- Email: [email protected]
Broadband stimulated Raman spectroscopy in the deep ultraviolet region
Chemical Physics Letters xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Chemical Physics Letters journal homepage: www.elsevier.com/locate/cplett
Research paper
Broadband stimulated Raman spectroscopy in the deep ultraviolet region Hikaru Kuramochi a,b,1, Tomotsumi Fujisawa a,1,2, Satoshi Takeuchi a,b, Tahei Tahara a,b,⇑ a b
Molecular Spectroscopy Laboratory, RIKEN, 2-1 Hirosawa, Wako 351-0198, Japan Ultrafast Spectroscopy Research Team, RIKEN Center for Advanced Photonics (RAP), 2-1 Hirosawa, Wako 351-0198, Japan
a r t i c l e
i n f o
Article history: Received 29 December 2016 In final form 7 February 2017 Available online xxxx Keywords: Stimulated Raman spectroscopy Deep ultraviolet Amino acid Ultrafast spectroscopy
a b s t r a c t We report broadband stimulated Raman measurements in the deep ultraviolet (DUV) region, which enables selective probing of the aromatic amino acid residues inside proteins through the resonance enhancement. We combine the narrowband DUV Raman pump pulse (<10 cm 1) at wavelengths as short as 240 nm and the broadband DUV probe pulse (>1000 cm 1) to realize stimulated Raman measurements covering a >1500 cm 1 spectral window. The stimulated Raman measurements for neat solvents, tryptophan, tyrosine, and glucose oxidase are performed using 240- and 290-nm Raman pump, highlighting the high potential of the DUV stimulated Raman probe for femtosecond time-resolved study of proteins. Ó 2017 Published by Elsevier B.V.
The function, activity and response of living organisms are closely related to the structural change of relevant proteins which takes place over a wide range of time scales. Therefore, it is of significant importance to track the structural change of proteins for elucidating the molecular mechanism behind the activation of biological functions. In photoreceptor proteins, the structural change of the protein is triggered by photoabsorption that induces a localized change in the embedded chromophore. This subtle, initial change brings about a chain of chemical reactions spanning a broad time range, which ultimately leads to the activation of a biological function [1–3]. Because the functional activation is synchronously triggered with light stimuli, photoreceptor proteins have been intensively studied with various time-resolved spectroscopic [4,5] and diffraction techniques [6–9] as the best system to investigate the structure–function relationship in proteins. Time-resolved spectroscopy has provided detailed insights into the primary response of various photoreceptor proteins with excellent temporal and structural information. In particular, femtosecond Raman spectroscopies have mapped out the primary structural events that occur on the femtosecond time scale for various photo-responsive proteins, such as rhodopsin [10], green fluorescent protein [11,12], phytochrome [13], and photoactive
⇑ Corresponding author at: Molecular Spectroscopy Laboratory, RIKEN, 2-1 Hirosawa, Wako 351-0198, Japan. E-mail address: [email protected] (T. Tahara). 1 H. K. and T. F. equally contributed to this work. 2 Present address: Department of Chemistry and Applied Chemistry, Graduate School of Science and Engineering, Saga University, Saga 840-8502, Japan.
yellow protein [14,15]. These studies successfully captured the primary structural dynamics of the chromophore in the proteins, which is actually the key initial process that triggers the activation of the biological function. On the other hand, information about the primary response of the surrounding amino acid residues has been rather limited. Because such a secondary response serves as the intermediary to the subsequent chain of chemical/biological reactions, it is very important to elucidate how the initial change in the chromophore propagates to the surrounding amino acid residues. It is one of the most important challenges to be tackled in protein science today, which will provide deep insights into the structural/functional cooperativity in proteins. Time-resolved ultraviolet (UV) resonance Raman spectroscopy has played an indispensable role in elucidating the structural dynamics in proteins on the picosecond and later time scales [16–18]. In this technique, after initiating the photoreaction by exciting the chromophore, the change in the vibrational spectrum of the surrounding aromatic amino acid residues is selectively monitored by UV resonance Raman probing at around 220 nm, by taking advantage of the resonance enhancement [19]. However, this spontaneous Raman approach is inherently not suitable for studying femtosecond structural dynamics because the time resolution is practically limited up to several picoseconds in exchange for obtaining frequency resolution as high as 10 cm 1. To overcome this limit, the timing of the Raman probing has to be determined with femtosecond accuracy with respect to the actinic excitation, which is not possible in the picosecond spontaneous Raman approach that utilizes only picosecond probe pulses. Actually, this has been realized with stimulated Raman techniques
http://dx.doi.org/10.1016/j.cplett.2017.02.015 0009-2614/Ó 2017 Published by Elsevier B.V.
Please cite this article in press as: H. Kuramochi et al., Chem. Phys. Lett. (2017), http://dx.doi.org/10.1016/j.cplett.2017.02.015
2
H. Kuramochi et al. / Chemical Physics Letters xxx (2017) xxx–xxx
in the frequency and time domains, i.e. femtosecond stimulated Raman spectroscopy (FSRS) [20–22] and time-resolved impulsive stimulated Raman spectroscopy (TR-ISRS) [23–25], respectively, which utilize femtosecond optical pulses. In FSRS, for example, the stimulated Raman process is induced by narrow-band picosecond Raman pump and broadband femtosecond probe pulses, which can define the timing to initiate the Raman transition with femtosecond temporal accuracy. Therefore, combined with the femtosecond actinic pump pulse, it enables tracking the change in the Raman spectrum on the femtosecond time scale. The advantage of FSRS is that it is relatively easy to perform experiments in various wavelength regions from UV (330–390 nm) [26–28] to near-IR (1190 nm) [29,30], although the inevitable background subtraction and inherent complex band shapes sometimes prevent straightforward interpretation. Further extending the spectral window of the FSRS measurements to the deeper UV region would allow us to track femtosecond dynamics of amino acid residues inside photoreceptor proteins. In this letter, we demonstrate broadband stimulated Raman spectroscopy in the deep UV region (DUV-SRS), which can be a technical basis of FSRS experiments for studying ultrafast structural dynamics of the aromatic amino acid residues in proteins. We realize stimulated Raman probing in the DUV region by introducing the Raman pump and probe pulses at wavelengths as short as 240 nm, thereby enabling selective probing of the aromatic amino acid residues in proteins, such as tryptophan and tyrosine. The steady-state stimulated Raman measurements are demonstrated for neat solvents, tryptophan, tyrosine, as well as glucose oxidase, indicating the high potential and feasibility of DUV-FSRS experiments for time-resolved studies of the aromatic amino acid residues inside photoreceptor proteins. A schematic diagram of the DUV-SRS setup is shown in Fig. 1. We used a Ti:Sapphire regenerative amplifier system (Legend Elite Duo, Coherent, 800 nm, 80 fs, 8 mJ, 1 kHz) as the light source. About 3.5 mJ of the total amplifier output was used for the DUVSRS experiments, which was further split into two portions to generate the Raman pump pulse and broadband probe pulse. The major portion (3 mJ) was first converted to a narrow-bandwidth picosecond pulse at 400 nm (<10 cm 1) with 1-mJ energy using a second-harmonic bandwidth compressor (SHBC, Light Conversion) [26,28,31–34]. This narrow-bandwidth 400-nm pulse was subsequently used to pump a narrow-bandwidth optical parametric amplifier (NB-OPA, Light Conversion), which was seeded by a white-light continuum. The NB-OPA delivered visible picosecond pulses (480–2400 nm, >150 lJ), and this output was further
Fig. 1. Schematic diagram of the DUV-SRS setup. OPA: optical parametric amplifier; SHBC: second-harmonic bandwidth compressor; NB-OPA: narrow-band optical parametric amplifier; NOPA: noncollinear optical parametric amplifier; HWP: half wave plate; CCD: Charge coupled device.
