Publication Date (Web): 10 Jul 2017 Downloaded from http://pubs.acs.org on July 13, 2017
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Reply to ”Comment on ‘Transient Conformational Changes of Sensory Rhodopsin II Investigated by Vibrational Stark Effect Probes’” Hendrik Mohrmann†, Joachim Heberle† †
Freie Universität Berlin, Department of Physics, Experimental Molecular Biophysics, Arnimallee 14, 14195 Berlin, Germany
In this comment, we respond to Boxer´s concern on the physical framework used to analyze the response of a Vibrational Stark Effect (VSE) probe. We´d like to point out here that our study 1 was aimed at experimentally resolving changes of a transmembrane protein in time and space. We applied time-resolved FTIR spectroscopy to trace the photoreaction of sensory rhodopsin II which was labeled by judiciously placed thiocyanide probes. These sites have first been identified by EPR spectroscopy using spin probes 2 and later confirmed by X-ray crystallography of the receptor/transducer complex 3 to be involved in intramembrane signal transduction from the receptor to the transducer. As conformational changes lead to electrostatic changes, we considered the use of a VSE probe a complementary approach to sense these changes with less impact on the protein structure then other probe labels like the larger EPR or fluorescence probes. Our study sets the basis for experiments that can provide higher temporal resolution enabling the observation of the onset of signal transfer in phototaxis as it is the case for Halobacteria. One may consider the gating of ion channels 4 or the activation of G-protein coupled receptors 5 to involve such conformational changes. These conformational changes are taking place on the microsecond-tomillisecond time scale amenable to novel IR sources like quantum cascade lasers 6. Our experimental approach is reminiscent to experiments on KSI performed by the Boxer group 7 where a ligand (coumarin) bound in the active site was photo-excited to mimic the change in charge distribution associated with the enzyme’s catalytic mechanism. The critical difference between the two approaches is that we have incorporated the VSE probe at the protein surface remote from the active center (the distance from C13 of the retinal to the thiocyanate probe was about 26 Å, Figure 1, left) whereas in the experiments on KSI the spectator SCN was placed close to the photo-excitable ligand (6.5 Å, Figure 1, right). This fact provides crucial implications for the interpretation of the spectroscopic response of the VSE probe. What Jha et al. have observed is the immediate response of the VSE to the photo-induced change in dipole moment of the ligand coumarin. In our experiments on SRII, the VSE probes the change in electric field of its immediate vicinity in the millisecond time range which is a result of a hierarchical sequence of temporal and structural transformations “shaking” the entire protein (protein quake 8) and triggered by the initial lightinduced isomerization (in the sub-picosecond time domain) of the chromophore retinal. Direct coupling between the chromophore´s dipolar change upon photo-excitation is not expected to be monitored by our approach due to the long distance and the late time scale of observation (milliseconds). As a consequence, the detected difference signal originates from a variety of interactions that can occur during the photocycle: (1) the probe may reside in multiple orientations (2) the orientation(s) relative to the protein can change (3) the local electric field amplitude can change (4) the local electric field direction can change. All this results in complex behavior and complex peak shape changes. The VSE probe has been employed to monitor these structural changes at various sites along the interaction interface between the photoreceptor SRII and its cognate transducer HtrII 1. Thus, it is not surprising that the spectral and temporal responses of the thiocyanate probes are different in the two experiments (Fig. 2).
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Figure 1: Structures of sensory rhodopsin II (PDB: 1H2S, left) and ketosteroid isomerase (PDB: 3OXA, right). The left figure highlights the distance from C13 of retinal (which experienced the largest electronic shift upon photonic excitation, see 9 for details) to the center of the nitrile bond of the thiocyanate at S158C of SRII by a dashed line. The right figure highlights the distances from the center of the electronic difference dipole of the cofactor coumarin in KSI to the center of the nitrile bond of the thiocyanate introduced at M116C (see the SI of 7 for details).
In an attempt to find a rationale for the peculiar line shape of the VSE probe in our experiments (Fig. 2, left panel), we analyzed the spectral response by a formalism that was derived by Boxer on the premise of an immobilized and isotropically distributed sample upon exposure to an external electric field. As discussed in Boxer´s comment, our experiment did neither match the requirement of immobilized sample nor the requirement of isotropy with regard to the oriented field change induced by conformational changes. We agree in this respect with the criticism raised by Boxer and appreciate the clarification in his comment.
