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2007, 111, 6293-6296 Published on Web 05/25/2007
Picosecond Protein Response to the Chromophore Isomerization of Photoactive Yellow Protein: Selective Observation of Tyrosine and Tryptophan Residues by Time-Resolved Ultraviolet Resonance Raman Spectroscopy Misao Mizuno,† Norio Hamada,‡ Fumio Tokunaga,§ and Yasuhisa Mizutani*,† Department of Chemistry, Graduate School of Science, Osaka UniVersity, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan, JST-CREST, Venture Business Laboratory, AdVanced Science InnoVation, Osaka UniVersity, 2-1 Yamada-oka, Suita, Osaka 565-0871, Japan, and Department of Earth and Space Science, Graduate School of Science, Osaka UniVersity, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan ReceiVed: April 16, 2007; In Final Form: May 15, 2007
Picosecond time-resolved ultraviolet resonance Raman (UVRR) spectra of photoactive yellow protein (PYP) were measured. UVRR bands attributed to the vibration of tyrosine and tryptophan residues showed a spectral change upon photoreaction. It was found that the hydrogen-bond strength between the chromophore and Y42 increases in the pG* state. The ultrafast change in the tryptophan band revealed that a photoinduced structural change of the chromophore had propagated to the W119 region, located 12 Å from the chromophore, within picoseconds.
Photosensors are a class of molecules that have attracted great interest owing to their ability to transform the small structural changes that occur in a photoreaction into large changes over the entire protein, thereby facilitating signal transduction. To understand the mechanism of signal transduction, it is important to show how changes in the chromophore trigger structural changes in the protein. Photoactive yellow protein (PYP) is a blue-light sensor considered to be responsible for the lightinduced negative phototaxis of bacteria. PYP is small, water soluble, easily crystallized, and photostable. It possesses the PAS (Per-Arnt-Sim) domain structural motif, which is widely involved in a number of sensory proteins, and thus serves as a useful structural prototype for the PAS superfamily as well as a good model for understanding the signal transduction of sensory proteins.1 Time-resolved and transient vibrational spectroscopy has given information on the photoinduced structural changes of PYP.2-10 To understand the signaling mechanism of sensory proteins, it is essential to investigate how the structural change propagates not only around the chromophore but also to functional sites away from the chromophore. Ultraviolet resonance Raman (UVRR) spectroscopy can probe changes in protein structure through the selective enhancement of vibrational bands from aromatic amino acid residues and from the polypeptide skeletal structure.11 Recently, UVRR spectroscopy was used to characterize structural changes in PYP during the formation of the pB state.6 In the present Letter, we investigate the ultrafast structural dynamics of aromatic amino acid residues in PYP using picosecond timeresolved UVRR spectroscopy. * To whom correspondence should be addressed. E-mail: mztn@ chem.sci.osaka-u.ac.jp. Phone: +81-6-6850-5776. Fax: +81-6-6850-5776. † Department of Chemistry, Graduate School of Science. ‡ JST-CREST. § Department of Earth and Space Science, Graduate School of Science.