Fig. 2. Typical spectra of the DUV Raman pump and probe pulses.
frequency-doubled in an 8-mm-thick b-barium borate (BBO, type I, h = 29.2°) crystal to generate narrow-bandwidth DUV Raman pump pulses (<10 cm 1, 20 lJ). The typical spectra of the Raman pump pulses are shown in Fig. 2. The remaining 0.5-mJ portion of the amplifier output was used for generating the broadband DUV pulse that was used as the Raman probe. Initially, we attempted to obtain a broadband DUV probe pulse by white-light continuum generation pumped by the second harmonic of the amplifier output as reported previously [27,28]. Although this approach enabled stimulated Raman measurements in the UV region (>330 nm), we found that the intensity of the DUV spectral component (<300 nm) of the white-light continuum was not high enough to perform stimulated Raman measurements with a reasonable accumulation time, keeping the signal-to-noise ratio high. Therefore, in the present work, we employed a more intense, broadband DUV pulse generated with a noncollinear optical parametric amplifier (NOPA [35–37]) as the Raman probe. The 0.5-mJ portion of the amplifier output drove a home-built single-stage NOPA. In this NOPA, a white-light seed pulse was generated in a 3-mm thick sapphire plate and was amplified in a 1.5-mm-thick BBO (type I, h = 31°) crystal, delivering tunable pulses in the visible range (480–750 nm, <10 lJ). After the rotation of the polarization, the NOPA output was frequency doubled in a 20-lm-thick BBO crystal (type I, h = 36.7°), generating a broadband UV pulse (240–375 nm) which was used as the Raman probe. For frequency doubling, the NOPA output was tightly focused to the BBO crystal (f = 76.2 mm) to broaden the spectrum of the second harmonic generated [38] while the focal position was carefully adjusted and placed slightly in front of the crystal to avoid the damage of the crystal. The typical DUV probe spectra are shown in Fig. 2. The full-width at the half maximum of the probe pulse exceeds 1000 cm 1, which is broad enough to simultaneously record all the stimulated Raman bands from 100 to 2000 cm 1. The generated broadband DUV pulse was spectrally separated from the visible pulse, and the chirp was compensated by a pair of fused silica prisms with the apex-to-apex distance of 25 cm. Chirp characteristic of the probe pulse was evaluated in situ by measuring the optical Kerr-effect (OKE) signal of water [39], as shown in Fig. 3. The gate pulse for the OKE measurement was generated by a femtosecond OPA (TOPAS-C, Light Conversion) which was pumped by the remaining 4-mJ output of the amplifier. This gate pulse is tunable in a wide frequency range (240–2600 nm) with <80-fs pulse duration, and it can be also used as the actinic pump
Please cite this article in press as: H. Kuramochi et al., Chem. Phys. Lett. (2017), http://dx.doi.org/10.1016/j.cplett.2017.02.015
H. Kuramochi et al. / Chemical Physics Letters xxx (2017) xxx–xxx
Fig. 3. (A) Typical spectrum of the Raman probe pulse centered at around 300 nm. (B) Group delay dispersion of the probe pulse shown in (A), which was evaluated by measuring the OKE signal of water.
pulse for femtosecond time-resolved SRS measurements, i.e. FSRS. The estimated group delay difference (GDD) of the typical probe pulse centered at 300 nm was 100 fs over the presented wavelength region, indicating that the dispersion in the DUV region was so large that the residual higher-order dispersion was not completely compensated only by the prism pair. Nevertheless, because this GDD was much smaller than the pulse duration of the Raman pump (3 ps), it did not cause a variation of the effective frequency resolution of individual stimulated Raman bands within the probe spectral range. For time-resolved measurements, the GDD of the probe can be post-corrected [39]. The probe intensity was attenuated by rotating the polarization of the visible NOPA output before the second harmonic generation. For the stimulated Raman measurements, the probe pulse passing through the sample was spectrally analyzed by a spectrograph (iHR320, Horiba) and then detected by a CCD (PIXIS 400F with UV coating, Princeton Instruments) with 3-ms exposure time. The stimulated Raman gain signal, which was induced by the Raman pump (and the probe pulse), was evaluated by mechanically chopping the Raman pump pulse. As the first sample of the DUV-SRS measurements, stimulated Raman spectra of neat solvents (ethanol and cyclohexane) were measured using the 290-nm Raman pump. The Raman pump and probe pulses were focused to a fused silica flow cell with a 1mm optical path length (0.2-mm window thickness), in which the sample solution was circulated. The Raman pump energy of 200 nJ was used, and the polarizations of the Raman pump and probe pulses were set parallel. For obtaining the spectra presented in the following, about 25,600 spectra were averaged, corresponding to the total acquisition time of 5 min. All the measurements were performed on the Stokes side. Fig. 4 shows the acquired stimulated Raman spectra of ethanol and cyclohexane. The spectra reveal all the relevant vibrational bands in a wide frequency range from 100 to 2000 cm 1, and they are in an excellent agreement with the reported spontaneous Raman spectra [40]. The bandwidth of the C-C stretch band of cyclohexane at 801 cm 1 indicates that the effective frequency resolution of the measurement is as good as 8 cm 1. In addition to the neat solvents, we also measured a stimulated Raman spectrum of tryptophan in a buffer solution (pH 7.0). The 290-nm Raman pump is pre-resonant with the two overlapping transitions to the La and Lb excited states of tryptophan (Fig. 5A) [41,42]. The stimulated Raman bands of tryptophan were observed at peak frequencies that are in agreement with those previously observed with the spontaneous resonance Raman
3
Fig. 4. Stimulated Raman spectra of cyclohexane, ethanol, and tryptophan in a TrisHCl buffer solution (2.9 mM, pH 7.0). The Raman pump wavelength was tuned to 290 nm.
