Active Site Structure of Photoactive Yellow Protein with a Locked

Letter pubs.acs.org/JPCL

Active Site Structure of Photoactive Yellow Protein with a Locked Chromophore Analogue Revealed by Near-Infrared Raman Optical Activity Kensuke Kubota,†,∥ Takahito Shingae,†,∥ Nicole D. Foster,‡ Masato Kumauchi,‡ Wouter D. Hoff,‡ and Masashi Unno*,† †

Department of Chemistry and Applied Chemistry, Graduate School of Science and Engineering, Saga University, Saga 840-8502, Japan ‡ Department of Microbiology, Molecular Genetics, Oklahoma State University, Stillwater, Oklahoma 74078, United States S Supporting Information *

ABSTRACT: Many biological cofactors, such as light-absorbing chromophores in photoreceptors, are intrinsically planar molecules. A protein environment, however, causes structural distortions of the cofactor, and such structural changes can lead to a modulation of chemical properties of the cofactor to maximize its biological activity. Here, we investigate the active site structure of photoactive yellow protein (PYP), a blue light photoreceptor that contains a p-coumaric acid (pCA) chromophore, by a near-infrared excited Raman optical activity (ROA). Specifically, we measured the ROA spectra of PYP, whose chromophore is replaced with a locked pCA analogue. Furthermore, we show that a spectral analysis based on quantum mechanical/molecular mechanical (QM/MM) calculations of the whole protein molecule is useful to obtain structural information from the observed ROA spectra. The use of the near-infrared ROA combined with QM/MM calculations is a novel and generally applicable spectroscopic tool to study the chromophore distortions within a protein environment. SECTION: Biophysical Chemistry and Biomolecules

M

signatures, each of which is a potential marker of molecular structure.13−15 We recently applied the near-infrared ROA to bacteriorhodopsin (BR)16 and photoactive yellow protein (PYP).17 These studies demonstrated that the near-infrared excitation allows us to measure the ROA spectra of a chromophore within a protein environment. In the case of BR, we have shown that the all-trans/13-cis isomerization of the retinal chromophore significantly affects the ROA spectra. On the other hand, PYP, from phototrophic bacterium Halorhodospira halophila, contains the p-coumaric acid (pCA) chromophore, which is covalently linked to Cys69 through a thiolester bond.18,19 PYP is a small water-soluble photoreceptor protein, and it has been an attractive model for studying protein structures and dynamics.20 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. As shown in Figure 1, the chromophore is stabilized in the trans configuration as a phenolate anion.3,21,22 The phenolate oxygen O1 of the chromophore hydrogen bonds with the hydroxyl group of Tyr42 and the protonated carboxyl group of Glu46. Because of an availability of high-resolution

any biological cofactors, such as light-absorbing chromophores in photoreceptors, carry a π-electron system and are planar molecules. The cofactors are, however, often nonplanar within a protein environment, and such structural distortions have been shown to be functionally important. For example, the disruption of planarity is one of the key factors controlling the absorption spectra of the chromophore in photoreceptors.1,2 Nonplanarity has been also correlated with enhanced or suppressed fluorescence3,4 as well as efficient photoisomerization of a chromophore.5−7 Additionally, in many photoreceptors, light absorption produces a high-energy intermediate with a structurally distorted chromophore.6−10 This distortion stores light energy that is used to drive subsequent protein conformational changes.11,12 Despite their functional importance, relatively small structural distortions are difficult to observe. For example, X-ray crystal structures with a fairly high resolution (∼1.5 Å) are not sufficient to accurately determine the out-of-plane distortion of a chromophore. Because the nonplanar structure makes the molecule chiral, vibrational optical activity, such as Raman optical activity (ROA), is expected to potentially yield a wealth of stereochemical information on the structural and conformational details of chromophores. A major advantage of ROA over electronic circular dichroism and optical rotatory dispersion is derived from the large number of vibrational © 2013 American Chemical Society

