Letter pubs.acs.org/JPCL
Cite This: J. Phys. Chem. Lett. 2019, 10, 5422−5427
Protein Dynamics Preceding Photoisomerization of the Retinal Chromophore in Bacteriorhodopsin Revealed by Deep-UV Femtosecond Stimulated Raman Spectroscopy Shinya Tahara,†,∥ Hikaru Kuramochi,†,‡,§ Satoshi Takeuchi,†,‡,⊥ and Tahei Tahara*,†,‡ †
Molecular Spectroscopy Laboratory, RIKEN, 2-1 Hirosawa, Wako 351-0198, Japan Ultrafast Spectroscopy Research Team, RIKEN Center for Advanced Photonics (RAP), 2-1 Hirosawa, Wako 351-0198, Japan § PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi 332-0012, Japan Downloaded via NOTTINGHAM TRENT UNIV on August 31, 2019 at 08:37:46 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
‡
S Supporting Information *
ABSTRACT: Bacteriorhodopsin is a prototypical photoreceptor protein that functions as a light-driven proton pump. The retinal chromophore of bacteriorhodopsin undergoes C13C14 trans-to-cis isomerization upon photoexcitation, and it has been believed to be the first event that triggers the cascaded structural changes in bacteriorhodopsin. We investigated the protein dynamics of bacteriorhodopsin using deep-ultraviolet resonance femtosecond stimulated Raman spectroscopy. It was found that the stimulated Raman signals of tryptophan and tyrosine residues exhibit significant changes within 0.2 ps after photoexcitation while they do not noticeably change during the isomerization process. This result implies that the protein environment changes first, and its change is small during isomerization. The obtained femtosecond stimulated Raman data indicate that ultrafast change is induced in the protein part by the sudden creation of the large dipole of the excited-state chromophore, providing an environment that realizes efficient and selective isomerization.
P
remarkable primary process of BR, various ultrafast spectroscopic studies have been carried out so far, and the details of the dynamics and mechanism of the photoisomerization of the retinal chromophore have been clarified.5,10−13 However, our knowledge on the ultrafast dynamics of the protein part is still limited. The picosecond-to-millisecond dynamics of the protein part has been studied by time-resolved ultraviolet resonance Raman (UVRR) spectroscopy.14−17 Because the Raman signals due to aromatic amino acids in proteins are enhanced with ultraviolet Raman excitation, UVRR has been used for site-specific observation of the changes in the protein part of rhodopsins.14−23 In a previous picosecond spontaneous UVRR study of BR,17 it was found that the resonance Raman signals due to the tryptophan and tyrosine residues change within the time resolution of the measurements (∼4 ps) and that the signal change largely recovers with a ∼30 ps time constant. The observed change of the UVRR signal was attributed to the change around the tryptophan and tyrosine residues that are located near the chromophore (Figure 1). However, it has not been yet clarified how this change of the protein part is related to the isomerization of the chromophore. In other words, we still have a central question, whether the protein part responds
hotoreceptor proteins are indispensable for life, and they convert light to chemical energy to initiate a variety of biological functions.1 The initial step in photoreceptor proteins is the absorption of light by a chromophore, which starts a photochemical reaction that triggers consecutive structural changes of the protein through allosteric interactions.2 The photochemical reaction of the chromophore is highly efficient and selective in the protein, which realizes the relevant biological function with a high quantum yield. Therefore, elucidation of the mechanism of the highly efficient photocycle of photoreceptor proteins is critically important for designing molecular systems that can efficiently convert light to more usable energy. Bacteriorhodopsin (BR) is the most intensively studied prototype of photoreceptor protein that functions as a lightdriven proton pump.3,4 BR possesses a retinal chromophore, and it undergoes photoisomerization on the subpicosecond time scale.5 This photoisomerization has been considered the first event in the photocycle, in which sequential structural changes occur in the protein to realize the proton pump.3 The photoisomerization quantum yield of the retinal chromophore in BR is much higher than that in solution (0.6 vs 0.2),6−8 and the isomerization exclusively occurs around the C13C14 bond, whereas it also occurs around other CC bonds in solution.9 Therefore, BR has been attracting much interest for decades as a prototypical system for which we can study how the protein realizes the efficient and selective photoreaction of the chromophore. In fact, to elucidate the mechanism of this © XXXX American Chemical Society
Received: August 5, 2019 Accepted: August 27, 2019
5422
DOI: 10.1021/acs.jpclett.9b02283 J. Phys. Chem. Lett. 2019, 10, 5422−5427
Letter
The Journal of Physical Chemistry Letters
Figure 1. Crystallographic structure around the chromophore of BR (PDB ID: 1KGB). The tryptophan (blue), tyrosine (red), retinal chromophore (yellow), water (green), and hydrogen bonds (green broken lines) are highlighted with color.
