Letter pubs.acs.org/JPCL
Raman Optical Activity Probing Structural Deformations of the 4‑Hydroxycinnamyl Chromophore in Photoactive Yellow Protein Takahito Shingae,†,¶ Kensuke Kubota,†,¶ Masato Kumauchi,‡ Fumio Tokunaga,§ and Masashi Unno*,†,∥ †
Department of Chemistry and Applied Chemistry, Graduate School of Science and Engineering, Saga University, Saga 840-8502, Japan ‡ Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, Oklahoma 74078, United States § Department of Earth and Space Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan ∥ PRESTO, JST, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan S Supporting Information *
ABSTRACT: Many biological cofactors, such as light-absorbing chromophores in photoreceptors, contain a π-electron system and are planar molecules. These cofactors are, however, usually nonplanar within a protein environment, and such structural distortions have been shown to be functionally important. Because the nonplanar structure makes the molecule chiral, Raman optical activity (ROA) provides a wealth of stereochemical information about the structural and conformational details of cofactors. The present study applied a near-infrared excited ROA to photoactive yellow protein, a blue light receptor. We successfully obtained the ROA spectra of the 4-hydroxycinnamyl chromophore embedded in a protein environment. Furthermore, calculations of the ROA spectra utilizing density functional theory provide detailed structural information, such as data on out-of-plane distortions of the chromophore. The structural information obtained from the ROA spectra includes the positions of hydrogen atoms, which are usually not detected in the crystal structures of biological samples. SECTION: Biophysical Chemistry and Biomolecules
resolution are usually not enough to accurately determine distortions. In fact, we have pointed out that the deformations of the retinal chromophore in bacteriorhodopsin (BR) vary significantly among the available crystal structures, even if structures whose resolutions are less than 2 Å are compared.13 It is therefore desirable to have a good spectroscopic method that explores structural distortions of cofactors. 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.13 As is the case with vibrational circular dichroism, a major advantage of ROA over electronic circular dichroism and optical rotatory dispersion derives from the large number of vibrational signatures, each of them being a potential marker of the molecular structure.14−16 Indeed, we recently showed that a near-infrared excited ROA can be used to explore the out-of-plane deformations of the retinal chromophore in BR.13 As described above, conformations of the retinal chromophore are distinctly different among the crystal structures. It was therefore difficult to verify the chromophore structure deduced from the ROA spectra with
Many protein molecules contain cofactors for their function. For heme proteins, iron−porphyrin complexes form active sites of many biologically important molecules. In the case of photoreceptor proteins, a small organic molecule called a chromophore is used to detect light.1 A light-induced local structural change in the chromophore drives larger structural changes in the protein moiety, leading ultimately to biological functions such as signal transduction. Most cofactors carry a πelectron system and are planar molecules. These cofactors are, however, usually nonplanar within a protein environment, and such structural distortion has been shown to be functionally important. In the case of photoreceptor proteins, for example, the disruption of planarity is one of the key factors controlling the absorption spectra of the chromophore.2,3 Nonplanarity has been also correlated with photophysical and photochemical properties of the chromophore, that is, the former involve enhanced or suppressed fluorescence,4,5 while the latter include efficient photoisomerization of a chromophore.6−8 In systems such as retinal proteins, photoactive yellow protein, and phytochrome,1 light illumination produces primary high-energy intermediates with structurally perturbed chromophores.9−11 This distortion stores light energy that is used to drive subsequent protein conformational changes.11,12 Although the structural distortion of a cofactor is believed to be important, it is not easy to observe experimentally. For example, NMR or X-ray crystal structures with moderate © XXXX American Chemical Society
Received: February 28, 2013 Accepted: April 4, 2013
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high-resolution crystal structures. Thus, in the present study, we applied the near-infrared ROA to photoactive yellow protein (PYP), for which ultrahigh-resolution (0.82 Å) crystallographic data are available.4 In addition, a highresolution neutron structure of PYP17 provides an opportunity to examine the positions of hydrogen atoms. PYP, from phototrophic bacterium Halorhodospira halophila, is a small water-soluble photoreceptor protein. It has been an attractive model for studying protein structures and dynamics.18 PYP gained further attention as a structural prototype for the PAS (Per-ARNT-Sim) and LOV (light, oxygen, or voltage) domains of a large class of receptor proteins. This protein has the 4-hydroxycinnamyl chromophore, which is covalently linked to Cys69 through a thiolester bond.19,20 As shown in Figure 1, the chromophore is stabilized in the trans
Figure 1. The structure and numbering of the 4-hydroxycinnamyl chromophore for PYP.