Received: July 31, 2013 Accepted: August 26, 2013 Published: August 26, 2013 3031

dx.doi.org/10.1021/jz4016266 | J. Phys. Chem. Lett. 2013, 4, 3031−3038

The Journal of Physical Chemistry Letters

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system, we started from the X-ray structure available for WT PYP (pdb code: 1NWZ).3 GaussView5 (Gaussian, Inc.) was used, in combination with visual inspection, to assign the protonation states of all titratable residues. Gln46 was protonated,28,29 whereas Arg52 was deprotonated.30 All MM calculations were performed using the Amber force field.31 All available force field parameters and types of each atom in each amino acid were taken from the Amber library,31 while atomic charges were calculated by the QEq charge equilibration scheme.32 The nonstandard Amber parameters used in this study are as given in the Supporting Information. In the QM/ MM calculation, the cofactor and its immediate environment are described with B3LYP/6-31+G**, and the rest of the protein moiety is described with the Amber force field.31 UV−Vis Absorption Spectra. Figure 2 compares the optical absorption spectra of pCA-PYP and locked-PYP. As displayed

Figure 1. The structures of the pCA chromophore and its trans-locked analogue. (A) 4-Hydroxy-cinnamic acid (pCA) and (B) 7-hydroxycoumarin-3-carboxylic acid (locked-pCA) chromophores. Atomic numbering for the locked-pCA is also shown.

crystal structures for PYP, quantum chemical calculations based on density functional theory (DFT) can be used to simulate the ROA spectra. These DFT studies demonstrated that the ROA spectra are highly sensitive to the out-of-plane deformations of the chromophore. Here, we have applied the near-infrared ROA to a PYP analogue that is reconstituted with a trans-locked pCA (lockedPYP) and compared it with a native pCA PYP (pCA-PYP) (Figure 1). Our strategy was to use the locked nature of the chromophore analogue to challenge the chromophore binding pocket by preventing specific dihedral distortions and to detect how this affects chromophore dihedral angles in the protein. Because this chromophore analogue is trans-locked via a covalent bond, the out-of-plane deformations of the lockedpCA are expected to be small compared to those of the native pCA. We therefore expected reduced ROA intensities for the locked-PYP analogue. Furthermore, we have performed quantum mechanical/molecular mechanical (QM/MM) calculations of the whole protein molecule to simulate the observed ROA spectra. The available high-resolution crystal structure of PYP allowed us to calculate a structural model for PYP containing the locked-pCA, and this model was validated by the agreement between the observed spectra and the spectra calculated using this model. These results indicate that the nearinfrared ROA combined with QM/MM calculations is a powerful spectroscopic tool for characterizing structures and conformational dynamics of cofactors in proteins. PYP from H. halophila was produced and isolated as described previously23 as hexahistidine tagged apoprotein in Escherichia coli. The apoprotein was reconstituted with the anhydride of the respective chromophore (i.e., 4-hydroxycinnamic acid (pCA) and 7-hydroxy-coumarin-3-carboxylic acid (locked-pCA; Molecular Probes, Invitrogen), as described.24 PYP was used in 10 mM tris-HCl, pH 7.5, without removal of the N-terminal hexahistidine tag. The ROA instrument used in this study is based on an incident circular polarization scheme described previously.16 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. The 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 small model systems. The calculated frequencies were scaled using a factor of 0.9648.26 QM/MM calculations were performed with the use of the two-layer ONIOM (QM/MM) scheme,27 in which the interface between the QM and MM regions is treated by hydrogen link atoms. Electrostatic interactions between two layers were calculated using an electronic embedding scheme, where they were calculated at the MM level. For setting up the

Figure 2. Optical absorption spectra of pCA-PYP and locked-PYP in 10 mM tris-HCl, pH 7.5.

in the figure, the absorption maximum of locked-PYP (λmax = 443 nm) is similar to that of pCA-PYP (λmax = 446 nm), but the PYP analogue has a much narrower visible absorption band. These spectral features are consistent with those reported previously.33 Because the molar extinction coefficient for locked-PYP has not yet been reported, we determined the value by the Bradford assay,34 as described in Figure S2 of the Supporting Information. Figure 2 illustrates that the molar extinction coefficient of the visible absorption maximum for locked-PYP (ε = 36.4 × 103 M−1 cm−1) is smaller than that for pCA-PYP (ε = 45.5 × 103 M−1 cm−1).35 Raman and ROA Spectra. Figure 3 shows the Raman and ROA spectra of pCA-PYP (traces a and d). The spectra for pCA-PYP are consistent with those reported previously.16 Although the PYP samples used in the present study contain an additional hexahistidine tag, the close similarity between the present and reported data indicates little influence of the protein modification on the active site structure. Figure 3 also shows the Raman and ROA spectra for the locked-PYP analogue (traces b, c, e, and f). In the case of the Raman spectra, the replacement of the pCA chromophore with the locked-pCA analogue drastically affects the spectrum (trace a → b). The Raman spectrum for pCA is characterized by intense bands at 1556, 1283, 1164, 985, and 539 cm−1, whereas the locked-pCA analogue exhibits main bands at 1561, 1337, 1032, 790, 737, 608, and 511 cm−1. In addition to the different frequencies, the replacement with the locked-pCA analogue reduces the Raman intensities for most of the chromophore bands. The band observed at 1667 cm−1 is assigned to amide I 3032