to the chromophore isomerization in a passive or static way or it plays a role in a more active and dynamic way. Femtosecond stimulated Raman spectroscopy (FSRS) is a powerful tool for investigating the structural dynamics on the femtosecond time scale.24,25 FSRS enables us to track changes of the stimulated Raman spectra with a femtosecond temporal accuracy while keeping frequency resolution as high as ∼10 cm−1. For example, this technique revealed the early structural dynamics of the chromophore of visual rhodopsin, which completes within a few picoseconds.26 FSRS is particularly useful for resonance Raman study in the wavelength region where it is difficult to carry out time-resolved impulsive stimulated Raman measurements with high time resolution.27−29 In fact, FSRS measurements can be performed in wavelength regions such as the UV30 and near-IR31 because they utilize a picosecond Raman pump pulse and a femtosecond broad-band Raman probe pulse that is not highly compressed. Extension of FSRS to the deep-UV region is highly desirable because it opens a possibility to examine femtosecond structural dynamics of the protein part of photoreceptor proteins. We recently realized steady-state stimulated Raman spectroscopy in the deep-UV region.32 In this study, we extend this method to femtosecond timeresolved measurement (DUV-FSRS) and apply it to BR to elucidate the protein dynamics on the femtosecond time scale. We excited the retinal chromophore of BR with a 100 fs actinic pump at 580 nm and acquired stimulated Raman spectra with a narrow-band 292 nm Raman pump and a broadband Raman probe centered at 302 nm (the experimental details are given in the Supporting Information). Figure 2 shows the DUV-FSRS spectra measured at various delay times. The spectrum at −100 ps is shown at the bottom, which corresponds to the steady-state stimulated Raman spectrum. In this spectrum, we clearly see the stimulated Raman bands of tryptophan residues at 756 (W18 mode), 1009 (W16), 1126 (W13), 1164 (W12), 1234 (W10), 1337 and 1361 (W7 doublet), 1560 (W3), and 1584 cm−1 (W2) and those of tyrosine residues at 814, 849 (Y1 doublet), and 1450 cm−1 (Y19b).33 These assignments were made by comparing the steady-state spectrum of BR with those of aqueous solutions of tryptophan and tyrosine measured under the same experimental conditions (Supporting Information). We note that a stimulated Raman band at 1528 cm−1 is not assigned to the
Figure 2. DUV-FSRS spectra of BR obtained with a 580 nm actinic pump and a 292 nm Raman pump. The purple spectrum at the bottom depicts the spectrum at −100 ps, which corresponds to the steady-state stimulated Raman spectrum. Other colored spectra depict the DUV-FSRS spectra at delay times from 0.2 to 200 ps, which are shown as the difference from the spectrum at −100 ps. Assignments of the vibrational modes are also given at the top.
vibration of tryptophan or tyrosine residues, and its assignment is not clear at the moment. The DUV-FSRS spectra from 0.2 to 200 ps are shown as the difference spectra from the spectrum at −100 ps. In these spectra, we clearly observe negative bands attributable to tryptophan or tyrosine, indicating that the intensity of the stimulated Raman bands of these residues significantly decreases with photoexcitation of the chromophore. This spectral change appears within 200 fs after photoexcitation, and no further substantial change is recognized in a few picosecond time range. These transient signals disappear on the time scale of a few tens of picoseconds, which agrees well with the results of the previous picosecond UVRR study.17 We note that the origin of these spectral changes is not selfabsorption due to the transient absorption. To check this, we measured DUV-FSRS spectra of BR in the presence of 0.2 M HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) as the intensity standard. The change in the stimulated Raman signal due to HEPES was negligible, indicating that the observed changes in the stimulated Raman signal do not arise from the change in the self-absorption due to the photoexcitation (the data are given in the Supporting Information). For quantitative discussion on the temporal change of the stimulated Raman signals, we analyzed the amplitude change of each Raman band. We evaluated the ratio between the area intensity of each band in the time-resolved (difference) spectra with that in the spectrum at −100 ps and obtained the temporal traces shown in Figure 3. In these plots, the time region from −0.5 to 0.1 ps is shaded and is not discussed hereafter because the spectra in this time region are largely disturbed by the perturbed free induction decay34 as well as the 5423
DOI: 10.1021/acs.jpclett.9b02283 J. Phys. Chem. Lett. 2019, 10, 5422−5427
Letter
The Journal of Physical Chemistry Letters