configuration as a phenolate anion.4,21,22 The phenolate oxygen O1 of the chromophore hydrogen bonds with the hydroxyl group of Tyr42 and the protonated carboxyl group of Glu46. In the present study, we demonstrate that the near-infrared ROA combined with quantum chemical calculations based on density functional theory (DFT) yields information about small dihedral twists of the chromophore. Furthermore, the present study shows that the ROA spectra are highly sensitive to the positions of the hydrogen atoms, which are not visible in most protein crystal structures. 13 C-labeled 4-hydroxycinnamic acids were prepared according to a method described previously.23,24 Production of wildtype PYP apoprotein from Escherichia coli, reconstitution of the holoprotein with the chromophore, and the subsequent protein purification were performed as described previously.23,24 PYPs with 13C-labeled chromophore were prepared by reconstitution of apoprotein with 4-hydroxycinnamic anhydride, whose carbonyl carbon atom (C9) or ring carbon atoms (C1−C6) were labeled with 13C. These labeled samples are denoted as 13 CO and 13C6 ring isotopomers, respectively. PYP in buffered D2O (90% D2O/10% H2O) was prepared by the proper dilution of a concentrated protein in 100 mM Tris-HCl buffer at pH 7.4 into D2O. The ROA instrument used in this study is based on an incident circular polarization scheme described previously.13 The 785 nm light from a diode laser excited the sample, which was contained in a quartz cuvette. The laser power at the sample was ∼200 mW, and acquisition times were about 48 h. A stability of the sample during the measurements was carefully checked by monitoring the observed spectra. Raman and ROA spectra were calculated using the DFT method via the Gaussian09 program.25 The hybrid functional B3LYP and the 6-31+G** basis set were used for these calculations. The calculated frequencies were scaled using a factor of 0.9648.26 Figure 2 shows the Raman and ROA spectra of PYP with 785 nm excitation. The Raman spectrum (trace a) is in agreement with the reported resonance Raman spectrum with 413.1 nm excitation (see Figure S1 in the Supporting Information).24 The
Figure 2. Raman and ROA spectra of PYP and its isotopomers in 10 mM Tris-HCl, pH 7.4. The sample concentration was 4−5 mM. The spectra were obtained with 785 nm excitation (∼200 mW). The spectra for (a,e) the natural abundance, (b,f) D2O, (c,g) 13CO, and (d,h) 13C6 ring samples are shown. The ROA spectra are magnified by a factor of 2000.