Fig. 5. Absorption (A) and stimulated Raman (B) spectra of glucose oxidase (0.2 mM) in a phosphate buffer solution (60 mM, pH 6.0) and aqueous solutions of FAD (1 mM), tryptophan (1 mM) and tyrosine (1 mM). The Raman pump wavelength was tuned at 240 nm.
spectroscopy using the 220-nm Raman pulse [19,43]. In contrast, the intensity pattern is largely different from these previous reports, which is attributable to the difference in the resonance condition. We note that the resonance Raman measurement of tryptophan with 290-nm excitation, which was carried out in this study, is extremely challenging with conventional spontaneous Raman measurements due to the intense fluorescence background.
Please cite this article in press as: H. Kuramochi et al., Chem. Phys. Lett. (2017), http://dx.doi.org/10.1016/j.cplett.2017.02.015
4
H. Kuramochi et al. / Chemical Physics Letters xxx (2017) xxx–xxx
In fact, in the previous reports of resonance Raman spectra of tryptophan, the Raman excitation wavelength was tuned at the blueedge of the absorption bands due to the transitions to the La and Lb excited states, for avoiding fluorescence [44,45]. To the best of our knowledge, the present study is the first report of the Raman spectrum of tryptophan under the pre-resonance condition. To further demonstrate the capability of SRS measurements in a deeper UV region and its applicability to protein systems, we measured stimulated Raman spectra of tyrosine and tryptophan, as well as glucose oxidase and its chromophore, flavine-adeninedinucleotide (FAD), with the 240-nm Raman pump. Fig. 5A shows the absorption spectra of tyrosine, tryptophan, FAD and glucose oxidase in a buffer solution (pH 6.0), representing the resonance condition for the measurements. The 240-nm Raman pump is pre-resonant with the transition to the Bb excited state of tryptophan and to the La excited state of tyrosine [41,42,46]. Glucose oxidase contains 27 tyrosine and 10 tryptophan residues, so that resonantly enhanced Raman signals from all these 37 aromatic amino acids are expected in the stimulated Raman spectrum. FAD also shows an intense absorption band in the probe region, thus, the FAD Raman bands may also appear. Fig. 5B shows the obtained stimulated Raman spectra of tryptophan, tyrosine, FAD and glucose oxidase measured with the 240-nm Raman pump. The Raman spectra of tyrosine and tryptophan are in agreement with those previously observed with spontaneous resonance Raman spectroscopy using the 220-nm excitation pulse [19,43]. The spectrum of glucose oxidase shows prominent Raman bands at 1009, 1173, 1557, and 1619 cm 1. The counterparts of these bands can be found in the stimulated Raman spectrum of tyrosine or tryptophan. For example, the 1557-cm 1 band observed for glucose oxidase can be assigned to the 1557-cm 1 band of tryptophan. The 1619 cm 1 band of glucose oxidase could be originated from the two overlapped contributions, i.e., the tyrosine band at 1621 cm 1 and tryptophan band 1625 cm 1. In contrast, Raman bands attributable to FAD were not clearly observed in the spectrum of glucose oxidase, probably due to the fact that the only one FAD molecule contributes to the spectrum, and its contribution is likely to be much smaller than those of tyrosine and tryptophan. We also note that Raman bands are not clearly observed even for FAD in a solution, suggesting its low resonance Raman enhancement. It is noteworthy that the peak frequencies of tyrosine and tryptophan Raman bands observed in the stimulated Raman spectrum of glucose oxidase are slightly shifted with respect to those in a buffer solution. It is well-known that some of the tyrosine and tryptophan Raman bands are sensitive to their microenvironment such as hydrophobicity, hydrogen-bonding condition or van der Waals interactions with surroundings. Therefore, the observed difference in the peak frequencies between protein and solution may be attributable to the difference in microenvironment. The wavy background feature, most pronounced in the tryptophan spectrum, is likely attributable to the etaloning effect due to the back reflection of the probe from the rear surface of the BBO crystal (for frequency-doubling) or the windows of the flow cell. Such a systematic noise, which cannot be simply averaged out, may be eliminated by a wavelength modulation technique [47,48]. In summary, we realized broadband stimulated Raman measurements in the DUV region for the first time and described the experimental procedure in detail. The DUV Raman probe pulse was generated as the second harmonic of the broadband NOPA output, and it has a sufficient bandwidth and intensity to perform stimulated Raman measurements with a reasonable accumulation time and a sufficient signal-to-noise ratio. We measured the stimulated Raman spectra of the standard solvents, tyrosine, tryptophan and glucose oxidase using the Raman pump pulses at 290 and 240 nm. The present measurements can be readily extended