dipole moment (>10 D37) of the excited-state chromophore which is generated with photoexcitation. Because such a drastic increase in the dipole moment of the chromophore occurs instantaneously with photoexcitation and it is expected to significantly affect the local environment around the chromophore, it is natural to consider that this is the origin of the Raman signal change observed in the present DUVFSRS experiments. Importantly, the transient DUV-FSRS signals remain even after the relaxation of the excited-state chromophore that occurs with the isomerization. This indicates that the observed change in the Raman signal is not merely due to the electronic response to the large dipole of the excited-state chromophore but due to the actual change of the protein part around the chromophore. Previous time-resolved absorption studies38−40 reported that the transient absorption signal in the UV region (260−320 nm) appears before the isomerization (instantaneously40 or within 0.2 ps38,39) and that the signal largely decays concomitantly with the relaxation of the excited-state chromophore (500 fs). They concluded that this transient signal arises from the Stark shift of Trp86, which is induced by the large dipole of the excited-state chromophore based on the results of mutagenesis experiments as well as theoretical modeling taking account of the dipole−dipole interaction between the chromophore and two adjacent tryptophan residues (Trp86 and Trp182). In sharp contrast, the DUVFSRS signal changes observed in this study do not decay with the decay of the excited-state chromophore but remain in a few tens of picoseconds. These contrasting results clearly demonstrate that they have different origins. As mentioned in the introduction, a previous picosecond UVRR study reported that the Raman bands due to the tryptophan and tyrosine residues exhibit significant change with excitation of the chromophore in BR.17 In the spectra measured with the 225 nm probe, for instance, a substantial intensity decrease was observed for the Raman bands, which were attributed to the change in the tryptophan and tyrosine residues around the chromophore, and the signal change was explained with the change of the local environment around the chromophore, i.e., the change in the hydrogen bond strength and hydrophobicity. In fact, in the picosecond UVRR spectra measured with the 238 nm probe, where the relative intensity of the tyrosine bands is enhanced, a frequency shift was observed for the Y7a band of tyrosine, which is known as a sensitive marker of the hydrogen bond strength of phenolic OH of tyrosine.41 In their experimental conditions, the Raman probe is in resonance with the Ba and Bb transitions of tryptophan and the La transition of tyrosine. Because the change of the local environment, i.e., the hydrogen bond strength and hydrophobicity, shifts the energies of these transitions,42 the observed change in the Raman intensity was explained by the decrease of the resonance enhancement due to the blue shift of the resonant electronic transitions that was caused by the change in the hydrogen bond strength and hydrophobicity of tryptophan and tyrosine around the chromophore.17 The resonance condition in the present DUV-FSRS experiments is different from those of the previous picosecond UVRR experiments because we used the 292 nm Raman pump. Nevertheless, the same argument is valid. The arguments of the picosecond UVRR study are based on the analysis by Chi et al.42 They examined solvatochromic shifts of the absorption band of skatole and p-cresol, which possess
Figure 3. Temporal changes of the DUV-FSRS signals of (a) tryptophan and (b) tyrosine. The shaded time region is disturbed by the coherent artifact and perturbed free induction decay. The horizontal time axis is shown on a logarithmic scale after 2 ps. The broken black lines depict the simulated curves, in which the Raman intensity decreases with 0.5 ps and recovers with 30 ps (see the main text).
coherent artifact (the data are shown in the Supporting Information). It has been considered that the protein part of BR is changed with the trigger by the isomerization of the chromophore (0.5 ps5) and exhibits subsequent change with K ⇀ KL conversion occurring with a time constant of 30 ps.17 Thus, we also plot the temporal change that is simulated for this expected protein dynamics with dotted lines, i.e., the signal appears with 0.5 ps and disappears with 30 ps. Obviously, the stimulated Raman signals change much faster than this expectation. The temporal change of the stimulated Raman bands reveals that the Raman signals due to the protein part change prior to the isomerization of the chromophore. This is very surprising because, if the isomerization is the first event that triggers all the following processes in the photocycle of BR, the change in the protein Raman signal should be slower than, or similar to, the isomerization process of the chromophore. As clearly seen in Figure 3, in contrast, the DUV-FSRS signals due to the tryptophan and tyrosine residues appear within the time resolution of the experiment (