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DFT and QM/MM Calculations. Next, we performed DFT calculations to obtain structural information from the ROA spectra. Figure 4 shows three models that we used for spectral

Figure 4. Optimized geometries of three models for the locked-PYP. Black, blue, red, and yellow represent carbon, nitrogen, oxygen, and sulfur atoms, respectively. In panel (C), only the active site of model 3 is shown. The QM region of the QM/MM calculation is illustrated by a ball and stick model. Figure 3. Raman and ROA spectra of PYP and its locked-PYP analogue in 10 mM tris-HCl, pH or pD 7.5. The sample concentration was 4−5 mM. The spectra were obtained with 785 nm excitation (∼200 mW). The spectra for (a,d) PYP with a native pCA chromophore, (b,e) PYP with a locked-pCA analogue, and (c,f) PYP with a locked-pCA analogue in the D2O buffer. The Raman spectra are arbitrarily normalized, and the ROA spectra are magnified by a factor of 2000.

simulations. We have employed the deprotonated 7-hydroxycoumarin-3-carboxyl methyl thiolester as a chromophore model (model 1). The calculated Raman and ROA spectra using model 1 are shown in Figure S3 in the Supporting Information (traces a and d). Because the molecule is planar (i.e., achiral), model 1 exhibits no ROA signals. In model 2, we consider methanol and acetic acid to mimic the hydrogen bonds of the phenolic O1 with Tyr42 and Glu46, 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. As illustrated in Figure S3 (Supporting Information), the hydrogen-bonding environments in model 2 affect both Raman and ROA spectra of the chromophore, and the calculated ROA intensities become nonzero. Because the locked-pCA chromophore in model 2 is essentially planar (Figure 4B), the calculated ROA intensities can be ascribed to the environmental chirality. However, there is apparently poor agreement between the experiments and calculations. The observed ROA spectrum exhibits a characteristic negative ROA band at 1337 cm−1, whereas the calculated spectrum only shows small positive features at around 1420 cm−1. These observations indicate that model 2 is not appropriate to simulate the ROA spectrum, implying that the locked-pCA chromophore is not planar within the protein environment. Therefore, we performed QM/MM calculations of the whole protein molecule to simulate the ROA spectra, as described below. Because the structure of the locked-pCA is similar to that of the trans form of pCA, overall protein structures are expected to be similar. We therefore used a high-resolution crystal structure of pCA-PYP3 as a starting structure and modified the chromophore to the locked-pCA. The QM part comprises the

(AmI) of the protein moiety,36 and the sharp band at 1003 cm−1 is due to a symmetric ring-stretching vibration of phenylalanine residues denoted F12.36 As seen in Figure 3, the intensities of these bands for locked-PYP are larger than those for pCA-PYP. This indicates that the locked-pCA analogue shows reduced Raman intensities compared to the native pCA. In Figure 3, we also compare the Raman spectra of locked-PYP in buffered H2O and D2O (traces b, c). The H/D exchange causes minor spectral changes, implying that the deprotonated phenolic OH group is like a native pCA chromophore.21,22 Figure 3 also compares the ROA spectra between pCA-PYP and locked-PYP (traces d and e). We previously showed that main ROA bands for pCA-PYP (985, 825, and 652 cm−1) mainly originate from HC7C8H hydrogen out-of-plane wagging modes of pCA, and the out-of-plane deformations of the chromophore are responsible for their large ROA intensities. Because the ethylenic −HC7C8− moiety is “locked” in the locked-pCA analogue, these low-frequency bands are expected to show reduced ROA intensities. In accordance with this idea, the low-frequency ROA bands are diminished in intensity in locked-PYP, while a sharp negative band is observed at 1337 cm−1. The H/D exchange did not induced significant changes in the ROA spectra (traces e and f). 3033