10.1021/jp072939d CCC: $37.00
The experimental setup for picosecond time-resolved UVRR measurements has already been described elsewhere.12 Briefly, the light source of the apparatus was an amplified picosecond Ti:sapphire laser. To generate the pump and probe pulses, the second harmonic of the laser output excited two Raman shifters separately. The pump pulses (446 and 460 nm, 5 µJ) were the first Stokes line generated from a CH4 Raman shifter. The probe pulses (236 and 230 nm, 0.5 µJ) were the second harmonic of the first Stokes line generated from H2 and CH4 Raman shifters, respectively. Under the present probe conditions, the saturation effect of the resonance Raman scattering was not observed. The pump and probe pulses were focused onto a flowing thin film of the sample solution formed by a wire-guided jet nozzle.13 The sample was prepared at a PYP concentration of 100 µM (236 nm probe) or 50 µM (230 nm probe) in 10 mM Tris-HCl buffer at pH 7.0. Figure 1 shows the 236 nm probe time-resolved UVRR difference spectrum of PYP measured at 3 ps (a) and UVRR spectra of dark-state PYP (b), tyrosine (c), and tryptophan (d). The mode assignments made by Harada and Takeuchi were adopted.14 The spectral changes in UVRR bands induced by light irradiation are so small that we discuss the changes in terms of difference spectra. Trace a shows that UVRR bands attributable to both the tyrosine and tryptophan residues exhibit spectral changes arising from photoinduced structural changes. Under the 236 nm probe conditions, it is suitable to observe the UVRR bands of tyrosine, because the intensity of the tyrosine bands is greater than those of tryptophan at this wavelength.15,16 In the figure, negative tyrosine bands were clearly observed at the Y8a, Y7a, and Y9a bands. The negative bands represent the intensity loss relative to the intensity in the dark state. The Y9a band exhibited a sigmoidal form attributable to an upshift in the Y9a band upon the photoreaction. For tryptophan, a small decrease in intensity was observed for the W3 and W7 bands. To © 2007 American Chemical Society
6294 J. Phys. Chem. B, Vol. 111, No. 23, 2007
Figure 1. (a) Time-resolved UVRR difference spectrum of PYP in 10 mM Tris-HCl buffer at pH 7.0 (concentration 100 µM, delay 3 ps). UVRR spectra of (b) dark-state PYP in 10 mM Tris-HCl buffer at pH 7.0 and (c) tyrosine and (d) tryptophan in water. The pump and probe wavelengths are 446 and 236 nm, respectively. The solvent spectrum has been subtracted.
Figure 2. (a) Time-resolved UVRR difference spectrum of PYP in 10 mM Tris-HCl buffer at pH 7.0 (concentration 50 µM, delay 3 ps). UVRR spectra of (b) dark-state PYP in 10 mM Tris-HCl buffer at pH 7.0 and (c) tyrosine and (d) tryptophan in water. The pump and probe wavelengths are 460 and 230 nm, respectively. The solvent spectrum has been subtracted.
distinctly observe changes of tryptophan bands, the 230 nm probe UVRR spectra were also measured (Figure 2). At this probe wavelength, the intensity of tryptophan UVRR bands, especially the totally symmetric bands, W3, W16, and W18, is strongly enhanced in resonance with the Bb transition. In Figure 2a, intensity loss was clearly observed for the tryptophan bands, W3, W7, W16, and W18, as well as tyrosine bands at 3 ps. The observed intensity change of UVRR bands of tyrosine and tryptophan residues is considered to not be directly due to changes in the electronic structure of the chromophore, pcoumaric acid. In UVRR spectra of myoglobin, the differences in tyrosine bands of the different ligation states of heme are not due to a tyrosine residue close to the heme but predominantly due to the farthest one, and thus, the electronic coupling between the chromophore and both the tyrosine and tryptophan residues is considered to negligibly affect the intensity change of the observed spectra.17 It is more reasonable to attribute the intensity changes to changes of the Raman excitation profiles in accordance with structural changes in the vicinity of these residues, such as changes in the hydrogen-bond strength and hydrophobicity, as the result of the propagation of the structural change in the protein. The probe wavelength is located at the red side
