Letter pubs.acs.org/JPCL
Ultrafast Structural Evolution of Photoactive Yellow Protein Chromophore Revealed by Ultraviolet Resonance Femtosecond Stimulated Raman Spectroscopy Hikaru Kuramochi,†,‡ Satoshi Takeuchi,† and Tahei Tahara*,† †
Molecular Spectroscopy Laboratory, RIKEN, 2-1 Hirosawa, Wako 351-0198, Japan Department of Chemistry and Materials Science, Tokyo Institute of Technology, 2-12-1 Ohokayama, Meguro-ku, Tokyo 152-8551, Japan
‡
S Supporting Information *
ABSTRACT: We studied ultrafast structural dynamics of the chromophore of photoactive yellow protein, trans-p-coumaric acid (pCA), using newly developed ultraviolet resonance femtosecond stimulated Raman spectroscopy (UV-FSRS). The UV-FSRS data of the anionic form (pCA−) in a buffer solution showed clear spectral changes within 1 ps, followed by a spectrally uniform decay with a time constant of 2.4 ps. The observed spectral change indicates that the structural change occurs in excited pCA− from the Franck−Condon state to the S1 potential minimum in the femtosecond time region. The S1 Raman spectra exhibit spectral patterns that are similar to the ground-state spectrum, suggesting that pCA− yet retains a planar-trans conformation throughout the S1 lifetime. We concluded that S1 pCA− undergoes a femtosecond in-plane deformation, rather than a substantial CetCet twist. With these femtosecond vibrational data, we discuss possible roles of the initial structural evolution of pCA in triggering the photoreceptive function when embedded in the protein. SECTION: Spectroscopy, Photochemistry, and Excited States first intermediate of the photocycle is formed with a time constant of ∼3 ps by femtosecond transient absorption in the visible and infrared regions, and it was assigned to the first ground-state intermediate having cis configuration, the socalled I0 state.9−11 However, it has not been clarified how the chromophore structure evolves in the photoexcited state and how it leads to the ground-state cis intermediate. There is a certain missing link between the initially prepared Franck− Condon (FC) state in the excited state and the experimentally observed I0 state in the ground state. Obviously, the information about the excited-state structural change is indispensable to fully understand how the pigment systems convert light stimuli into a structural signal, which eventually provokes a biological response. In fact, this is a key to elucidate how a high signaling efficiency is achieved in the organism. To clarify the initial events in PYP at the molecular level, the elucidation of the excited-state dynamics of the chromophore itself, pCA, is indispensable. Therefore, a large number of experimental and theoretical studies have been performed for pCA and analogues in solution to obtain detailed information about the primary process of the photocycle.12−14 Precedent femtosecond transient absorption and fluorescence upconversion studies on pCA in aqueous buffer solutions showed that
P
hotoactive yellow protein (PYP), discovered in Halorhodospira halophila, is a relatively small cytosolic protein (14 kDa; 125 amino acids) and is widely considered to function as a blue-light photoreceptor for the negative phototaxis of this organism.1 The function of this protein is realized with a photocycle, which is driven by photoinduced trans-to-cis isomerization of the embedded chromophore, trans-p-coumaric acid (pCA). This subtle structural change of the chromophore subsequently induces a global change of the protein that triggers the biological response. A number of intermediates (I0, I0‡, pR, and pB) appear over a wide time scale from femtoseconds to milliseconds in the photocycle.1 Since the discovery of PYP,2 a great deal of effort has been made to elucidate the photocycle and to characterize intermediates, using various experimental techniques such as FT-IR,3,4 resonance Raman,5,6 X-ray crystallography,7 and solution NMR.8 These studies provided detailed insights into the structure of each intermediate, that is, the conformation of the chromophore, the hydrogen bonding between the chromophore and surrounding amino acid residues, and so forth. However, these experimental approaches can provide information about the events that take place on time scales longer than ∼100 ps. Therefore, in contrast to the wellcharacterized slow dynamics, the primary processes of PYP in the femto- to picosecond region have been still veiled largely. In particular, the most intriguing initial trans-to-cis isomerization process still remains a task to elucidate. It was reported that the © 2012 American Chemical Society
Received: May 1, 2012 Accepted: June 21, 2012 Published: June 22, 2012 2025
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difference is recognized in the time-resolved spectra within 1 ps, presumably because of the improved time resolution of the present experiment. A weak transient absorption signal remains in the UV region even after 10 ps, and it could be due to the anion radical generated by two-photon excitation.30