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are indicated in the figure. Here, we use the mode labels defined for the deprotonated 7-hydroxy-coumarin-3-carboxyl methyl thiolester (model 1), and νi and γi correspond to inplane and out-of-plane vibrations, respectively. Figure 6 presents atomic displacements for selected normal modes of model 1. In this figure, we use a simple model 1 because vibrational couplings with the protein moiety complicate some of the vibrational modes for models 2 and 3. In addition to the observed frequencies, Table 1 summarizes the computed frequencies as well as approximate descriptions of the vibrational modes. Thus, the combined use of Raman, ROA, and computational approaches reported here allows the unambiguous assignment of most observed vibrational modes without isotopic labeling. The value of ROA in obtaining reliable vibrational assignments may also be of value for vibrational spectroscopy of a range of other chromophoric proteins. The most intense negative ROA band at 1337 cm−1 is assigned to ν15, which is calculated at 1325 cm−1 for model 3. Although ν15 mainly involves the in-plane CH rocking vibrations, the deformation of the chromophore adds the outof-plane character to ν15.40 This accounts for the large ROA intensity for ν15. In addition, the out-of-plane deformation increases the ν15 frequency from 1291 to 1325 cm−1 (model 2 → 3), which is close to the observed frequency of 1337 cm−1. A negative ROA band at 790 cm−1 is assigned to ν26, which is the ring-breathing vibration of the locked-pCA chromophore. This mode also gains the out-of-plane character due to the structural distortion of the chromophore like ν15. In the low-frequency region below 1000 cm−1, there are several ROA features that are ascribed to the out-of-plane vibrations of the chromophore. The ROA band at 657 cm−1 is attributable to the out-of-plane C9O2 wagging mode γ14. A similar C9O2 wagging vibration of the pCA chromophore also showed a negative ROA band at 651 cm−1.17,37 This observation suggests a similar structural distortion around the C9O2 moiety between the native and locked-pCA chromophores. In accordance with this idea, the present QM/MM calculation predicts the out-of-plane distortion of the C9O2 moiety (Figure 4C), and its corresponding dihedral angle τ(C7−C8−C9-O2) = −10.6° is close to the value (−11.5°) reported for the crystal structures for pCA-PYP (Table 2).3,30 The consistency of these two independently determined dihedral angles provides evidence for the accuracy of the ROA approach to obtain specific structural information. A small negative ROA feature at 737 cm−1 can be correlated to γ12, which is mainly allocated to the out-of-plane motion of the C1−C6 ring (Figure 6). The pCA chromophore in WT PYP exhibits a similar ROA band at 733 cm−1.17 We previously showed that a sharp Raman band at 1003 cm−1 is assigned to a ring symmetric stretching vibration of phenylalanine residues denoted F12.36 However, the negative ROA band at 1003 cm−1 for locked-PYP can be ascribed to the C7−H out-of-plane wagging mode γ6 that is overlapped with the F12 band because this mode shows a negative ROA band at 985 cm−1 in the calculated spectrum. The DFT calculations show that the 1450−1600 cm−1 region contains normal modes that are assigned to CC stretching modes of the C1−C6 ring and the C7C8 group. Negative ROA bands at 1605, 1561, and 1486 cm−1 are assigned to ν6, ν8, and ν10, respectively. In addition to the CC stretching vibration, the C9O2 stretching motion also contributes to the high-frequency ν6 mode. Although these modes are

locked-pCA chromophore along with the covalently bound Cys69 moiety. In addition, nearby amino acid side chains (i.e., Tyr42 and Glu46) are also treated as the QM region, resulting in a total of 53 atoms including the link atoms. A geometry optimization from this QM/MM calculation leads to model 3, which is illustrated in Figure 4C. Analogous to the case of pCAPYP, the phenolate O1 and the carbonyl O2 of the locked-pCA form hydrogen bonds with surrounding protein moiety. In contrast, the O3 and O4 oxygen atoms of the linker fragment do not form hydrogen bonds because the chromophore binding pocket is mostly hydrophobic. A comparison between models 2 and 3 shows that the locked-pCA in model 3 is clearly nonplanar within a protein environment. This result is understandable because the C−C(−S−)O fragment is not locked in the locked-pCA. Figure 5 compares the observed

Figure 5. Observed and calculated Raman and ROA spectra of the locked-PYP. The observed (a) Raman and (b) ROA spectra of the locked-PYP are shown. The calculated (c) Raman and (d) ROA spectra for model 3 are also displayed.