close similarity between the nonresonance and resonance Raman spectra indicates that most of the observed bands in Figure 2 are attributable to the chromophore. An exception, the Raman band at 1667 cm−1, which is absent in the 413.1 nm spectrum, is assigned to amide I of the protein moiety.27 The major contributions of the chromophore vibrations are further illustrated by the effects of isotopic substitutions. We measured the spectrum of PYP in buffered D2O solution, where the exchangeable protons are replaced by deuterons (trace b). The Raman spectra are also shown for the 13CO (trace c) and 13 C6 ring (trace d) isotopomers. The effects of these isotopic substitutions are consistent with the previous resonance Raman data,24 confirming the observations of the Raman bands from the chromophore. In Figure 2, we also demonstrate the ROA spectra of PYP, and examination of the spectra reveals that many of their features coincide with the Raman bands of the chromophore. This implies that most of the ROA bands are attributable to the chromophore vibrations. This conclusion is confirmed by the effects of the isotopic substitutions on the ROA spectra (traces f−h). In addition, the ROA spectrum of a PYP analogue reconstituted with a fluorinated chromophore demonstrates a major contribution of the chromophore to the ROA spectra (Figure S2, Supporting Information). We note that a 1323
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and Figure 4 compares the observed (panel A) and calculated (panel B) spectra. As the figure shows, there is apparently poor
comparison of the ROA spectra between PYP and lysozyme further supports the idea (Figure S3, Supporting Information). These observations allow us to assign most of the observed ROA bands, and their mode labels are indicated in the figure. Here, we use the mode labels defined previously,23,24 and νi and γi correspond to in-plane and out-of-plane vibrations, respectively. As indicated in Figure 2, ROA bands are ascribed mainly to out-of-plane vibrations such as γ2, γ8, and γ10. Although the low-frequency region (below ∼550 cm−1) of the ROA spectrum for the 13CO isotopomer (trace g) is noisy, the negative feature at 631 cm−1 can be assigned to γ10. The natural abundance sample showed the Raman band at 539 cm−1 (trace a), whereas the ROA band was observed at 535 cm−1 (trace e). We consider that the Raman band at 539 cm−1 consists of ν36 and γ11, and the 535 cm−1 ROA band is mainly due to γ11. Here, we note that most of the observed Raman bands, including γ2, γ8, and γ10, are not strongly polarized (Figure S4, Supporting Information). Because strongly polarized Raman bands tend to give spurious artifact signals in ROA spectra,28 we can rule out the possibility that the ROA signals for PYP are attributable to polarization artifacts. In Figure S5 of the Supporting Information, we display the spectra of deprotonated trans-4-hydroxycinnamic acid. Because of the absence of the protein environment, no ROA signal was observed. Thus, the observation of the ROA spectra for PYP is a clear indication that the achiral 4-hydroxycinnamyl chromophore becomes chiral within a protein environment. Next, we performed DFT calculations to consider what structural information we could get from the ROA spectra. Figure 3 shows the active site of the crystal structure of PYP4 as
Figure 4. Observed and calculated Raman and ROA spectra of PYP. (A) Observed spectra. (B) Simulated spectra from model 1. (C) Simulated spectra from models 2 (black) and 3 (red). (D) Simulated spectra from model 4. For simulated Raman and ROA spectra in panels B−D, the intensities for ν11, ν13, and ν23 are reduced by factors of 10, 10, and 4, respectively. The asterisk indicates the ROA band due to a methyl deformation mode of methanol, which is a mimic of Tyr42.
agreement between the experiments and calculations. The observed ROA spectrum is characterized mainly by negative bands, whereas the calculated spectrum exhibits many positive bands. These observations imply that model 1 is not appropriate to simulate the ROA spectrum, although it serves as a reasonable model with which to assign the chromophore vibrations of PYP.24 The lower parts of panels A and B in Figure 3 illustrate side views of the chromophore for the crystal structure and model 1, respectively. In model 1, a full geometry optimization without constraints results in a planar structure. In contrast, the chromophore in the crystal structure is clearly nonplanar, and Table 1 gives selected dihedral angles that characterize the structural distortions. For instance, the C9O2 carbonyl moiety of the chromophore is out-of-plane distorted, and its corresponding dihedral angle τ(C7−C8−C9-O2) is about −10° in the crystal structures.4,17 In addition, the ethylenic −CHCH− moiety is also distorted by τ(C4−C7−C8-C9) = 165−168°. To examine the effects of the structural distortions, we consider model 2, where six dihedral angles are fixed on the basis of crystal structures. We also examine model 3, where each of the six dihedral angles is fixed with the same amounts, but their signs are reversed compared to model 2 (Table 1). Black traces in Figure 4C are the calculated spectra obtained using model 2. The predicted spectra are clearly improved and are similar to the experimental spectra. On the other hand, model 3 produces the calculated spectra shown in red in the
Figure 3. A crystal structure of wild-type PYP (ref 4) and optimized geometries of three active site models.