the excited-state dynamics of the chromophore is similar to that in PYP.13−16 Its isomerization yield (0.47)17 was also comparable to that of PYP.18,19 These results ensure that the study of free trans-pCA in solution can provide relevant information about the dynamics of the chromophore in PYP. However, the nature of the observed subpicosecond dynamics and structural changes of photoexcited pCA in solution are not yet clarified, as the chromophore in PYP itself. Therefore, it is highly desirable to obtain structural information about the free trans-pCA in the initial stage after photoexcitation. In the past decade, femtosecond stimulated Raman spectroscopy (FSRS) has emerged as a powerful method to study structural changes that proceed on the femtosecond time scale.20−22 Taking advantage of resonance enhancement, FSRS enables us to selectively obtain vibrational spectra of specific transients in a wide frequency range, which is not possible in femtosecond infrared experiments. The use of the stimulated Raman process with femtosecond excitation enables us to track the change of the vibrational spectra with a femtosecond accuracy of the delay time, keeping the frequency resolution high (∼15 cm−1). These advantages of FSRS have made it possible to obtain crucial information about the structural change of molecules in the femtosecond time region.23−25 In fact, tracking of the structural evolution of molecules is very important to deepen our understanding about reactions, including new insight into transition states.26−28 In this Letter, we report on an ulraviolet resonance FSRS (UV-FSRSa) study of the free chromophore in anionic form, pCA−, in aqueous buffer solution. Because pCA− exhibits excited-state absorption only in the UV region,14 the UV resonance is essential to obtain vibrational spectra of the excited state. The obtained UV-FSRS spectra clearly show a large spectral change in the femtosecond time region, indicating ultrafast structural evolution of the pCA− chromophore in the excited state.
Figure 1. Spectral data of pCA− in phosphate buffer solution (pH = 7). (A) Steady-state absorption and emission spectra. (B) Femtosecond time-resolved absorption spectra measured at selected delays with 315 nm excitation. The Raman pump wavelength used in the UVFSRS experiment (λRp = 375 nm) is indicated by a dotted line. (C) Temporal behavior of the integrated intensity of the transient absorption signal between 325 and 550 nm. The best biexponential fit is also shown (black solid line).
To analyze the temporal behaviors of transients more quantitatively, we integrated the transient absorption intensity 550 over the entire spectral range (∫ 325 (ΔOD)/(λ) dλ) and plotted the integrated intensity as a function of the delay time in Figure 1C. The temporal trace exhibits a biexponential decay having time constants of 450 fs and 2.5 ps. The 450 fs component corresponds to the initial dynamics associated with the spectral shift, whereas the 2.5 ps decay represents the population decay of the S1(ππ*) state. Although the experiment is carried out in a polar aqueous solution, the 450 fs component cannot be attributed to the solvent response (solvation dynamics) because the band integrated intensity is, in principle, insensitive to the solvation process. (The band integrated intensity is proportional to the square of the transition dipole moment and the population of the relevant electronic state.31) Therefore, this result manifests that ultrafast processes other than the solvation process occur within 1 ps in the excited pCA− molecule, regardless of whether or not the solvation process takes place on the same time scale. In order to examine the initial relaxation process from the structural viewpoint, we measured femtosecond time-resolved Raman spectra by UV-FSRS. The Raman pump pulse was set at 375 nm (the peak wavelength of the ESA1 band) to gain resonance enhancement for the excited state. The UV-FSRS spectra of pCA− observed over the decay time range of the excited-state absorption are shown in Figure 2, in which the ground-state Raman spectrum is also shown for comparison. The ground-state spectrum exhibits the CetCet stretch band at 1636 cm−1, which can be a marker for the isomerization
Scheme 1. Structural Change in trans-to-cis Photoisomerization of the p-Coumaric Acid Anion (pCA−) in Aqueous Buffer Solution at pH = 7