to time-resolved measurements, i.e., FSRS, by introducing the femtosecond actinic pump pulse. Thus, this work paves a way for tracking the ultrafast structural dynamics of the amino acid residues inside the photoreceptor proteins. DUV-FSRS measurements of the photoreceptor proteins are now underway in our laboratory. Acknowledgement This work was partly supported by JSPS KAKENHI Grant Numbers JP16H04102 to S. T. and JP25104005 to T. T. H. K. and T. F. acknowledge RIKEN Special Postdoctoral Researchers (SPDR) program. References [1] M.A. van der Horst, K.J. Hellingwerf, Acc. Chem. Res. 37 (2003) 13. [2] W.R. Briggs, J.L. Spudich, Handbook of Photosensory Receptors, John Wiley & Sons, 2005. [3] A. Möglich, X. Yang, R.A. Ayers, K. Moffat, Annu. Rev. Plant Biol. 61 (2010) 21. [4] J.T.M. Kennis, M.-L. Groot, Curr. Opin. Struct. Biol. 17 (2007) 623. [5] V. Sundström, Annu. Rev. Phys. Chem. 59 (2008) 53. [6] S. Westenhoff, E. Nazarenko, E. Malmerberg, J. Davidsson, G. Katona, R. Neutze, Acta Crystallogr. Sect. A: Found. Crystallogr. 66 (2010) 207. [7] R. Neutze, K. Moffat, Curr. Opin. Struct. Biol. 22 (2012) 651. [8] K. Moffat, Philos. Trans. Roy. Soc. B: Biol. Sci. 369 (2014) 20130568. [9] R. Neutze, Philos. Trans. Roy. Soc. B: Biol. Sci. 369 (2014) 20130318. [10] P. Kukura, D.W. McCamant, S. Yoon, D.B. Wandschneider, R.A. Mathies, Science 310 (2005) 1006. [11] C. Fang, R.R. Frontiera, R. Tran, R.A. Mathies, Nature 462 (2009) 200. [12] T. Fujisawa, H. Kuramochi, H. Hosoi, S. Takeuchi, T. Tahara, J. Am. Chem. Soc. 138 (2016) 3942. [13] J. Dasgupta, R.R. Frontiera, K.C. Taylor, J.C. Lagarias, R.A. Mathies, Proc. Natl. Acad. Sci. USA 106 (2009) 1784. [14] M. Creelman, M. Kumauchi, W.D. Hoff, R.A. Mathies, J. Phys. Chem. B 118 (2013) 659. [15] H. Kuramochi, S. Takeuchi, K. Yonezawa, H. Kamikubo, M. Kataoka, T. Tahara, Nat. Chem. (in press). http://dx.doi.org/10.1038/NCHEM.2717. [16] J.E. Kim, D. Pan, R.A. Mathies, Biochemistry 42 (2003) 5169. [17] A. Sato, Y. Mizutani, Biochemistry 44 (2005) 14709. [18] A. Sato, Y. Gao, T. Kitagawa, Y. Mizutani, Proc. Natl. Acad. Sci. USA 104 (2007) 9627. [19] R.P. Rava, T.G. Spiro, J. Am. Chem. Soc. 106 (1984) 4062. [20] M. Yoshizawa, M. Kurosawa, Phys. Rev. A 61 (1999) 013808. [21] D.W. McCamant, P. Kukura, S. Yoon, R.A. Mathies, Rev. Sci. Instrum. 75 (2004) 4971. [22] P. Kukura, D.W. McCamant, R.A. Mathies, Annu. Rev. Phys. Chem. 58 (2007) 461. [23] S. Fujiyoshi, S. Takeuchi, T. Tahara, J. Phys. Chem. A 107 (2003) 494. [24] S. Takeuchi, S. Ruhman, T. Tsuneda, M. Chiba, T. Taketsugu, T. Tahara, Science 322 (2008) 1073. [25] H. Kuramochi, S. Takeuchi, T. Tahara, Rev. Sci. Instrum. 87 (2016) 043107. [26] S. Laimgruber, H. Schachenmayr, B. Schmidt, W. Zinth, P. Gilch, Appl. Phys. B 85 (2006) 557. [27] J.M. Rhinehart, J.R. Challa, D.W. McCamant, J. Phys. Chem. B (2012). [28] H. Kuramochi, S. Takeuchi, T. Tahara, J. Phys. Chem. Lett. 3 (2012) 2025. [29] T. Takaya, K. Iwata, J. Phys Chem. A 118 (2014) 4071. [30] T. Takaya, K. Iwata, Analyst 141 (2016) 4283. [31] F. Raoult, A.C.L. Boscheron, D. Husson, C. Sauteret, A. Modena, V. Malka, F. Dorchies, A. Migus, Opt. Lett. 23 (1998) 1117. [32] S.A. Kovalenko, A.L. Dobryakov, N.P. Ernsting, Rev. Sci. Instrum. 82 (2011) 063102. [33] M. Nejbauer, C. Radzewicz, Opt. Express 20 (2012) 2136. [34] L. Zhu, W. Liu, C. Fang, Appl. Phys. Lett. 105 (2014) 041106. [35] T. Wilhelm, J. Piel, E. Riedle, Opt. Lett. 22 (1997) 1494. [36] G. Cerullo, M. Nisoli, S. Stagira, S. De Silvestri, Opt. Lett. 23 (1998) 1283. [37] A. Shirakawa, T. Kobayashi, Appl. Phys. Lett. 72 (1998) 147. [38] S.H. Ashworth, M. Joschko, M. Woerner, E. Riedle, T. Elsaesser, Opt. Lett. 20 (1995) 2120. [39] S. Yamaguchi, H. Hamaguchi, Appl. Spectrosc. 49 (1995) 1513. [40] B. Schrader, W. Meier, Raman/Infrared Atlas of Organic Compounds, VCH Weinheim, 1989. [41] I. Harada, H. Takeuchi, Adv. Infrared Raman Spectrosc. 13 (1986) 113. [42] J.A. Sweeney, S.A. Asher, J. Phys Chem. 94 (1990) 4784. [43] M. Mizuno, N. Hamada, F. Tokunaga, Y. Mizutani, J. Phys. Chem. B 111 (2007) 6293. [44] R.P. Rava, T.G. Spiro, J. Phys. Chem. 89 (1985) 1856. [45] S.A. Asher, M. Ludwig, C.R. Johnson, J. Am. Chem. Soc. 108 (1986) 3186. [46] M. Ludwig, S.A. Asher, J. Am. Chem. Soc. 110 (1988) 1005. [47] A. Weigel, N.P. Ernsting, J. Phys. Chem. B 114 (2010) 7879. [48] M. Kloz, R.v. Grondelle, J.T.M. Kennis, Phys. Chem. Chem. Phys. 13 (2011) 18123.