well as several models that we used for spectral simulations. We have employed deprotonated trans-4-hydroxycinnamyl methyl thiolester as a chromophore model. In model 1, we also consider methanol and acetic acid to mimic the hydrogen bonds of the phenolic O1 with Tyr42 and Gln46, respectively. Furthermore, the carbonyl O2 forms a hydrogen bond with the amide nitrogen of methylamine, which is a model of the backbone amide of Cys69. These components were arranged on the basis of the crystal structure4 and subsequently optimized to yield the structures illustrated in Figure 3B. This model has been shown to reproduce the main features of the observed Raman spectra, such as the observed isotope shifts.24 Thus, we used this model to simulate the ROA spectra, 1324
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Table 1. Structures and relative energies of the active site models for PYP 1NWZa C3−C4−C7−C8 C4−C7−C8−C9 C7−C8−C9−O2 C1−C2−C3−H3 C3−C4−C7−H7 H7−C7−C8−H8 ΔE a
−2.5 165.1 −11.5 nac na na
2ZOIb −4.7 168.2 −10.0 −169.4 −179.8 −172.5
model 1 Dihedral Angle (deg) −0.1 −180.0 0.275 −179.9 −179.9 179.8 Energy (kJ mol−1) 0
model 2
model 3
model 4
−10.0 170.0 −10.0 −170.0 −179.8 −170.0
10.0 −170.0 10.0 170.0 179.8 170.0
−10.0 170.0 −10.0 −170.0 167.3 174.3
25.1
24.9
6.7
X-ray crystal structure (PDB code: 1NWZ) from ref 4. bNeutron crystal structure (PDB code: 2ZOI) from ref 17. cna = not available.
figure. Although the calculated Raman spectra using models 2 and 3 are almost superimposable, the predicted ROA spectra are essentially mirror images of each other. Note that the positive ROA band at 1428 cm−1 in Figure 4B−D is due to the methyl deformation mode of methanol, which is a mimic of Tyr42, and is therefore not present in the observed spectrum. The agreement between the calculated and experimental results is improved particularly in the range of 600−1000 cm−1, with model 2 correctly predicting the main ROA bands, such as γ2, γ8, and γ10. Figure S6 in the Supporting Information illustrates atomic displacements for the important normal modes. The highest-frequency out-of-plane mode γ2 is observed at 984 cm−1 and involves the Au type of HC7C8H hydrogen out-of-plane wagging. A negative ROA band at 825 cm−1 is assigned to γ8, which is a C−H wagging of the aromatic ring as well as the C8−H moiety of the chromophore.24 The ROA band at 651 cm−1 is attributable to the out-of-plane C9O2 wagging mode γ10. These results indicate that the appearance of the out-of-plane vibrations in the ROA spectrum reflects the out-of-plane distortions of the chromophore. Furthermore, the signs of the ROA spectra indicate the direction of the structural distortions. In the region above 1000 cm−1, the observed ROA spectrum exhibits relatively small features. The calculated spectrum with model 2, however, reproduces several observed features for ν13, ν14, ν17, ν23, and ν25. Although these modes are assigned to in-plane vibrations, they have some out-of-plane characters due to the structural distortions of the chromophore. As mentioned above, we made constraints for six dihedral angles listed in Table 1 to describe the structural distortions. Three dihedral angles, τ(C3−C4−C7−C8), τ(C4−C7−C8− C9), and τ(C3−C4−C9−O2), represent distortions of the skeletal structure, whereas the positions of the hydrogen atoms H3, H7, and H8 are defined by the remaining three parameters. Although these values are based on crystal structures (4, 17), we note an exception for τ(C3−C4−C7−C8) = −10°, which is appreciably different from those seen in the crystal structures (−2.5 to −5°). Because the value at around −10° is needed to account for an intense ROA band for γ2, we suggest that τ(C3− C4−C7−C8) for PYP under physiological conditions is about −10°. In addition to the out-of-plane modes, here, we note a possible implication of the negative ROA band at around 1556 cm−1. As shown in Figure 2, the ν13 ROA band shows a clear downshift upon 13C6 ring substitution (trace e−h). Because ν13 is largely due to the CC stretching vibrations of the aromatic ring, the observation of the negative ν13 band may reflect structural distortions of the phenolic ring moiety. Finally, we discuss the influence of the positions of hydrogen atoms on the ROA spectra. Now, we consider model 4, where