Letters of the peak wavelengths (around 225 nm) of the excitation profiles of tyrosine and tryptophan,15,16 so that the loss in intensity of the tyrosine and tryptophan bands arises from a blue shift in the excitation profiles.11,18,19 It has been reported that an increase in hydrogen-bond strength causes a blue shift in the excitation profiles of tyrosine UVRR bands.11,19 The intensity loss of the tyrosine bands suggests that the strength of the hydrogen bond which the tyrosine residue forms increases at 3 ps. The frequency of the Y9a band is an indicator of the hydrogen-bond strength,11,20 and thereby, its upshift supports our interpretation that the intensity loss of the tyrosine bands results from the stronger hydrogen-bond formation. In addition, the Y9a frequency is correlated to the dihedral angle between the COH and benzene planes in a tyrosine molecule.21 We cannot rule out the possibility that the OH hydrogen atom displaces responding to the structural change of the chromophore. The blue shift in the Raman excitation profile of the tryptophan residue can be attributed to a reduction in hydrophobicity in the region surrounding the residue.11,18 Such bleach in UVRR signals attributable to the environmental changes surrounding the tyrosine and tryptophan residues upon photoreaction have been reported for myoglobin12 and rhodopsin.22 We need to assign the amino acid residues that show spectral changes in the spectra to investigate the signal transduction mechanism. PYP possesses a single tryptophan residue, W119;23 hence, the spectral change in the tryptophan band is definitely attributed to a structural change in W119. For tyrosine, on the other hand, there are five residues in a PYP molecule: Y42, Y76, Y94, Y98, and Y118.23 The difference spectra indicate the change of the hydrogen-bond strength. It is highly likely that the spectral changes observed for tyrosine bands arise from structural changes in the hydrogen-bonded interior residues, Y42 and Y118. On the basis of the present UVRR spectra, however, the number of the tyrosine residues affecting the spectral change is not conclusively determined. Y42 is the most probable, because it is directly hydrogen-bonded to the chromophore, whose structure changes considerably during trans/cis isomerization, which occurs in picoseconds. Y118 is hydrogen-bonded to D116 and is a neighbor of W119 which undergoes the structural change. Indeed, the Y9a band seems to be the sigmoidal and negative feature under the 236 and 230 nm probe conditions, respectively. There are two possibilities of the tyrosine residue(s) which contributes to the spectral changes: only Y42 or both Y42 and Y118. Three tyrosines of Y76, Y94, and Y98 are on the protein surface and are not expected to contribute to spectral changes.24 To elucidate the picosecond structural dynamics, timeresolved UVRR spectra of PYP were measured (Figure 3a). Figure 3b shows the temporal intensity change of the Y8a band. It was found that the intensity of the Y8a band decreased instantaneously (within the instrument response time), partially recovered with the single-exponential kinetics with a time constant of 8 ps, and maintained a constant negative value up to 1 ns. Time-resolved absorption spectroscopy revealed that the primary isomerization event in PYP is finished in a few picoseconds, proceeding from the electronic excited state, called pG*, to the early red-shifted states with similar absorption properties, named I0 and I0q. After a few nanoseconds, I0q thermally relaxes to the pR intermediate (also referred to as I1 or PYPL), which finally converts on a submillisecond time scale to a signaling state of PYP called pB (also referred to as I2 or PYPM).25 The observed spectral change up to 1 ns is attributed to the structural change of the tyrosine residue in the pG*, I0,
Letters
Figure 3. (a) Picosecond time-resolved UVRR difference spectra of PYP (pump 446 nm, probe 236 nm). (b) Temporal intensity change of the Y8a band relative to the intensity in the probe-only spectrum. The cross-correlation time between pump and probe pulses is 2.8 ps.