Raman and ROA spectra with the calculated ones using model 3. In contrast to model 2, model 3 exhibits several intense ROA bands in the calculated ROA spectrum (see also Figure S3, Supporting Information). In fact, the predicted spectra are clearly improved and are similar to the experimental spectra. Especially, the agreements between the observed and calculated frequencies are mostly within ±10 cm−1. This indicates that model 3 is a reasonable structural model for locked-PYP. We will discuss the details of the assignments in the next section. Assignments. Because the Raman spectra of PYP containing native and locked pCA are quite different (Figure 3), the spectra for locked-PYP represent a novel band assignment problem. Usually, such assignments require laborious experiments involving the use of isotopically labeled chromophore derivatives, as we have performed for pCA-PYP.16,22,37−39 However, the results shown in Figure 5 allow us to assign most of the observed Raman and ROA bands, and their mode labels 3034



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Figure 6. Atomic displacement vectors for selected vibrational modes of 7-hydroxy-coumarin-3-carboxyl methyl thiolester (model 1).

Table 1. Observed and Calculated Vibrational Frequencies (cm−1) of Locked-PYP and Its Model νcalb νobs

a

1725 1605 1561 1486 1445 1405 1337 1242 1179 1125 1032 1003 892 790 737 657 608 511

model 1

model 2

model 3

1693 1611 1595 1575 1519 1498 1443 1398 1292 1261 1135 1086 1010 946 884 753 691 631 586 496

1710 1605 1590 1551 1496 1480 1445 1399 1291 1282 1146 1104 1007 953 897 764 694 632 595 512

1716 1614 1598 1553 1505 1487 1447 1399 1325 1231 1155 1109 1021 985 902 774 714 637 599 506

assignmentc ν5 ν6 ν7 ν8 ν9 ν10 ν11 ν12 ν15 ν16 ν19 ν20 ν21 γ6 ν24 ν26 γ12 γ14 ν30 ν31

νC10O3 νC9O2, νCC νCC, νC9O2 νCC, νC1O1, νC9O2 νC7C8 νC2C3, νC5C6 νCC, δCH νCC, δCH νCC, δCH νCC, δCH νC−C, νC−O, δCH δCH νC10−O4, νC8−C9, δCH γC7−H δCCC νCC (rings breathing) γCH γC9O2 δC4C7C8, δC3O4C10 δC2C1C6, δC3C4C5

a

Observed vibrational frequencies of the locked-PYP. bCalculated vibrational frequencies of models 1, 2, and 3. cThe observed Raman and ROA bands are assigned to the calculated in-plane (νi) or out-of-plane (γi) normal modes. Approximate descriptions of the calculated normal modes are provided.

As discussed above, many of the Raman and ROA bands for locked-PYP are ascribed to the chromophore. We should note, however, that the spectra for locked-PYP show relatively intense protein bands compared to those for pCA-PYP. For example, Figure 3 illustrates that the intensities of the Raman as well as ROA bands for the AmI mode at around 1670 cm−1 are larger in locked-PYP than those in pCA-PYP. Analogously, locked-PYP exhibits intense bands near 1300 cm−1, which are assigned to amide III (AmIII). 36 The larger spectral contributions from the protein moiety imply lower preresonance Raman enhancement of the chromophore bands in locked-PYP. This result is reasonable because the molar extinction coefficient of locked-PYP is smaller than that of pCA-PYP (Figure 2).

basically in-plane vibrations, the deformation of the locked-pCA chromophore again explains their ROA activity. Here, we note that a small feature, which can be discerned at 1725 cm−1 in the Raman spectrum (Figure 5, trace a), is assigned to the C10 O3 stretching mode ν5. In accordance with this assignment, the corresponding Raman band was not observed in the pCA chromophore of WT PYP (see traces a and b in Figure 3). Note that the comparison between models 1 and 2 indicates an opposite effect of the hydrogen-bonding interactions at the O1 and O2 oxygen atoms on the CO stretching frequencies. The formation of the hydrogen bonds (model 1 → 2) upshifts the C10O3 stretching mode ν5 by 17 cm−1, whereas the C1 O1/C9O2 stretching modes (ν6−ν8) are downshifted by 5− 24 cm−1. 3035