the constraints for the dihedral angles defining the positions of the hydrogen atoms are removed. Table 1 compares the relative energies ΔE of models 2−4 compared to model 1, which is the most stable structure. Because of the structural constraints with six dihedral angles, models 2 and 3 are higher in energy by about 25 kJ mol−1. On the other hand, model 4 is relatively stable compared to models 2 and 3 (ΔE = ∼7 kJ mol−1). Figure 4D shows the simulated Raman and ROA spectra using model 4. A comparison with the calculated spectra shown in Figure 4C demonstrates distinctly different simulated spectra between models 2 and 4. For instance, the relative ROA intensity between γ2 and γ8 differs significantly, and an incorporation of the constraints for the positions of the hydrogen atoms (model 4 → 2) leads to better agreement with the experimental ROA spectrum. As illustrated in Figure 5, a hydrogen bonding
Figure 5. Optimized geometries of two active site models and the neutron crystal structure of PYP (ref 17.).
interaction with a backbone amide of Cys69 causes an out-ofplane distortion of the carbonyl C9O2 moiety, and this structural distortion favors the out-of-plane positions of the ethylenic hydrogen atoms (H7, H8) in model 4. However, energetically higher in-plane positions of the ethylenic hydrogen atoms are observed in the neutron crystal structure of PYP (Figure 5C).17 A possible reason for the unstable structure is steric interactions with a nearby protein moiety, such as Phe96, which directly faces the chromophore in the active site.29 Thus, the present result indicates that the nearinfrared ROA, combined with density functional calculations, provides detailed structural information, including data about the positions of hydrogen atoms. In conclusion, we have reported the first measurement of ROA spectra of PYP, a blue light receptor. The measurements using isotopically labeled samples establish that a near-infrared excitation enables us to measure the ROA spectra of the chromophore within a protein environment. Furthermore, calculations of the ROA spectra utilizing DFT provide detailed structural information, such as data about out-of-plane 1325
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(10) Kukura, P.; McCamant, D. W.; Yoon, S.; Wadschneider, D. B.; Mathies, R. A. Structural Observation of the Primary Isomerization in Vision with Femtosecond-Stimulated Raman. Science 2005, 310, 1006−1009. (11) Sudo, Y.; Furutani, Y.; Wada, A.; Ito, M.; Kamo, N.; Kandori, H. Steric Constraint in the Primary Photoproduct of an Archaeal Rhodopsin from Regiospecific Perturbation of C−D Stretching Vibration of the Retinyl Chromophore. J. Am. Chem. Soc. 2005, 127, 16036−16037. (12) Warshel, A.; Barboy, N. Energy Storage and Reaction Pathways in the First Step of the Vision Process. J. Am. Chem. Soc. 1982, 104, 1469−1476. (13) Unno, M.; Kikukawa, T.; Kumauchi, M.; Kamo, N. Exploring the Active Site Structure of a Photoreceptor Protein by Raman Optical Activity. J. Phys. Chem. B 2013, 117, 1321−1325. (14) Nafie, L. A. Infrared and Raman Vibrational Optical Activity: Theoretical and Experimental Aspects. Annu. Rev. Phys. Chem. 1997, 48, 357−386. (15) Hug, W. Handbook of Vibrational Spectroscopy; John Wiley & Sons: Chichester, U.K., 2002, Vol. 1. (16) Blanch, E. W.; Hecht, K.; Barron, L. D. Vibrational Raman Optical Activity of Proteins, Nucleic Acids, and Viruses. Methods 2003, 29, 196−209. (17) Yamaguchi, S.; Kamikubo, H.; Kurihara, K.; Kuroki, R.; Niimura, N.; Shimizu, N.; Yamazaki, Y.; Kataoka, M. Low-Barrier Hydrogen Bond in Photoactive Yellow Protein. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 440−444. (18) van der Horst, M. A.; Hendriks, J.; Vreede, J.; Yeremenko, S.; Crielaard, W.; Hellingwerf, K. J. Handbook of Photosensory Receptors; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, Germany, 2005. (19) Baca, M.; Borgstahl, G. E. O.; Boissinot, M.; Burke, P. M.; Williams, D. R.; Slater, K. A.; Getzoff, E. D. Complete Chemical Structure of Photoactive Yellow Protein: Novel Thioester-Linked 4Hydroxycinnamyl Chromophore and Photocycle Chemistry. Biochemistry 1994, 33, 14369−14377. (20) Hoff, W. D.; Dux, P.; Devreese, B.; Roodzant-Nugteren, I. M.; Crielaard, W.; Boelens, R.; Kaptein, R.; Van Beeumen, J.; Hellingwerf, K. J. Thiol Ester-Linked p-Coumaric Acid as a New Photoactive Prosthetic Group in a Protein with Rhodopsin-Like Photochemistry. Biochemistry 1994, 33, 13959−13962. (21) Kim, M.; Mathies, R. A.; Hoff, W. D.; Hellingwerf, K. J. Resonance Raman Evidence That the Thioester-Linked 4-Hydroxycinnamyl Chromophore of Photoactive Yellow Protein Is Deprotonated. Biochemistry 1995, 34, 12669−12672. (22) Unno, M.; Kumauchi, M.; Sasaki, J.; Tokunaga, F.; Yamauchi, S. Resonance Raman Spectroscopy and Quantum Chemical Calculations Reveal Structural Changes in the Active Site of Photoactive Yellow Protein. Biochemistry 2002, 41, 5668−5674. (23) Unno, M.; Kumauchi, M.; Sasaki, J.; Tokunaga, F.; Yamauchi, S. Assignment of Resonance Raman Spectrum of Photoactive Yellow Protein in Its Long-Lived Blue-Shifted Intermediate. J. Phys. Chem. B 2003, 107, 2837−2845. (24) Unno, M.; Kumauchi, M.; Tokunaga, F.; Yamauchi, S. Vibrational Assignment of the 4-Hydroxycinnamyl Chromophore in Photoactive Yellow Protein. J. Phys. Chem. B 2007, 111, 2719−2726. (25) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A. et al. Gaussian 09, revision A.2; Gaussian, Inc.: Wallingford, CT, 2009. (26) Merrick, J. P.; Moran, D.; Radom, L. An Evaluation of Harmonic Vibrational Frequency Scale Factors. J. Phys. Chem. A 2007, 111, 11683−11700. (27) Kitagawa, T.; Hirota, S. Handbook of Vibrational Spectroscopy; John Wiley & Sons: Chichester, U.K., 2002, Vol. 5. (28) Barron, L. D.; Bogaard, M. P.; Buckingham, A. D. Raman Scattering of Circularly Polarized Light by Optically Active Molecules. J. Am. Chem. Soc. 1973, 95, 603−605. (29) Morishita, T.; Harigai, M.; Yamazaki, Y.; Kamikubo, H.; Kataoka, M.; Imamoto, Y. Array of Aromatic Amino Acid Side Chains
distortions of the chromophore. Because such data can be obtained for a wide range of chromophoric proteins, the approach should be broadly applicable.
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ASSOCIATED CONTENT
S Supporting Information *
Resonance Raman spectra of PYP, Raman and ROA spectra of F-PYP and 4-hydroxycinnamic acid, discussion and comparison of ROA spectra for PYP and lysozyme, depolarization ratios, and atomic displacement vectors for some vibrational modes. 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]. Author Contributions ¶
T.S. and K.K. contributed equally.
Funding
This study was supported by KAKENHI (23550019 to M.U.) and the Mitsubishi Foundation (M.U.). A part of the computations was performed using the Research Center for Computational Science, Okazaki, Japan. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank W. D. Hoff for the helpful discussions. REFERENCES
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