I0q, and pR states of the chromophore. On the basis of the PYP photocycle, the initial process showing the intensity loss of the tyrosine bands corresponds to the structural change of Y42 due to the excitation of the chromophore to the pG* state. The subsequent partial intensity recovery in Figure 3 results from the formation of the I0 state. However, the time constant is longer than the I0-formation time from the pG* state reported by the absorption study.25 This is reasonably explained if we postulate the recovery is related to the structural change of Y42 upon that of the chromophore; namely, the protein backbone including the moiety around Y42 changes slowly responding to the chromophore isomerization. In this temporal region, another intermediate of the chromophore called the “groundstate intermediate” was reported. This intermediate is necessary to explain the complex kinetics of the observed time-resolved signals and directly recovers to the dark state.26 In the present data for tyrosine dynamics, however, the possibility of the contribution of this intermediate might be small, because the recovery process showed single-exponential kinetics and the bleach signal was still observed after the partial recovery was completed (>10 ps). In the I0, I0q, and pR states, little structural change of Y42 gives rise to the constant intensity of the Y8a band. Consequently, the present study showed that the hydrogenbond strength between Y42 and the chromophore becomes strong in the pG* state and then weakens in the I0, I0q, and pR states but is still stronger than in the dark state. The spectral pattern of the UVRR difference spectrum due to the pB intermediate is distinct from that observed in the present study.6 This is because the structure around the chromophore in the pB state is considerably different from that in the I0, I0q, and pR states due to the chromophore protonation.2
J. Phys. Chem. B, Vol. 111, No. 23, 2007 6295 Stark spectroscopy demonstrated that the photoexcitation induces the negative charge transfer and the following charge redistribution on the chromophore, resulting in the formation of a carbonyl-like oxygen on the phenolate, which strengthens the hydrogen bond.27 This is consistent with our interpretation of the present UVRR data. On the other hand, ultrafast infrared spectroscopy showed the picosecond spectral dynamics of the carbonyl band of the E46, and revealed that the hydrogen bond between the chromophore and E46 is weakened in the pG* state and recovers in the I0, I0q, and pR states.4 To account for changes in the hydrogen-bond strength in the pG* state observed in both the time-resolved infrared4 and present UVRR studies, we need to assume changes in the distance between the phenolate oxygen on the chromophore and hydroxy hydrogen atoms on both Y42 and E46. Motion of the chromophore in the region between Y42 and E46 would cause the subsequent structural changes around the chromophore, such as the proton transfer from E46 to the chromophore2 from the pR-state structure. It is surprising that the structural change in W119 was observed at 3 ps, although the distance between the chromophore and W119 is 12 Å.23 This implies that the photoinduced structural change in the chromophore gives rise to the structural change of the relatively distant region around W119 via changes in the protein structure (e.g., the central β-sheets) within picoseconds. Such an ultrafast propagation of structural changes has been observed in PYP for the first time. As mentioned above, the intensity loss of the tryptophan UVRR bands originates from a reduction in hydrophobicity in the vicinity of the tryptophan residue.11,18 In addition, there is a possibility that the band intensity is affected by the change of the orientation of the unit including W119 in accordance with the structural change in picoseconds. To conclude, the protein dynamics of PYP in the early picoseconds region were elucidated by time-resolved UVRR spectroscopy. Ultrafast spectral changes on both the tyrosine and tryptophan bands were selectively observed for the first time. Spectral changes in bands of the tyrosine residue revealed that the hydrogen bond between Y42 and the chromophore becomes strong in the pG* state. An observed loss in intensity of the tryptophan bands indicated that the structural change had propagated to W119, 12 Å distant from the chromophore, within picoseconds. Acknowledgment. The authors thank Mr. Kazuhiro Kobayashi of Nagoya University for helpful advice in constructing the sample flow system. M.M. is indebted to Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists. This work was supported by a Grant-in-Aid for Scientific Research (B) (Grant No. 17350009) from the Japan Society for the Promotion of Science to Y.M. References and Notes (1) Cusanovich, M. A.; Meyer, T. E. Biochemistry 2003, 42, 4759. (2) Xie, A.; Kelemen, L.; Hendriks, J.; White, B. J.; Hellingwerf, K. J.; Hoff, W. D. Biochemistry 2001, 40, 1510. (3) Heyne, K.; Mohammed, O. F.; Usman, A.; Dreyer, J.; Nibbering, E. T. J.; Cusanovich, M. A. J. Am. Chem. Soc. 2005, 127, 18100. (4) Groot, M. L.; van Wilderen, L. J. G. W.; Larsen, D. S.; van der Horst, M. A.; van Stokkum, I. H. M.; Hellingwerf, K. J.; van Grondelle, R. Biochemistry 2003, 42, 10054. (5) Unno, M.; Kumauchi, M.; Sasaki, J.; Tokunaga, F.; Yamauchi, S. J. Phys. Chem. B 2003, 107, 2837. (6) El-Mashtoly, S. F.; Yamauchi, S.; Kumauchi, M.; Hamada, N.; Tokunaga, F.; Unno, M. J. Phys. Chem. B 2005, 109, 23666. (7) Pan, D.; Philip, A.; Hoff, W. D.; Mathies, R. A. Biophys. J. 2004, 86, 2374. (8) Zhou, Y.; Ujj, L.; Meyer, T. E.; Cusanovich, M. A.; Atkinson, G. H. J. Phys. Chem. A 2001, 105, 5719.