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structural distortions in the chromophore is reasonable because the estimated energy of ∼9 kJ mol−1 is smaller than a typical hydrogen bond energy of 20 kJ mol−1. This analysis demonstrates that ROA is capable of detecting the fairly subtle structural distortions of the chromophore that are accessible on a realistic energy scale similar to that of a single hydrogen bond. These results also suggest that the main causes of the out-ofplane distortions are the three hydrogen-bonding interactions at the O1 and O2 oxygen atoms of the locked-pCA. In summary, due to its sensitivity to chirality, ROA is a powerful probe of molecular structure in solution. In recent studies,16,17 we have applied the near-infrared excited ROA to photoreceptor proteins and successfully measured the ROA spectra of a chromophore within a protein environment. Furthermore, calculations of the ROA spectra utilizing DFT provide detailed structural information, such as data about outof-plane distortions of the chromophore. These findings extend the applicability of ROA to many biological cofactors. Here, we have measured the near-infrared ROA spectra of the PYP analogue, which is reconstituted with a trans-locked pCA. The observed spectral differences between native pCA and lockedpCA demonstrate a high structural sensitivity of the ROA spectra to the chromophore distortions. Our results also illustrate how ROA spectra can greatly aid the process of vibrational assignment. Furthermore, we obtained the structural information of the locked-pCA from the QM/MM calculations. These studies demonstrate that the near-infrared ROA combined with QM/MM calculations will be useful to explore the active site structure of cofactor-containing proteins. One of the interesting applications may be a photointermediate of a photoreceptor protein. Particularly, the capability of ROA combined with DFT calculations to extract the energy stored in specific dihedral angles reported here promises to provide accurate information on how photointermediates use chromophore distortions to store energy.

Table 2. Selected Dihedral Angles (deg) and Relative Energies (kJ mol−1) of the Models for Locked-PYP and pCAPYPa

model 1 model 2 model 3 model 4 model 5 model 6 model 7 model 8 pCAPYPb

C2−C3− C4−C7

C4−C7− C8−C9

C7−C8− C9−O2

O4−C10− C8−C9

180.0 180.0 −171.5 −171.5 −171.5 −179.8 −149.6 −176.2 −175.7

180.0 −179.9 165.3 165.3 −179.9 165.3 −178.6 169.1 165.1

0.0 −0.8 −10.6 −10.6 −1.3 3.3 −10.6 0.8 −11.5

180.0 180.0 −159.9 −159.9 −177.8 −168.2 179.6 −159.9

ΔE 0 9.3 1.5 4.8 1.1 6.0

a Dihedral angles that are fixed during the geometry optimizations are shown in bold italics. bA crystal structure of pCA-PYP (ref 3).

As discussed above, many of the Raman and ROA bands for locked-PYP are ascribed to the chromophore. We should note, however, that the spectra for locked-PYP show relatively intense protein bands compared to those for pCA-PYP. For example, Figure 3 illustrates that the intensities of the Raman as well as ROA bands for the AmI mode at around 1670 cm−1 are larger in locked-PYP than those in pCA-PYP. Analogously, locked-PYP exhibits intense bands near 1300 cm−1, which are assigned to AmIII.36 The larger spectral contributions from the protein moiety imply lower preresonance Raman enhancement of the chromophore bands in locked-PYP. This result is reasonable because the molar extinction coefficient of lockedPYP is smaller than that of pCA-PYP (Figure 2). Distortion Energies. Finally, we discuss the energy stored in the distorted locked-pCA chromophore. To estimate the energy changes associated with the out-of-plane distortions of each of the four dihedral angles involved, we modified model 2 to make models 4−8, as described below. Table 2 lists selected dihedral angles that characterize the out-of-plane distortions of the locked-pCA in models 1−8. The dihedral angles τ(C2−C3− C4−C7), τ(C4−C7−C8−C9), τ(C7−C8−C9−O2), and τ(O4−C10−C8−C9) are selected because these deviate more than 10° from planarity for model 3. In model 4, these dihedral angles were fixed with the values for model 3 in the geometry optimization. In addition, one of the four dihedral angles was fixed at selected values to obtain models 5−8. As illustrated in Figure S4 in the Supporting Information, the calculated Raman and ROA spectra for model 4 are similar to those for model 3. The spectra for models 5−8 are also shown in Figure S4 (Supporting Information). The figure demonstrates that the calculated ROA spectra are highly sensitive to the out-of-plane distortions, while the calculated Raman spectra for models 2 and 4−8 differ only slightly. This result of Figure S4 (Supporting Information) clearly illustrates a high sensitivity of the ROA spectra to small distortions of specific dihedral angles. In Table 2, we also compare the relative energies ΔE of models 4−8 compared to those of model 2. The dihedral twists that explain the main ROA signatures for model 3 correspond to the distortion energy of ∼9 kJ mol−1 (model 4). The energies associated with a single dihedral twist can be estimated by ΔE for models 5−8. As listed in Table 2, most of the total distortion energy can be correlated to dihedral twists of τ(C4− C7−C8−C9) = 165° and τ(O4−C10−C8−C9) = −160° (models 6 and 8, respectively). This energy scale for the

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ASSOCIATED CONTENT

S Supporting Information *

Additional AMBER parameters, results for the Bradford assay, and calculated Raman and ROA spectra. 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 ∥

K.K. and T.S. contributed equally.

