竹内 佐年

Abstract The multi-ionization processes of Kr and Xe in the K-edge photon energy region were investigated using short-pulse x-rays and time-of-flight apparatus. The generation of Kr¹³⁺ ions in this photon energy region was observed for the first time. The distribution characteristics observed in the experiment, such as the production of Kr¹³⁺ ions, high production ratios of Kr⁴⁺, Kr⁵⁺, and Kr⁶⁺ ions, and the existence of a small peak for Kr⁸⁺, were quantitatively reproduced by the Monte Carlo simulation for the charge distribution of Kr ions following 1s of inner shell ionization. The highly charged ions were suppressed in the experiments compared with those in the simulation, probably owing to the inhibition of certain Coster-Kronig processes in the highly charged ions, which were not considered for analysis. The charge distribution of Xe ions following 1s of inner shell ionization was also investigated. A similar charge distribution was observed when the photon energy was located between the K and L edges. This is because the Xe 1s hole state mainly undergoes ultrafast relaxation to the 2p hole state owing to the strong dipole transition moment between the 1s and 2p states.

A light-accessed scanning tunneling microscope (STM) is a powerful spectroscopic tool that enables chemical analysis at the single molecular level, but it requires highly precise optical alignments to pinpoint the nano-scale tunneling gap, leaving experimental challenges. Here we present straightforward procedures to align the optical setup for STM-luminescence and STM-based tip-enhanced Raman spectroscopy (TERS) performed with a reflection geometry in an ultrahigh vacuum chamber. Observing real-space images of the metal tip apex through a spectrograph set to the zeroth-order diffraction enables “ in situ” optimization of the detection path and introduction of the excitation light of TERS to the nanogap. The best spatial overlap with the nanogap can be achieved by monitoring plasmon-enhanced, low-frequency inelastic scattering of the metal. This protocol allows us to overcome such difficulties in STM-based spectroscopy and facilitates physicochemical study of single adsorbates on nontransparent substrates.

Excited state dynamics of a rotaxane-based molecular shuttle were examined using femtosecond time-resolved spectroscopy. Results suggest two energetic barriers in the excited state along the isomerization coordinate precede the shuttling motion.

We studied ultrafast structural dynamics of photoactive yellow protein (PYP) using ultraviolet femtosecond stimulated Raman spectroscopy. By employing the Raman pump and probe pulses in the ultraviolet region, resonantly enhanced, rich vibrational features of the excited-state chromophore were observed in the fingerprint region. In contrast to the marked spectral change reported for the excited-state chromophore in solution, in the protein, all of the observed Raman bands in the fingerprint region did not show any noticeable spectral shifts nor band shape changes during the excited-state lifetime of PYP. This indicates that the significant skeletal change does not occur on the chromophore in the excited state of PYP and that the trans conformation is retained in its lifetime. Based on the femtosecond Raman data of PYP obtained so far, we discuss a comprehensive picture of the excited-state structural dynamics of PYP.

Femtosecond time-resolved absorption and picosecond time-resolved emission measurements were carried out for highly concentrated aqueous solutions of K2[Pt(CN)4] to investigate excited-state dynamics of the [Pt(CN)42−] oligomers formed with metallophilic interactions. Time-resolved absorption spectra exhibit complicated dynamics that are represented with five time constants. Among them, the 90-ps and 400-ps dynamics were assigned to the S1 → T1 intersystem crossing of the trimer and tetramer coexisting in the solution by comparison with the fluorescence decays. Clear oscillations of transient absorption were observed in the first few picoseconds, and the frequency-detected-wavelength 2D analysis revealed that the 135-cm−1 and 65-cm−1 oscillations arise from the Pt–Pt stretch motions of the S1 trimer and S1 tetramer, respectively. The obtained time-resolved spectroscopic data provide a clear view of the excited-state dynamics of the [Pt(CN)42−] oligomers in the femto-/picosecond time region.

Combining surface-enhanced Raman scattering (SERS) with the coherent nonlinear Raman technique is a promising route for achieving higher sensitivity and time-resolved SERS measurements, yet such attempts have just been started. Here, we report time-domain Raman measurements of trans-1,2-bis(4-pyridyl)ethylene (BPE) adsorbed on gold nanoparticle assemblies (GNAs), which were carried out with impulsive stimulated Raman spectroscopy using sub-8 fs pulses. We observe coherent nuclear wavepacket motion of BPE on GNAs with drastic enhancement through the surface plasmon resonance, which provides information on the Raman-active vibrations in the time domain. Through Fourier transform of the measured time-domain Raman data, we obtained SERS spectra of BPE on GNAs with enhancement factors as high as 105-106. The present study not only demonstrates applicability of time-domain nonlinear Raman techniques in SERS, i.e., surface-enhanced impulsive stimulated Raman spectroscopy (SE-ISRS), but also provides a technical basis for femtosecond time-resolved SE-ISRS experiments to track ultrafast dynamics of the adsorbates.

