Exploring the Active Site Structure of a Photoreceptor Protein by

Article pubs.acs.org/JPCB

Exploring the Active Site Structure of a Photoreceptor Protein by Raman Optical Activity Masashi Unno,*,†,‡ Takashi Kikukawa,§ Masato Kumauchi,∥ and Naoki Kamo¶ †

Department of Chemistry and Applied Chemistry, Graduate School of Science and Engineering, Saga University, Saga 840-8502, Japan ‡ PRESTO, JST, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan § Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810, Japan ∥ Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, Oklahoma 74078, United States ¶ College of Pharmaceutical Sciences, Matsuyama University, Matsuyama 790-8578, Japan S Supporting Information *

ABSTRACT: We have developed a near-infrared excited Raman optical activity (ROA) spectrometer and report the first measurement of near-infrared ROA spectra of a light-driven proton pump, bacteriorhodopsin. Our results demonstrate that a near-infrared excitation enables us to measure the ROA spectra of the chromophore within a protein environment. Furthermore, the ROA spectra of the all-trans, 15-anti and 13-cis, 15-syn isomers differ significantly, indicating a high structural sensitivity of the ROA spectra. We therefore expect that future applications of the near-infrared ROA will allow the experimental elucidation of the active site structures in other proteins as well as reaction intermediates.

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INTRODUCTION Many biological cofactors, such as light-absorbing chromophores in photoreceptors or iron porphyrin in heme proteins, each carry a π-electron system and are a planar molecule. These cofactors are, however, usually nonplanar within a protein environment, and such structural distortions have been shown to be functionally important.1−6 Additionally, in the case of sensor proteins, for example, an external stimulus such as light absorption produces a high-energy intermediate with a structurally distorted chromophore.7−9 Despite their functional importance, relatively small structural distortions are difficult to observe. For example, X-ray crystal structures with a moderate resolution (∼1.5 Å) are not enough to accurately determine the out-of-plane distortion of a chromophore (see below). Because the nonplanar structure makes the molecule chiral, vibrational optical activity, including Raman optical activity (ROA) and vibrational circular dichroism, is expected to potentially yield a wealth of stereochemical information about the structural and conformational details of cofactors. Like vibrational circular dichroism, a major advantage of ROA over electronic circular dichroism and optical rotatory dispersion is derived from the large number of vibrational signatures, each of which is a potential marker of molecular structure.10−13 However, the application of ROA to cofactor molecules is very limited,14,15 because most of these molecules are strong fluorophores with a commonly used visible excitation. Another reason would be the photodegradation of the sample, which absorbs excitation light. We report here the first measurements of ROA spectra of a chromoprotein, called bacteriorhodopsin (BR), using nearinfrared laser excitation at 785 nm, which enables us to measure © 2013 American Chemical Society

the ROA spectra of a cofactor molecule within protein environments. The motivation for near-infrared ROA measurements is the widespread success in recent years of near-infrared excited Raman spectroscopy. That is, excitation in the nearinfrared region dramatically reduces interfering fluorescence from most samples. This interference is a problem in obtaining a Raman spectrum. In addition, photodegradation is not a problem for many systems because there is no light absorption in the near-infrared region.16−18 BR is a photoreactive membrane protein found in the purple membrane of Halobacterium salinarum.19 The light-sensitive chromophore in BR is the protonated Schiff base of retinal that is covalently bound to Lys216 within the protein interior. In the light-adapted state (BR568), this protein exhibits a broad visible absorption maximum at 568 nm. Absorption of a photon by BR568 initiates a cyclic photoreaction that drives the transport of protons across the bacterial cell membrane. In the dark, BR converts to a dark-adapted state (BR560), which contains a mixture of BR568 and BR548. These isomers are thermally interconvertible, and BR568 contains all-trans, 15-anti retinal (Figure 1A), whereas the retinal in BR548 is 13-cis, 15-syn (Figure 1B).20 The atomic structures of the two isomers have been reported,21−30 and their structural comparison indicates that the retinal moiety C1−C11 moves only slightly when the retinal isomerization around both the C13C14 and C15N double bonds proceeds. On the other hand, the out-of-plane Received: January 4, 2013 Revised: January 12, 2013 Published: January 14, 2013 1321