In a phosphate buffer solution at pH = 7, pCA exists as the anionic form (pCA−) in which the carboxylic hydrogen is deprotonated (Scheme 1).13 This anionic form shows the lowest-energy absorption band in the 250−330 nm region (Figure 1A). Figure 1B shows femtosecond time-resolved absorption spectra of pCA− measured at selected time delays after excitation at 315 nm. As seen in this figure, an excitedstate absorption band with a peak maximum at 375 nm (ESA1) is observed immediately after the photoexcitation. This transient rapidly blue shifts within 1 ps, forming an excitedstate absorption band peaked at 355 nm (ESA2). A stimulated emission band also appears at 430 nm (SE). These ESA2 and SE bands decay together with a time constant of 2.5 ps while showing a clear isosbestic point at 392 nm. The simultaneous decay of ESA2 and SE allows us to assign this 2.5 ps kinetics to the population decay from the S1(ππ*) state that emits fluorescence. The time scale of the observed dynamics is in good agreement with a previous report,14 although a minor 2026
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process. The CphCph stretching bands (1606 and 1589 cm−1, poorly resolved) and a CO stretching band (1536 cm−1) also appear in the CC stretch region, forming a set of congested high-frequency bands.32 The UV-FSRS spectra are characterized by the immediate appearance of transient bands in the 700−1750 cm−1 region upon photoexcitation. Furthermore, a significant spectral change is observed in the first 1 ps, as shown in Figure 2. The bands observed initially at 812 and 1573 cm−1 exhibit large frequency upshifts by ∼20 cm−1, and remarkable intensity growths are observed for the 1150 and 1475 cm−1 bands. Actually, the 1150 cm−1 band gradually appears with the disappearance of the 1180 cm−1 band that is clearly recognized in the 50 fs spectrum. (Probably, these changes in the spectral feature are more easily seen in the contour representation shown at the bottom in Figure 2.) These prominent spectral changes in 1 ps unequivocally indicate that the structure of excited-state pCA− changes on the femtosecond time scale after photoexcitation. From 1 ps onward, all of the transient bands decay synchronously with a time constant of ∼3 ps. This time constant agrees well with the lifetime of the relaxed S1(ππ*) state, 2.5 ps, which was determined by the transient absorption measurement. Therefore, we can safely assign the UV-FSRS spectra observed after 1 ps to the relaxed S1(ππ*) state.
in Figure 3. The two time constants were also determined in this analysis as τ1 = ∼0.3 ps and τ2 = 2.4 ps. These two time constants agree well with the values obtained in the timeresolved absorption measurements (450 fs and 2.5 ps), within the experimental uncertainty.
Figure 3. Results of the SVD analysis of the UV-FSRS data. (A) Reconstructed spectra of the two states having lifetimes of ∼0.3 and 2.4 ps. (B) Temporal profiles of the population of the two states with the best global fits (dotted curves).
The success of the spectral decomposition supports our interpretation that the optically generated FC state is converted to the relaxed S1 state in several hundreds of femtoseconds. Moreover, this analysis indicates that the Raman spectra of the FC and relaxed S1 states are distinctly different, as shown in Figure 3A. This demonstrates that excited-state pCA− undergoes substantial structural change in the femtosecond time region. Comparing the spectra in Figure 2, we can notice that the overall spectral pattern of the UV-FSRS spectra at the late time delays, which correspond to the relaxed S1(ππ*) state, has a high similarity to the spectral pattern of the S0 Raman. In fact, the transient bands at around 820 and 970 cm−1 have clear counterparts in the S0 Raman spectrum at 812 (with a shoulder at 856 cm−1) and 978 cm−1, respectively. The transient bands in the 1100−1200 cm−1 region look to correspond to the 1174 cm−1 band in the S0 Raman. Similar correspondence between UV-FSRS and S0 Raman spectra is also seen in the highestfrequency region (green shaded area in Figure 2). The similarity in the high-frequency region, in particular, strongly suggests that the CetCet stretch (1636 cm−1) and CphCph stretch (1606, 1589 cm−1) bands in the S0 Raman are shifted into a bunch of transient bands at around 1550 cm−1 in the UVFSRS spectra. This means that the key CetCet stretching vibration of the S1 pCA− has a quite high frequency, implying that the CetCet bond of pCA− still possesses a double-bond character in the S1 state. On the basis of this CetCet frequency as well as the overall spectral similarity between the UV-FSRS and the ground-state spectra, we conclude that the molecular structure of pCA− yet retains a planar-trans conformation at the relaxed S1(ππ*) state, as in the ground state. It should be noted that the strongest transient Raman signal that appears at around 1255 cm−1 exhibits a rather dispersive band shape at the early
Figure 2. UV-FSRS spectra of pCA− in phosphate buffer solution (pH = 7) obtained at various delay times. Actinic pump and Raman pump wavelengths were tuned to 300 and 375 nm, respectively. The groundstate Raman spectrum is also shown for comparison at the top. The contour representation of the UV-FSRS data (after smoothing) is given at the bottom.