Please cite this article in press as: H. Kuramochi et al., Chem. Phys. Lett. (2017), http://dx.doi.org/10.1016/j.cplett.2017.02.015
Contents lists available at ScienceDirect
Chemical Physics Letters journal homepage: www.elsevier.com/locate/cplett
Research paper
Broadband stimulated Raman spectroscopy in the deep ultraviolet region Hikaru Kuramochi a,b,1, Tomotsumi Fujisawa a,1,2, Satoshi Takeuchi a,b, Tahei Tahara a,b,⇑ a b
Molecular Spectroscopy Laboratory, RIKEN, 2-1 Hirosawa, Wako 351-0198, Japan Ultrafast Spectroscopy Research Team, RIKEN Center for Advanced Photonics (RAP), 2-1 Hirosawa, Wako 351-0198, Japan
a r t i c l e
i n f o
Article history: Received 29 December 2016 In final form 7 February 2017 Available online xxxx Keywords: Stimulated Raman spectroscopy Deep ultraviolet Amino acid Ultrafast spectroscopy
a b s t r a c t We report broadband stimulated Raman measurements in the deep ultraviolet (DUV) region, which enables selective probing of the aromatic amino acid residues inside proteins through the resonance enhancement. We combine the narrowband DUV Raman pump pulse (<10 cm 1) at wavelengths as short as 240 nm and the broadband DUV probe pulse (>1000 cm 1) to realize stimulated Raman measurements covering a >1500 cm 1 spectral window. The stimulated Raman measurements for neat solvents, tryptophan, tyrosine, and glucose oxidase are performed using 240- and 290-nm Raman pump, highlighting the high potential of the DUV stimulated Raman probe for femtosecond time-resolved study of proteins. Ó 2017 Published by Elsevier B.V.
The function, activity and response of living organisms are closely related to the structural change of relevant proteins which takes place over a wide range of time scales. Therefore, it is of significant importance to track the structural change of proteins for elucidating the molecular mechanism behind the activation of biological functions. In photoreceptor proteins, the structural change of the protein is triggered by photoabsorption that induces a localized change in the embedded chromophore. This subtle, initial change brings about a chain of chemical reactions spanning a broad time range, which ultimately leads to the activation of a biological function [1–3]. Because the functional activation is synchronously triggered with light stimuli, photoreceptor proteins have been intensively studied with various time-resolved spectroscopic [4,5] and diffraction techniques [6–9] as the best system to investigate the structure–function relationship in proteins. Time-resolved spectroscopy has provided detailed insights into the primary response of various photoreceptor proteins with excellent temporal and structural information. In particular, femtosecond Raman spectroscopies have mapped out the primary structural events that occur on the femtosecond time scale for various photo-responsive proteins, such as rhodopsin [10], green fluorescent protein [11,12], phytochrome [13], and photoactive
⇑ Corresponding author at: Molecular Spectroscopy Laboratory, RIKEN, 2-1 Hirosawa, Wako 351-0198, Japan. E-mail address: [email protected] (T. Tahara). 1 H. K. and T. F. equally contributed to this work. 2 Present address: Department of Chemistry and Applied Chemistry, Graduate School of Science and Engineering, Saga University, Saga 840-8502, Japan.
yellow protein [14,15]. These studies successfully captured the primary structural dynamics of the chromophore in the proteins, which is actually the key initial process that triggers the activation of the biological function. On the other hand, information about the primary response of the surrounding amino acid residues has been rather limited. Because such a secondary response serves as the intermediary to the subsequent chain of chemical/biological reactions, it is very important to elucidate how the initial change in the chromophore propagates to the surrounding amino acid residues. It is one of the most important challenges to be tackled in protein science today, which will provide deep insights into the structural/functional cooperativity in proteins. Time-resolved ultraviolet (UV) resonance Raman spectroscopy has played an indispensable role in elucidating the structural dynamics in proteins on the picosecond and later time scales [16–18]. In this technique, after initiating the photoreaction by exciting the chromophore, the change in the vibrational spectrum of the surrounding aromatic amino acid residues is selectively monitored by UV resonance Raman probing at around 220 nm, by taking advantage of the resonance enhancement [19]. However, this spontaneous Raman approach is inherently not suitable for studying femtosecond structural dynamics because the time resolution is practically limited up to several picoseconds in exchange for obtaining frequency resolution as high as 10 cm 1. To overcome this limit, the timing of the Raman probing has to be determined with femtosecond accuracy with respect to the actinic excitation, which is not possible in the picosecond spontaneous Raman approach that utilizes only picosecond probe pulses. Actually, this has been realized with stimulated Raman techniques
http://dx.doi.org/10.1016/j.cplett.2017.02.015 0009-2614/Ó 2017 Published by Elsevier B.V.
Please cite this article in press as: H. Kuramochi et al., Chem. Phys. Lett. (2017), http://dx.doi.org/10.1016/j.cplett.2017.02.015
2
H. Kuramochi et al. / Chemical Physics Letters xxx (2017) xxx–xxx
in the frequency and time domains, i.e. femtosecond stimulated Raman spectroscopy (FSRS) [20–22] and time-resolved impulsive stimulated Raman spectroscopy (TR-ISRS) [23–25], respectively, which utilize femtosecond optical pulses. In FSRS, for example, the stimulated Raman process is induced by narrow-band picosecond Raman pump and broadband femtosecond probe pulses, which can define the timing to initiate the Raman transition with femtosecond temporal accuracy. Therefore, combined with the femtosecond actinic pump pulse, it enables tracking the change in the Raman spectrum on the femtosecond time scale. The advantage of FSRS is that it is relatively easy to perform experiments in various wavelength regions from UV (330–390 nm) [26–28] to near-IR (1190 nm) [29,30], although the inevitable background subtraction and inherent complex band shapes sometimes prevent straightforward interpretation. Further extending the spectral window of the FSRS measurements to the deeper UV region would allow us to track femtosecond dynamics of amino acid residues inside photoreceptor proteins. In this letter, we demonstrate broadband stimulated Raman spectroscopy in the deep UV region (DUV-SRS), which can be a technical basis of FSRS experiments for studying ultrafast structural dynamics of the aromatic amino acid residues in proteins. We realize stimulated Raman probing in the DUV region by introducing the Raman pump and probe pulses at wavelengths as short as 240 nm, thereby enabling selective probing of the aromatic amino acid residues in proteins, such as tryptophan and tyrosine. The steady-state stimulated Raman measurements are demonstrated for neat solvents, tryptophan, tyrosine, as well as glucose oxidase, indicating the high potential and feasibility of DUV-FSRS experiments for time-resolved studies of the aromatic amino acid residues inside photoreceptor proteins. A schematic diagram of the DUV-SRS setup is shown in Fig. 1. We used a Ti:Sapphire regenerative amplifier system (Legend Elite Duo, Coherent, 800 nm, 80 fs, 8 mJ, 1 kHz) as the light source. About 3.5 mJ of the total amplifier output was used for the DUVSRS experiments, which was further split into two portions to generate the Raman pump pulse and broadband probe pulse. The major portion (3 mJ) was first converted to a narrow-bandwidth picosecond pulse at 400 nm (<10 cm 1) with 1-mJ energy using a second-harmonic bandwidth compressor (SHBC, Light Conversion) [26,28,31–34]. This narrow-bandwidth 400-nm pulse was subsequently used to pump a narrow-bandwidth optical parametric amplifier (NB-OPA, Light Conversion), which was seeded by a white-light continuum. The NB-OPA delivered visible picosecond pulses (480–2400 nm, >150 lJ), and this output was further
Fig. 1. Schematic diagram of the DUV-SRS setup. OPA: optical parametric amplifier; SHBC: second-harmonic bandwidth compressor; NB-OPA: narrow-band optical parametric amplifier; NOPA: noncollinear optical parametric amplifier; HWP: half wave plate; CCD: Charge coupled device.