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Figure 1. The structure and numbering of the protonated (A) all-trans and (B) 13-cis retinal Schiff base chromophore for BR. Front and top views of the chromophores in the crystal structures for (C) BR568 (1iw6) and (D) BR548 (1x0s). Black and blue balls represent carbon and nitrogen atoms, respectively.

deformation of the Schiff base moiety is clearly different between the two isomers (Figure 1, parts C and D). In the present study, we demonstrate that the ROA spectra of the retinal chromophore of BR can be measured with 785 nm excitation. Furthermore, we found that the ROA spectrum of the 13-cis, 15-syn retinal in BR548 exhibits distinctly larger signals compared to that of the all-trans, 15-anti form, suggesting a structural distortion of the chromophore. This work presents a new and powerful tool for characterizing structures and conformational dynamics of cofactors in proteins.

Figure 2. Raman and ROA spectra of (a, c) BR560 and (b, d) BR568. The spectra were obtained with 785 nm excitation (200 mW).

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(Figure S2 in the Supporting Information). Although the spectrum of BR568 is similar to that of BR560, there are some distinct differences. The most clear difference is a lack of a Raman band at 800 cm−1 for BR568. In Figure 2, we also demonstrate the ROA spectra of BR560 (trace c) and BR568 (trace d), and an inspection reveals that all of the features in the ROA spectra coincide with the Raman bands of the retinal chromophore. This implies that the observed ROA bands are due to the chromophore vibrations. In contrast to the Raman spectra, the ROA spectra are dramatically different between BR568 and BR560. Since BR560 is a mixture of BR548 and BR568,16,17,20 we calculated the Raman and ROA spectra of BR548 in Figure 3. As reported previously,16,17 the Raman spectrum of BR548 is calculated by subtracting the normalized BR568 spectrum from the BR560 one. The normalization factor was determined by minimizing the positive or negative 882 cm−1 intensity, which has been assigned to the C14H out-of-plane wagging mode for BR568 (trace b).34 We used the normalization factor of 0.65 in Figure 3, and the estimated Raman spectrum agrees well with those reported previously.16,17,34,35 We used the same normalization factor to obtain the ROA spectrum of BR548 (trace d). As illustrated in Figure 3, the ROA spectra of BR548 and BR568 differ significantly; most of the ROA bands are intensified upon the formation of BR548 from BR568. In addition, the ROA bands for the C−C stretching (1150−1250 cm−1) and methyl rocking (∼1010 cm−1) modes change in sign. Here we note that none of the observed Raman bands are strongly polarized (see Figure S3 in the Supporting Information). Since strongly polarized Raman bands tend to give a spurious artifact signal in ROA spectra,36 we can rule out the possibility that polarization artifacts give the ROA signals for BR. The results shown in Figure 3 indicate a high structural sensitivity of the ROA spectra. Because BR568 and BR548

EXPERIMENTAL SECTION BR, a purple membrane, was isolated from Halobacterium salinarum strain S9 by the established standard method. HR protein expression and purification were reported previously.31 The ROA instrument used in this study is based on an incident circular polarization scheme, and the performance of the spectrometer was examined by measuring several standard samples (see Figure S1 in the Supporting Information). We measured the Raman and ROA spectra of BR568 under continuous illumination with an orange LED light. Raman and ROA spectra were calculated by using the density functional theory (DFT) method via the Gaussian09 program.32 The hybrid functional B3LYP and the 6-31+G** basis set were used for these calculations. The calculated frequencies were scaled by a factor of 0.9648.33

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RESULTS AND DISCUSSION Figure 2 shows the Raman spectra of BR with 785 nm excitation. The spectrum of BR560 (trace a) is dominated by vibrations of the retinylidene chromophore of BR, in particular by CC (1527 cm−1) and C−C stretches (1170 and 1203 cm−1); CCH rocking modes (1273, 1324, and 1345 cm−1); and methyl rocking mode (1008 and 1382 cm−1). Nearly all of the observed bands were observed in the resonance Raman spectra with 514.5 nm excitation,34,35 indicating a preresonance condition for Raman excitation at 785 nm. These observations are in agreement with previous near-infrared Raman studies with 840 or 1064 nm excitation.16,17 Upon illumination with an orange light (∼590 nm), BR560 is converted to BR568, and Figure 2 shows the Raman spectrum of BR568 (trace b). The Raman spectrum of BR568 with 785 nm excitation also agrees well with those obtained at 514.534,35 or 532 nm excitation 1322