Funding

This study was supported by KAKENHI (23550019 to M.U.) and the Mitsubishi Foundation (M.U.) and by NSF Grant MCB-1051590 to W.D.H. 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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REFERENCES

(1) Rocha-Rinza, T.; Sneskov, K.; Christiansen, O.; Ryde, U.; Kongsted, J. Unraveling the Similarity of the Photoabsorption of Deprotonated p-Coumaric Acid in the Gas Phase and within the Photoactive Yellow Protein. Phys. Chem. Chem. Phys. 2011, 13, 1585− 1589. 3036



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(2) Sekharan, S.; Morokuma, K. QM/MM Study of the Structure, Energy Storage, and Origin of the Bathochromic Shift in Vertebrate and Invertebrate Bathorhodopsins. J. Am. Chem. Soc. 2011, 133, 4734− 4737. (3) Getzoff, E. D.; Gutwin, K. N.; Genick, U. K. Anticipatory ActiveSite Motions and Chromophore Distortion Prime Photoreceptor PYP for Light Activation. Nat. Struct. Biol. 2003, 10, 663−668. (4) Conyard, J.; Kondo, M.; Heisler, I. A.; Jones, G.; Baldridge, A.; Tolbert, L. M.; Solntsev, K. M.; Meech, S. R. Chemically Modulating the Photophysics of the GFP Chromophore. J. Phys. Chem. B 2011, 115, 1571−1577. (5) Sugihara, M.; Buss, V.; Entel, P.; Elstner, M.; Frauenheim, T. 11cis-Retinal Protonated Schiff Base: Influence of the Protein Environment on the Geometry of the Rhodopsin Chromophore. Biochemistry 2002, 41, 15259−15266. (6) Yamada, A.; Ishikura, T.; Yamato, T. Role of Protein in the Primary Step of the Photoreaction of Yellow Protein. Proteins 2004, 55, 1063−1069. (7) Gromov, E. V.; Burghardt, I.; Köppel, H.; Cederbaum, L. S. Photoinduced Isomerization of the Photoactive Yellow Protein (PYP) Chromophore: Interplay of Two Torsions, a HOOP Mode and Hydrogen Bonding. J. Phys. Chem. A 2011, 107, 2837−2845. (8) Ihee, H.; Rajagopal, S.; Srajer, V.; Pahl, R.; Anderson, S.; Schmidt, M.; Schotte, F.; Anfinrud, P. A.; Wulff, M.; Moffat, K. Visualizing Reaction Pathways in Photoactive Yellow Protein from Nanoseconds to Seconds. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 7145−7150. (9) 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. (10) Schobert, B.; Cupp-Vickery, J.; Hornak, V.; Smith, S. O.; Lanyi, J. K. Crystallographic Structure of the K Intermediate of Bacteriorhodopsin: Conservation of Free Energy after Photoisomerization of the Retinal. J. Mol. Biol. 2002, 321, 715−726. (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) Nafie, L. A. Infrared and Raman Vibrational Optical Activity: Theoretical and Experimental Aspects. Annu. Rev. Phys. Chem. 1997, 48, 357−386. (14) Hug, W. Handbook of Vibrational Spectroscopy; John Wiley & Sons: Chichester, U.K.; 2002, Vol. 1. (15) Blanch, E. W.; Hecht, K.; Barron, L. D. Vibrational Raman Optical Activity of Proteins, Nucleic Acids, and Viruses. Methods 2003, 29, 196−209. (16) 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. (17) Shingae, T.; Kubota, K.; Kumauchi, M.; Tokunaga, F.; Unno, M. Raman Optical Activity Probing Structural Deformations of the 4Hydroxycinnamyl Chromophore in Photoactive Yellow Protein. J. Phys. Chem. Lett. 2013, 4, 1322−1327. (18) 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. (19) 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.