Fig. 2. Typical spectra of the DUV Raman pump and probe pulses.
frequency-doubled in an 8-mm-thick b-barium borate (BBO, type I, h = 29.2°) crystal to generate narrow-bandwidth DUV Raman pump pulses (<10 cm 1, 20 lJ). The typical spectra of the Raman pump pulses are shown in Fig. 2. The remaining 0.5-mJ portion of the amplifier output was used for generating the broadband DUV pulse that was used as the Raman probe. Initially, we attempted to obtain a broadband DUV probe pulse by white-light continuum generation pumped by the second harmonic of the amplifier output as reported previously [27,28]. Although this approach enabled stimulated Raman measurements in the UV region (>330 nm), we found that the intensity of the DUV spectral component (<300 nm) of the white-light continuum was not high enough to perform stimulated Raman measurements with a reasonable accumulation time, keeping the signal-to-noise ratio high. Therefore, in the present work, we employed a more intense, broadband DUV pulse generated with a noncollinear optical parametric amplifier (NOPA [35–37]) as the Raman probe. The 0.5-mJ portion of the amplifier output drove a home-built single-stage NOPA. In this NOPA, a white-light seed pulse was generated in a 3-mm thick sapphire plate and was amplified in a 1.5-mm-thick BBO (type I, h = 31°) crystal, delivering tunable pulses in the visible range (480–750 nm, <10 lJ). After the rotation of the polarization, the NOPA output was frequency doubled in a 20-lm-thick BBO crystal (type I, h = 36.7°), generating a broadband UV pulse (240–375 nm) which was used as the Raman probe. For frequency doubling, the NOPA output was tightly focused to the BBO crystal (f = 76.2 mm) to broaden the spectrum of the second harmonic generated [38] while the focal position was carefully adjusted and placed slightly in front of the crystal to avoid the damage of the crystal. The typical DUV probe spectra are shown in Fig. 2. The full-width at the half maximum of the probe pulse exceeds 1000 cm 1, which is broad enough to simultaneously record all the stimulated Raman bands from 100 to 2000 cm 1. The generated broadband DUV pulse was spectrally separated from the visible pulse, and the chirp was compensated by a pair of fused silica prisms with the apex-to-apex distance of 25 cm. Chirp characteristic of the probe pulse was evaluated in situ by measuring the optical Kerr-effect (OKE) signal of water [39], as shown in Fig. 3. The gate pulse for the OKE measurement was generated by a femtosecond OPA (TOPAS-C, Light Conversion) which was pumped by the remaining 4-mJ output of the amplifier. This gate pulse is tunable in a wide frequency range (240–2600 nm) with <80-fs pulse duration, and it can be also used as the actinic pump
Please cite this article in press as: H. Kuramochi et al., Chem. Phys. Lett. (2017), http://dx.doi.org/10.1016/j.cplett.2017.02.015
H. Kuramochi et al. / Chemical Physics Letters xxx (2017) xxx–xxx
Fig. 3. (A) Typical spectrum of the Raman probe pulse centered at around 300 nm. (B) Group delay dispersion of the probe pulse shown in (A), which was evaluated by measuring the OKE signal of water.
pulse for femtosecond time-resolved SRS measurements, i.e. FSRS. The estimated group delay difference (GDD) of the typical probe pulse centered at 300 nm was 100 fs over the presented wavelength region, indicating that the dispersion in the DUV region was so large that the residual higher-order dispersion was not completely compensated only by the prism pair. Nevertheless, because this GDD was much smaller than the pulse duration of the Raman pump (3 ps), it did not cause a variation of the effective frequency resolution of individual stimulated Raman bands within the probe spectral range. For time-resolved measurements, the GDD of the probe can be post-corrected [39]. The probe intensity was attenuated by rotating the polarization of the visible NOPA output before the second harmonic generation. For the stimulated Raman measurements, the probe pulse passing through the sample was spectrally analyzed by a spectrograph (iHR320, Horiba) and then detected by a CCD (PIXIS 400F with UV coating, Princeton Instruments) with 3-ms exposure time. The stimulated Raman gain signal, which was induced by the Raman pump (and the probe pulse), was evaluated by mechanically chopping the Raman pump pulse. As the first sample of the DUV-SRS measurements, stimulated Raman spectra of neat solvents (ethanol and cyclohexane) were measured using the 290-nm Raman pump. The Raman pump and probe pulses were focused to a fused silica flow cell with a 1mm optical path length (0.2-mm window thickness), in which the sample solution was circulated. The Raman pump energy of 200 nJ was used, and the polarizations of the Raman pump and probe pulses were set parallel. For obtaining the spectra presented in the following, about 25,600 spectra were averaged, corresponding to the total acquisition time of 5 min. All the measurements were performed on the Stokes side. Fig. 4 shows the acquired stimulated Raman spectra of ethanol and cyclohexane. The spectra reveal all the relevant vibrational bands in a wide frequency range from 100 to 2000 cm 1, and they are in an excellent agreement with the reported spontaneous Raman spectra [40]. The bandwidth of the C-C stretch band of cyclohexane at 801 cm 1 indicates that the effective frequency resolution of the measurement is as good as 8 cm 1. In addition to the neat solvents, we also measured a stimulated Raman spectrum of tryptophan in a buffer solution (pH 7.0). The 290-nm Raman pump is pre-resonant with the two overlapping transitions to the La and Lb excited states of tryptophan (Fig. 5A) [41,42]. The stimulated Raman bands of tryptophan were observed at peak frequencies that are in agreement with those previously observed with the spontaneous resonance Raman
3
Fig. 4. Stimulated Raman spectra of cyclohexane, ethanol, and tryptophan in a TrisHCl buffer solution (2.9 mM, pH 7.0). The Raman pump wavelength was tuned to 290 nm.