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cm−1 band.35 The C8−C9 stretch is localized at 1203 cm−1, but this band also involves a contribution from the C14−C15 stretching vibration. The observation of distinct ROA bands at both 1170 and 1203 cm−1 suggests a structural distortion around the C14−C15 bond. This is further supported by the observation of the ROA band at 800 cm−1, which has been assigned to the C14−H hydrogen out-of-plane wagging mode (14H wag). Table 1 compares the selected dihedral angles of Table 1. Selected Dihedral Angles of the Retinal Chromophore for BR568 and BR548

Figure 3. Raman and ROA spectra of BR548 (a, d), BR568 (b, e), and HR578 (c, f). The spectra of BR548 were obtained by subtracting the spectra of BR568 multiplied by a factor of 0.65 from those of BR560.

a

PDB

res.a

3ns0 2ntu 1py6 1m01 1kgb 1qhj 1c3w 1iw6

1.78 1.53 1.80 1.47 1.65 1.90 1.55 2.30

1x0s 1r84

2.50 b

C8−C9

C13−C14

C14−C15

dihedral angle (deg) for BR568 175.2 −169.4 156.0 −171.4 −155.9 −176.0 −179.6 −179.5 −179.8 −173.6 −153.5 176.7 −179.7 −167.4 −173.7 −175.7 −178.1 158.5 −173.7 −156.9 178.9 −176.7 −172.7 176.0 dihedral angle (deg) for BR548 −178.9 1.7 −151.8 −178.9 25.8 −172.2

C15−N

ref

−156.2 −173.1 −145.3 −171.5 −156.5 −149.5 −162.7 −164.1

21 22 23 24 25 26 27 28

3.6 10.1

29 30

Resolution in Å. bNMR structure.

the retinal chromophore found in X-ray crystallographic and solution NMR structures.21−30 Although the available two structures for BR548 are somewhat different, either the dihedral angle τ(C12−C13−C14−C15) or τ(C13−C14−C15−N) demonstrates a significant deviation from planarity. This would explain the high ROA intensities of BR548. In the case of BR568, more than 10 crystal structures are available, and Table 1 lists the structures whose resolutions are better than 2 Å. This table also includes the 2.3 Å structure, pdb entry 1iw6,22 because it was used for a comparison with BR548.29 As can be seen in the table, the reported dihedral angles vary distinctly, implying that the available crystal structures are inadequate to determine the out-of-plane distortion of the retinal chromophore. On the other hand, the present ROA study found relatively low ROA intensities of BR 568 , suggesting smaller structural distortions of the chromophore. Alternatively, the structural flexibility of the retinal chromophore in BR568 might be sufficient to allow multiple conformations, which would lead to a decrease in the average ROA intensity. To interpret the observed ROA spectra in a quantitative way, a quantum chemical calculation based on DFT is expected to be useful.42−45 We therefore performed DFT calculations to analyze the ROA spectrum of BR548. We employed a 13-cis retinal protonated Schiff base as a simple chromophore model (see Figure S4 in the Supporting Information), and Figures 4 and S5 (Supporting Information) demonstrate that the ROA spectra in the 800−1280 cm−1 region are highly sensitive to the out-of-plane distortions of the chromophore. For instance, the dihedral twist about the C8−C9 bond increases the ROA intensities for the CH3 rock (989 cm−1) and C14−C15 stretch (1168 cm−1) bands, and the signs of the ROA bands reflect the direction of the twist (Supporting Information, Figure S5, traces c and d). In addition, dihedral twists at distinct positions affect the ROA spectra in different ways, and the effects of these structural distortions on the spectra are roughly additive (panel