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Figure 2: Responses of VSE probes introduced into SRII (left) and into KSI (right). The left panel displays the time-resolved FTIR difference spectra of thiocyanate introduced at S158C of SRII (see Fig. 1, left) recorded at various times as indicated after pulsed excitation. The inset displays the frequency shift of the maximum intensity vs. (logarithmic) time scale. The right graph shows timeresolved transient IR absorption spectra of the 13C15N-labeled M116C variant of KSI after the photoexcitation of the bound coumarin (see Fig. 1, right). The figure has been reproduced from 7. Still, as demonstrated in our and other experiments 5, 10-12, the VSE probe senses the changes in electric fields induced by conformational changes during signal transduction. These conformational changes are comprised of classical Stark effects and chemical effects like hydrogen bonding. The latter is most relevant in proteins and one of the contributions to the signal change in protein-bound VSE probes 13. As the hydrogen bonding interaction contains significant amount of electrostatics, a clear-cut separation of the two effects is difficult. Thus, a rigorous spectral analysis of the VSE response must involve quantum-mechanical treatment. As has been previously demonstrated by hybrid QM/MM simulations 14, the simulated spectral response of the VSE probe agree with the experimental data. Thus, such simulations are meaningful and we set out to perform QM/MM simulations on VSE-labeled SRII in the ground and in the signaling state. We thank Dr. Steven G. Boxer for his valuable comments and discussions.
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References: 1. Mohrmann, H.; Kube, I.; Lorenz-Fonfria, V. A.; Engelhard, M.; Heberle, J. Transient Conformational Changes of Sensory Rhodopsin II Investigated by Vibrational Stark Effect Probes. J Phys Chem B 2016, 120, 4383-7. 2. Wegener, A. A.; Klare, J. P.; Engelhard, M.; Steinhoff, H. J. Structural insights into the early steps of receptor-transducer signal transfer in archaeal phototaxis. EMBO J 2001, 20, 53125319. 3. Moukhametzianov, R.; Klare, J. P.; Efremov, R.; Baeken, C.; Goppner, A.; Labahn, J.; Engelhard, M.; Buldt, G.; Gordeliy, V. I. Development of the signal in sensory rhodopsin and its transfer to the cognate transducer. Nature 2006, 440, 115-119. 4. Lorenz-Fonfria, 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 U S A 2015, 112, E5796-804. 5. 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. Nature 2010, 464, 1386-9. 6. Resler, T.; Schultz, B. J.; Lorenz-Fonfria, V. A.; Schlesinger, R.; Heberle, J. Kinetic and vibrational isotope effects of proton transfer reactions in channelrhodopsin-2. Biophys J 2015, 109, 287-97. 7. Jha, S. K.; Ji, M.; Gaffney, K. J.; Boxer, S. G. Direct measurement of the protein response to an electrostatic perturbation that mimics the catalytic cycle in ketosteroid isomerase. Proc Natl Acad Sci U S A 2011, 108, 16612-7. 8. Ansari, A.; Berendzen, J.; Bowne, S. F.; Frauenfelder, H.; Iben, I. E.; Sauke, T. B.; Shyamsunder, E.; Young, R. D. Protein states and proteinquakes. Proc Natl Acad Sci U S A 1985, 82, 5000-5004. 9. Xu, D.; Martin, C.; Schulten, K. Molecular dynamics study of early picosecond events in the bacteriorhodopsin photocycle: dielectric response, vibrational cooling and the J, K intermediates. Biophys J 1996, 70, 453-460. 10. Fafarman, A. T.; Webb, L. J.; Chuang, J. I.; Boxer, S. G. Site-specific conversion of cysteine thiols into thiocyanate creates an IR probe for electric fields in proteins. J Am Chem Soc 2006, 128, 13356-7. 11. Zimmermann, J.; Thielges, M. C.; Seo, Y. J.; Dawson, P. E.; Romesberg, F. E. Cyano groups as probes of protein microenvironments and dynamics. Angew Chem Int Ed Engl 2011, 50, 8333-7. 12. Alfieri, K. N.; Vienneau, A. R.; Londergan, C. H. Using infrared spectroscopy of cyanylated cysteine to map the membrane binding structure and orientation of the hybrid antimicrobial peptide CM15. Biochemistry 2011, 50, 11097-108. 13. Fafarman, A. T.; Sigala, P. A.; Herschlag, D.; Boxer, S. G. Decomposition of vibrational shifts of nitriles into electrostatic and hydrogen-bonding effects. J Am Chem Soc 2010, 132, 128113. 14. Layfield, J. P.; Hammes-Schiffer, S. Calculation of vibrational shifts of nitrile probes in the active site of ketosteroid isomerase upon ligand binding. J. Am. Chem. Soc. 2013, 135, 717-25.
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