Fig. 5. Absorption (A) and stimulated Raman (B) spectra of glucose oxidase (0.2 mM) in a phosphate buffer solution (60 mM, pH 6.0) and aqueous solutions of FAD (1 mM), tryptophan (1 mM) and tyrosine (1 mM). The Raman pump wavelength was tuned at 240 nm.
spectroscopy using the 220-nm Raman pulse [19,43]. In contrast, the intensity pattern is largely different from these previous reports, which is attributable to the difference in the resonance condition. We note that the resonance Raman measurement of tryptophan with 290-nm excitation, which was carried out in this study, is extremely challenging with conventional spontaneous Raman measurements due to the intense fluorescence background.
Please cite this article in press as: H. Kuramochi et al., Chem. Phys. Lett. (2017), http://dx.doi.org/10.1016/j.cplett.2017.02.015
4
H. Kuramochi et al. / Chemical Physics Letters xxx (2017) xxx–xxx
In fact, in the previous reports of resonance Raman spectra of tryptophan, the Raman excitation wavelength was tuned at the blueedge of the absorption bands due to the transitions to the La and Lb excited states, for avoiding fluorescence [44,45]. To the best of our knowledge, the present study is the first report of the Raman spectrum of tryptophan under the pre-resonance condition. To further demonstrate the capability of SRS measurements in a deeper UV region and its applicability to protein systems, we measured stimulated Raman spectra of tyrosine and tryptophan, as well as glucose oxidase and its chromophore, flavine-adeninedinucleotide (FAD), with the 240-nm Raman pump. Fig. 5A shows the absorption spectra of tyrosine, tryptophan, FAD and glucose oxidase in a buffer solution (pH 6.0), representing the resonance condition for the measurements. The 240-nm Raman pump is pre-resonant with the transition to the Bb excited state of tryptophan and to the La excited state of tyrosine [41,42,46]. Glucose oxidase contains 27 tyrosine and 10 tryptophan residues, so that resonantly enhanced Raman signals from all these 37 aromatic amino acids are expected in the stimulated Raman spectrum. FAD also shows an intense absorption band in the probe region, thus, the FAD Raman bands may also appear. Fig. 5B shows the obtained stimulated Raman spectra of tryptophan, tyrosine, FAD and glucose oxidase measured with the 240-nm Raman pump. The Raman spectra of tyrosine and tryptophan are in agreement with those previously observed with spontaneous resonance Raman spectroscopy using the 220-nm excitation pulse [19,43]. The spectrum of glucose oxidase shows prominent Raman bands at 1009, 1173, 1557, and 1619 cm 1. The counterparts of these bands can be found in the stimulated Raman spectrum of tyrosine or tryptophan. For example, the 1557-cm 1 band observed for glucose oxidase can be assigned to the 1557-cm 1 band of tryptophan. The 1619 cm 1 band of glucose oxidase could be originated from the two overlapped contributions, i.e., the tyrosine band at 1621 cm 1 and tryptophan band 1625 cm 1. In contrast, Raman bands attributable to FAD were not clearly observed in the spectrum of glucose oxidase, probably due to the fact that the only one FAD molecule contributes to the spectrum, and its contribution is likely to be much smaller than those of tyrosine and tryptophan. We also note that Raman bands are not clearly observed even for FAD in a solution, suggesting its low resonance Raman enhancement. It is noteworthy that the peak frequencies of tyrosine and tryptophan Raman bands observed in the stimulated Raman spectrum of glucose oxidase are slightly shifted with respect to those in a buffer solution. It is well-known that some of the tyrosine and tryptophan Raman bands are sensitive to their microenvironment such as hydrophobicity, hydrogen-bonding condition or van der Waals interactions with surroundings. Therefore, the observed difference in the peak frequencies between protein and solution may be attributable to the difference in microenvironment. The wavy background feature, most pronounced in the tryptophan spectrum, is likely attributable to the etaloning effect due to the back reflection of the probe from the rear surface of the BBO crystal (for frequency-doubling) or the windows of the flow cell. Such a systematic noise, which cannot be simply averaged out, may be eliminated by a wavelength modulation technique [47,48]. In summary, we realized broadband stimulated Raman measurements in the DUV region for the first time and described the experimental procedure in detail. The DUV Raman probe pulse was generated as the second harmonic of the broadband NOPA output, and it has a sufficient bandwidth and intensity to perform stimulated Raman measurements with a reasonable accumulation time and a sufficient signal-to-noise ratio. We measured the stimulated Raman spectra of the standard solvents, tyrosine, tryptophan and glucose oxidase using the Raman pump pulses at 290 and 240 nm. The present measurements can be readily extended