contain all-trans, 15-anti (Figure 1A) and 13-cis, 15-syn (Figure 1B) retinal chromophores, respectively,16,17,34,35 the significant variation of the ROA spectra can be related to the isomerization state of the chromophore. In fact, previous crystallographic and solution NMR studies on BR568 and BR548 have shown only small differences in the protein moiety.29,30 To further examine the influence of the retinal isomerization state, we measured the Raman and ROA spectra of halorodopsin (HR) from Natronomonas pharaonis. HR binds retinal covalently as a protonated Schiff base to Lys256 and acts as a light-driven chloride pump.37 The X-ray crystal structures of HR under the dark state (HR578) indicate that the transmembrane region of HR is structurally very similar to that of BR.38,39 In addition, the retinal chromophore is in an all-trans, 15-anti configuration like BR568.38−40 As displayed in Figure 3, the Raman spectrum of HR578 (trace c) is remarkably similar to that of BR568 (trace b). This observation is consistent with the results of a previous resonance Raman study.41 Figure 3 further illustrates the close similarity between the ROA spectra of HR578 and those of BR568 (traces e and f). As described above, the ROA spectra are remarkably sensitive to the isomerization state of the retinal chromophore. Because a planar molecule is not chiral, the high ROA intensities may be attributable to the out-of-plane deformation of the chromophore. In the case of BR, the vibrational modes associated with the nonconjugated β-ionone ring and lysine side chain are almost absent in the Raman spectra.34,35 Thus the significant difference in the ROA spectra can be correlated with the structural distortion of the ethylenic/Schiff base moieties. For BR548, the 1150−1250 cm−1 C−C stretching region exhibits three resolved Raman bands, at 1170, 1184, and 1203 cm −1 . Although these modes are highly mixed combinations of the C−C stretches and CCH rocks, the C14−C15 stretching vibration contributes mainly to the 1170 1323

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ASSOCIATED CONTENT

S Supporting Information *

Materails and methods details and Raman and ROA spectra and the structure of the 13-cis retinal protonated Schiff base. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank T. Shingae for helping us construct the ROA spectrometer, and W. D. Hoff for the helpful discussions. This study was supported by KAKENHI (23550019 to M.U.) and the Mitsubishi Foundation (M.U.). Some of the computations were performed using the Research Center for Computational Science, Okazaki, Japan.

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REFERENCES

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Figure 4. Calculated Raman (a) and ROA (b−f) spectra of the 13-cis retinal protonated Schiff base. The ROA spectra are multiplied by a factor of 2000 compared to the Raman spectra. The following dihedral angles were fixed to obtain optimized geometries for ROA spectra shown as traces c−f and their Raman spectra: (c) τ(C7−C8−C9− C10) = 160°, (d) τ(C12−C13−C14−C15) = 30°, (e) τ(C13−C14− C15−N) = 170°, (f) τ(C7−C8−C9−C10) = 160°, τ(C12−C13− C14−C15) = 30°, τ(C13−C14−C15−N) = 170°.

B in Figure S5, Supporting Information). Thus the main features of the observed ROA spectrum can be reproduced by a combination of three dihedral twists about the C8−C9 (160°), C13−C14 (30°), and C14−C15 (170°) bonds. As illustrated as trace f in Figure 4, the chromophore with these distortions explains the negative ROA bands for the CH3 rock and the C14−C15 stretch as well as the positive band for the C14−H wag. This structure seems to be similar to the NMR structure30 where the C13−C14 bond is distorted (∼26°), although the distortion around the C8−C9 bond was not observed (Table 1). At this stage, however, we should note that the present analysis requires further improvement for better understanding the observed ROA spectra. For example, the present analysis does not involve the effects of the protein environment such as hydrogen bonding at the Schiff base moiety. Therefore, further DFT calculations are currently in progress. In conclusion, we have developed a near-infrared excited ROA spectrometer and report the first measurement of nearinfrared ROA spectra of a light-driven proton pump, BR. Our results demonstrate that a near-infrared excitation enables us to measure the ROA spectra of the chromophore within a protein environment. Furthermore, the ROA spectra of the all-trans, 15-anti and 13-cis, 15-syn isomers differ significantly, indicating a high structural sensitivity of the ROA spectra. We therefore expect that future applications of the near-infrared ROA will allow the experimental elucidation of the active site structures in other proteins as well as reaction intermediates. 1324

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