To present the spectral evolution in a more intuitive manner, we performed the singular value decomposition (SVD) analysis of the UV-FSRS data.33 The SVD gave two predominant principal values, so that the two components were taken into consideration in the subsequent spectral reconstruction, which we assigned to the FC state and relaxed S1 states.35 Assuming a τ1 τ2 simple cascaded relaxation scheme (FC → relaxed S1 → S0 ), we obtained the reconstructed spectra of the FC and relaxed S1 states, as well as temporal profiles of their population, as shown 2027
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intensively studied as one of the key factors determining the subsequent protein dynamics.39,40 Finally, we note that the pCA chromophore, when embedded in the protein pocket, exhibits excited-state absorption exclusively in the UV region, as in solution.14,19 The amino acid residues surrounding the chromophore also show specific absorption bands in the UV region. Therefore, UV-FSRS with a tunable Raman pump source can be a powerful method to investigate femtosecond structural dynamics of the protein as well as the chromophore− protein interactions in the initial stage of the photoreception.
delay time. As shown in the Supporting Information, this band shape significantly changes depending on the Raman pump wavelength, so that we consider that this feature likely arises from a third-order process using S0 ← S1 resonance.34 So far, the excited-state dynamics of the free pCA in the neutral, anionic, and dianionic forms has been investigated by femtosecond transient absorption.14 The experiment has revealed the femtosecond dynamics that corresponds to the 450 fs component observed in the present time-resolved absorption measurement, although the assignment of the observed dynamics remained ambiguous. In fact, Vengris et al. discussed the possibility of the vibrational cooling and internal conversion process between closely lying excited states but argued that neither of these possibilities could reasonably account for the experimental observations.14 The present UVFSRS measurements clearly showed that the Raman spectrum of excited-state pCA− changes drastically on the femtosecond time scale. It strongly suggests that the femtosecond dynamics corresponds to the ultrafast structural evolution from the initially prepared FC state to the relaxed S1(ππ*) state. Because the UV-FSRS spectral pattern indicates that the planar-trans conformation is retained in the S1 state throughout its lifetime, it is highly likely that the structural change in the femtosecond region is a rapid in-plane deformation rather than a substantial twist about the CetCet bond. It is worth mentioning that this conclusion is consistent with the result of recent ab initio calculations on a PYP model chromophore.35 The calculation suggested that the photoexcited FC molecule undergoes a structural relaxation within the planar geometry and reaches the S1(ππ*) local potential minimum, where the Cph−CetCet angle is smaller than that at the FC state. It was also suggested that the Cph−Cet and CetCet torsional motions play an important role in the subsequent relaxation from the S1 state to the ground state.35,36 QM//MM simulations on PYP by Groenhof et al. provided similar findings as well.37 These theoretical studies concluded that the Cph−Cet torsion leads the S1 molecule back to the S0 state in the original trans configuration, while the CetCet torsion gives the cis product. Furthermore, it was proposed that the CetCet torsion is more favored than the Cph−Cet torsion in the protein (PYP) due to the restriction of the environment.37 All of these results indicate that the shape of the multidimensional S1 potential surface, especially along the two torsional coordinates, is an essential factor for efficient entry into the photocycle when pCA is embedded in the protein. The present UV-FSRS study provides the first vibrational information about the early time photodynamics of the PYP chromophore. It was indicated that the initial structural evolution in excited-state pCA− is not directly correlated to the CetCet torsional motion. Instead, the data suggested that the rapid in-plane Cph−CetCet deformation takes place upon photoexcitation and that pCA− still retains the planar-trans conformation all through the S1(ππ*) lifetime. Although the protonation site of pCA− is different from that in the pG state of PYP, the findings obtained in the present study can provide new insights into the understanding of the structural dynamics in PYP. A possible occurrence of the similar in-plane deformation in the protein as well as its role in the primary events of PYP, such as the efficient isomerization and successful entry into the photocycle, is an intriguing subject.38 Moreover, the in-plane deformation is highly expected to perturb the hydrogen bond network among pCA and surrounding amino acid residues (Tyr42, Glu46, and Thr50), which was recently
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ASSOCIATED CONTENT
* Supporting Information S
Description of the sample materials, experimental setups, and supplementary transient absorption and UV-FSRS data. 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 This work was supported by a Grant-in-Aid for Scientific Research on Priority Area “Molecular Science for Supra Functional Systems” (No. 19056009) from MEXT and Grant-in-Aid for Scientific Research (A)(No. 22245005) from JSPS.
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REFERENCES
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