to time-resolved measurements, i.e., FSRS, by introducing the femtosecond actinic pump pulse. Thus, this work paves a way for tracking the ultrafast structural dynamics of the amino acid residues inside the photoreceptor proteins. DUV-FSRS measurements of the photoreceptor proteins are now underway in our laboratory. Acknowledgement This work was partly supported by JSPS KAKENHI Grant Numbers JP16H04102 to S. T. and JP25104005 to T. T. H. K. and T. F. acknowledge RIKEN Special Postdoctoral Researchers (SPDR) program. References [1] M.A. van der Horst, K.J. Hellingwerf, Acc. Chem. Res. 37 (2003) 13. [2] W.R. Briggs, J.L. Spudich, Handbook of Photosensory Receptors, John Wiley & Sons, 2005. [3] A. Möglich, X. Yang, R.A. Ayers, K. Moffat, Annu. Rev. Plant Biol. 61 (2010) 21. [4] J.T.M. Kennis, M.-L. Groot, Curr. Opin. Struct. Biol. 17 (2007) 623. [5] V. Sundström, Annu. Rev. Phys. Chem. 59 (2008) 53. [6] S. Westenhoff, E. Nazarenko, E. Malmerberg, J. Davidsson, G. Katona, R. Neutze, Acta Crystallogr. Sect. A: Found. Crystallogr. 66 (2010) 207. [7] R. Neutze, K. Moffat, Curr. Opin. Struct. Biol. 22 (2012) 651. [8] K. Moffat, Philos. Trans. Roy. Soc. B: Biol. Sci. 369 (2014) 20130568. [9] R. Neutze, Philos. Trans. Roy. Soc. B: Biol. Sci. 369 (2014) 20130318. [10] P. Kukura, D.W. McCamant, S. Yoon, D.B. Wandschneider, R.A. Mathies, Science 310 (2005) 1006. [11] C. Fang, R.R. Frontiera, R. Tran, R.A. Mathies, Nature 462 (2009) 200. [12] T. Fujisawa, H. Kuramochi, H. Hosoi, S. Takeuchi, T. Tahara, J. Am. Chem. Soc. 138 (2016) 3942. [13] J. Dasgupta, R.R. Frontiera, K.C. Taylor, J.C. Lagarias, R.A. Mathies, Proc. Natl. Acad. Sci. USA 106 (2009) 1784. [14] M. Creelman, M. Kumauchi, W.D. Hoff, R.A. Mathies, J. Phys. Chem. B 118 (2013) 659. [15] H. Kuramochi, S. Takeuchi, K. Yonezawa, H. Kamikubo, M. Kataoka, T. Tahara, Nat. Chem. (in press). http://dx.doi.org/10.1038/NCHEM.2717. [16] J.E. Kim, D. Pan, R.A. Mathies, Biochemistry 42 (2003) 5169. [17] A. Sato, Y. Mizutani, Biochemistry 44 (2005) 14709. [18] A. Sato, Y. Gao, T. Kitagawa, Y. Mizutani, Proc. Natl. Acad. Sci. USA 104 (2007) 9627. [19] R.P. Rava, T.G. Spiro, J. Am. Chem. Soc. 106 (1984) 4062. [20] M. Yoshizawa, M. Kurosawa, Phys. Rev. A 61 (1999) 013808. [21] D.W. McCamant, P. Kukura, S. Yoon, R.A. Mathies, Rev. Sci. Instrum. 75 (2004) 4971. [22] P. Kukura, D.W. McCamant, R.A. Mathies, Annu. Rev. Phys. Chem. 58 (2007) 461. [23] S. Fujiyoshi, S. Takeuchi, T. Tahara, J. Phys. Chem. A 107 (2003) 494. [24] S. Takeuchi, S. Ruhman, T. Tsuneda, M. Chiba, T. Taketsugu, T. Tahara, Science 322 (2008) 1073. [25] H. Kuramochi, S. Takeuchi, T. Tahara, Rev. Sci. Instrum. 87 (2016) 043107. [26] S. Laimgruber, H. Schachenmayr, B. Schmidt, W. Zinth, P. Gilch, Appl. Phys. B 85 (2006) 557. [27] J.M. Rhinehart, J.R. Challa, D.W. McCamant, J. Phys. Chem. B (2012). [28] H. Kuramochi, S. Takeuchi, T. Tahara, J. Phys. Chem. Lett. 3 (2012) 2025. [29] T. Takaya, K. Iwata, J. Phys Chem. A 118 (2014) 4071. [30] T. Takaya, K. Iwata, Analyst 141 (2016) 4283. [31] F. Raoult, A.C.L. Boscheron, D. Husson, C. Sauteret, A. Modena, V. Malka, F. Dorchies, A. Migus, Opt. Lett. 23 (1998) 1117. [32] S.A. Kovalenko, A.L. Dobryakov, N.P. Ernsting, Rev. Sci. Instrum. 82 (2011) 063102. [33] M. Nejbauer, C. Radzewicz, Opt. Express 20 (2012) 2136. [34] L. Zhu, W. Liu, C. Fang, Appl. Phys. Lett. 105 (2014) 041106. [35] T. Wilhelm, J. Piel, E. Riedle, Opt. Lett. 22 (1997) 1494. [36] G. Cerullo, M. Nisoli, S. Stagira, S. De Silvestri, Opt. Lett. 23 (1998) 1283. [37] A. Shirakawa, T. Kobayashi, Appl. Phys. Lett. 72 (1998) 147. [38] S.H. Ashworth, M. Joschko, M. Woerner, E. Riedle, T. Elsaesser, Opt. Lett. 20 (1995) 2120. [39] S. Yamaguchi, H. Hamaguchi, Appl. Spectrosc. 49 (1995) 1513. [40] B. Schrader, W. Meier, Raman/Infrared Atlas of Organic Compounds, VCH Weinheim, 1989. [41] I. Harada, H. Takeuchi, Adv. Infrared Raman Spectrosc. 13 (1986) 113. [42] J.A. Sweeney, S.A. Asher, J. Phys Chem. 94 (1990) 4784. [43] M. Mizuno, N. Hamada, F. Tokunaga, Y. Mizutani, J. Phys. Chem. B 111 (2007) 6293. [44] R.P. Rava, T.G. Spiro, J. Phys. Chem. 89 (1985) 1856. [45] S.A. Asher, M. Ludwig, C.R. Johnson, J. Am. Chem. Soc. 108 (1986) 3186. [46] M. Ludwig, S.A. Asher, J. Am. Chem. Soc. 110 (1988) 1005. [47] A. Weigel, N.P. Ernsting, J. Phys. Chem. B 114 (2010) 7879. [48] M. Kloz, R.v. Grondelle, J.T.M. Kennis, Phys. Chem. Chem. Phys. 13 (2011) 18123.
Please cite this article in press as: H. Kuramochi et al., Chem. Phys. Lett. (2017), http://dx.doi.org/10.1016/j.cplett.2017.02.015