(20) 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. (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) Kort, R.; Hoff, W. D.; van West, M.; Kroon, A. R.; Hoffer, S. M.; Vlieg, K. H.; Crielaand, W.; van Beeumen, J. J.; Hellingwerf, K. J. The Xanthopsins: A New Family of Eubacterial Blue-Light Photoreceptors. EMBO J. 1996, 15, 3209−3218. (24) Imamoto, Y.; Ito, T.; Kataoka, M.; Tokunaga, F. Reconstitution of Photoactive Yellow Protein from Apoprotein and p-Coumaric Acid Derivatives. FEBS Lett. 1995, 374, 157−160. (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) Vreven, T.; Byun, K. S.; Komáromi, I.; Dapprich, S.; Montgomery, J. A., Jr.; Morokuma, K.; Frisch, M. J. Combining Quantum Mechanics Methods with Molecular Mechanics Methods in ONIOM. J. Chem. Theory Comput. 2006, 2, 815−826. (28) Borgstahl, G. E. O.; Williams, D. R.; Getzoff, E. D. 1.4 Å Structure of Photoactive Yellow Protein, a Cytosolic Photoreceptor: Unusual Fold, Active Site, and Chromophore. Biochemistry 1995, 34, 6278−6287. (29) Imamoto, Y.; Mihara, K.; Hisatomi, O.; Kataoka, M.; Tokunaga, F.; Bojkova, N.; Yoshihara, K. Evidence for Proton Transfer from Glu46 to the Chromophore during the Photocycle of Photoactive Yellow Protein. J. Biol. Chem. 1997, 272, 12905−12908. (30) 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. (31) Cornell, W. D.; Cieplak, P.; Bayly, C. I.; Gould, I. R.; Merz, K. M., Jr.; Ferguson, D. M.; Spellmeyer, D. C.; Fox, T.; Caldwell, J. W.; Kollman, P. A. A Second Generation Force Field for the Simulation of Proteins, Nucleic Acids, and Organic Molecules. J. Am. Chem. Soc. 1995, 117, 5179−5197. (32) Rappé, A. K.; Goddard, W. A., III. Charge Equilibration for Molecular-Dynamics Simulations. J. Phys. Chem. 1991, 95, 3358−3363. (33) Cordfunke, R.; Kort, R.; Pierik, A.; Gobets, B.; Koomen, G. J.; Verhoeven, J. W.; Hellingwerf, K. J. trans/cis (Z/E) Photoisomerization of the Chromophore of Photoactive Yellow Protein Is Not a Prerequisite for the Initiation of the Photocycle of This Photoreceptor Protein. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 7396−7401. (34) Bradford, M. M. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein−Dye Binding Reproduction. Anal. Biochem. 1976, 72, 248−254. (35) Meyer, T. E.; Tollin, G.; Hazzard, J. H.; Cusanovich, M. A. Photoactive Yellow Protein from the Purple Phototrophic Bacterium, Ectothiorhodospira halophila. Quantum Yield of Photobleaching and Effects of Temperature, Alcohols, Glycerol, and Sucrose on Kinetics of Photobleaching and Recovery. Biophys. J. 1989, 56, 559−564. (36) Kitagawa, T.; Hirota, S. Handbook of Vibrational Spectroscopy; John Wiley & Sons: Chichester, U.K.; 2002, Vol. 5. (37) 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. (38) Unno, M.; Kumauchi, M.; Sasaki, J.; Tokunaga, F.; Yamauchi, S. Assignment of Resonance Raman Spectrum of Photoactive Yellow 3037



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Protein in its Long-Lived Blue-Shifted Intermediate. J. Phys. Chem. B 2003, 107, 2837−2845. (39) Unno, M.; Kumauchi, M.; Sasaki, J.; Tokunaga, F.; Yamauchi, S. Evidence for a Protonated and cis Configuration Chromophore in the Photobleached Intermediate of Photoactive Yellow Protein. J. Am. Chem. Soc. 2000, 122, 4233−4234. (40) A further analysis that consists of the decomposition of the total ROA intensity of each vibrational mode into quasi-atomic and/or quasi-group contributions may be useful to understand the sign and intensity of a specific ROA band.41 Such an analysis, however, is beyond the scope of the present study. (41) Hug, W. Visualizing Raman and Raman Optical Activity Generation in Polyatomic Molecules. Chem. Phys. 2001, 264, 53−69.

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Active Site Structure of Photoactive Yellow Protein with a Locked

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