6296 J. Phys. Chem. B, Vol. 111, No. 23, 2007 (9) Brudler, R.; Rammelsberg, R.; Woo, T. T.; Getzoff, E. D.; Gerwert, K. Nat. Struct. Biol. 2001, 8, 265. (10) Unno, M.; Kumauchi, M.; Hamada, N.; Tokunaga, F.; Yamauchi, S. J. Biol. Chem. 2004, 279, 23855. (11) Kitagawa, T.; Hirota, S. Raman Spectroscopy of Proteins. In Handbook of Vibrational Spectroscopy; Chalmers, J. M., Griffiths, P. R., Eds.; John Wiley & Sons: Chichester, U.K., 2002; Vol. 5, p 3426. (12) Sato, A.; Mizutani, Y. Biochemistry 2005, 44, 14709. (13) Tauber, M. J.; Mathies, R. A.; Chen, X.; Bradforth, S. E. ReV. Sci. Instrum. 2003, 74, 4958. (14) Harada, I.; Takeuchi, H. Raman and Ultraviolet Resonance Raman Spectra of Proteins and Related Compounds. In Spectroscopy of Biological Systems; Clark, R. J. H., Hester, R. E., Eds.; John Wiley & Sons: Chichester, U.K., 1986; p 113. (15) Ludwig, M.; Asher, S. A. J. Am. Chem. Soc. 1988, 110, 1005. (16) Sweeney, J. A.; Asher, S. A. J. Phys. Chem. 1990, 94, 4784. (17) Haruta, N.; Aki, M.; Ozaki, S.; Watanabe, Y.; Kitagawa, T. Biochemistry 2001, 40, 6956. (18) Matsuno, M.; Takeuchi, H. Bull. Chem. Soc. Jpn. 1998, 71, 851. (19) Chi, Z.; Asher, S. A. J. Phys. Chem. B 1998, 102, 9595.
Letters (20) Rodgers, K. R.; Su, C.; Subramaniam, S.; Spiro, T. G. J. Am. Chem. Soc. 1992, 114, 3697. (21) Takeuchi, H.; Watanabe, N.; Satoh, Y.; Harada, I. J. Raman Spectrosc. 1989, 20, 233. (22) Kim, J. E.; Pan, D.; Mathies, R. A. Biochemistry 2003, 42, 5169. (23) Baca, M.; Borgstahl, G. E. O.; Boissinot, M.; Burke, P. M.; Williams, D. R.; Slater, K. A.; Getzoff, E. D. Biochemistry 1994, 33, 14369. (24) Althogh Y98 is connected to the chromophore region via T50 and R52 in the crystal structure, the phenolic OH group is exposed to the solvent (PDB ID: 2PHY, from ref 23), and is expected to be hydrogen-bonded to water molecules. Even if the protein structure changes, the environment around the phoenolic OH group of Y98 would not change. (25) Ujj, L.; Devanathan, S.; Meyer, T. E.; Cusanovich, M. A.; Tollin, G.; Atkinson, G. H. Biophys. J. 1998, 75, 406. (26) Larsen, D. S.; van Stokkum, I. H. M.; Vengris, M.; van der Horst, M. A.; de Weerd, F. L.; Hellingwerf, K. J.; van Grondelle, R. Biophys. J. 2004, 87, 1858. (27) Premvardhan, L. L.; van der Horst, M. A.; Hellingwerf, K. J.; van Grondelle, R. Biophys. J. 2003, 84, 3226.