Real-time observation of chemical bond formation and subsequent nuclear rearrangements is an ultimate goal of chemical science. Yet, such attempts have been hampered by the technical difficulty of triggering bond formation at well-defined, desired timing. The trimer of dicyanoaurate complex ([Au(CN)2-]3) is an ideal system for achieving this aim because the tight covalent Au-Au bonds are formed upon photoexcitation. Despite the apparent simplicity of the system, however, recent time-resolved studies failed to construct a consistent picture of its ultrafast dynamics. Here, we report femtosecond time-domain Raman tracking of ultrafast structural dynamics of the [Au(CN)2-] trimer upon photoinduced Au-Au bond formation. The obtained Raman data reveal that the Au-Au breathing vibration at â¼90 cm-1 exhibits a gradual frequency upshift in a few picoseconds, demonstrating a continuous bent-to-linear structural change on the triplet-state potential energy surface upon the Au-Au bond formation. The comprehensive ultrafast spectroscopic study settles the controversy on this prototypical molecular assembly.

Photodynamic therapy (PDT) utilizes photoirradiation in the presence of photosensitizers to ablate cancer cells via generation of singlet oxygen (O-1(2)), but it is important to minimize concomitant injury to normal tissues. One approach for achieving this is to use activatable photosensitizers that can generate O-1(2) only under specific conditions. Here, we report a novel photosensitizer that is selectively activated under hypoxia, a common condition in solid tumors. We found that introducing an azo moiety into the conjugated system of a seleno-rosamine dye effectively hinders the intersystem crossing process that leads to O-1(2) generation. We show that the azo group is reductively cleaved in cells under hypoxia, enabling production of O-1(2) to occur. In PDT in vitro, cells under mild hypoxia, within the range typically found in solid tumors (up to about 5% O-2), were selectively ablated, leaving adjacent normoxic cells intact. This simple and practical azobased strategy should be widely applicable to design a range of activatable photosensitizers.

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. (C) 2017 Elsevier B.V. All rights reserved.

Unveiling the nuclear motions of photoreceptor proteins in action is a crucial goal in protein science in order to understand their elaborate mechanisms and how they achieve optimal selectivity and efficiency. Previous studies have provided detailed information on the structures of intermediates that appear during the later stages (>ns) of such photoreception cycles, yet the initial events immediately after photoabsorption remain unclear because of experimental challenges in monitoring nuclear rearrangements on ultrafast timescales, including protein-specific low-frequency motions. Using time-domain Raman probing with sub-7-fs pulses, we obtain snapshot vibrational spectra of photoactive yellow protein and a mutant with high sensitivity, providing insights into the key responses that drive photoreception. Our data show a drastic intensity drop of the excited-state marker band at 135 cm(-1) within a few hundred femtoseconds, suggesting a rapid weakening of the hydrogen bond that anchors the chromophore. We also track formation of the first ground-state intermediate over the first few picoseconds and fully characterize its vibrational structure, revealing a substantially-twisted cis conformation.

Enzyme/substrate pairs, such as beta-galactosidase with chromogenic x-gal substrate, are widely used as reporters to monitor biological events, but there is still a requirement for new reporter systems, which may be orthogonal to existing systems. Here, we focused on azoreductase (AzoR). We designed and synthesized a library of azo-rhodamine derivatives as candidate fluorogenic substrates. These derivatives were nonfluorescent, probably due to ultrafast conformational change around the N=N bond after photoexcitation. We found that AzoR-mediated reduction of the azo bond of derivatives bearing an electron donating group on the azobenzene moiety was followed by nonenzymatic cleavage to afford highly fluorescent 2-methyl-rhodamine green (2-Me RG), which was well retained in cells. We show that the AzoR/compound 9 reporter system can detect azoreductase-expressing live cells at the single cell level.

The local environment within a recently developed anthracene-shelled micelle (ASM), which is a micellelike nanocapsule composed of anthracene-embedded amphiphiles, was investigated by steady-state and time-resolved spectroscopy of an encapsulated solvatochromic fluorescent probe molecule, coumarin 153 (C153). The absorption maximum of encapsulated C153 (452 nm) is more red-shifted than that of free C153 in water, indicating a highly polar environment inside the micelle. Despite this, the fluorescence Stokes shift of encapsulated C153 (similar to 3700 cm(-1))is substantially smaller than that of free C153 in water. Femtosecond time-resolved broadband fluorescence measurements further showed that the dynamic Stokes shift is completed within 1 ps, revealing that the reorganization of the micelle interior following photoexcitation of the C153 probe is characterized by a sub-picosecond, limited-amplitude response. The femtosecond fluorescence anisotropy data showed that the orientational diffusion of the host-guest complex is slower (860 ps) than that of the empty micelle (510 ps), suggesting that the micelle structure is flexible enough to expand when the guest molecule is accommodated and that the micelle rotates with the encapsulated guest molecule. This softness of the micelles further allows some of them to simultaneously encapsulate two C153 molecules, as evidenced by the appearance of blue-shifted, H-dimer-like absorption and fluorescence bands. Based on these steady-state and femtosecond timeresolved spectroscopic data, we discuss the electronic state of C153 and micelle structure as well as the host-guest interaction in this novel flexible synthetic nanocapsule.

We describe details of the setup for time-resolved impulsive stimulated Raman spectroscopy (TR-ISRS). In this method, snapshot molecular vibrational spectra of the photoreaction transients are captured via time-domain Raman probing using ultrashort pulses. Our instrument features transform-limited sub-7-fs pulses to impulsively excite and probe coherent nuclear wavepacket motions, allowing us to observe vibrational fingerprints of transient species from the terahertz to 3000-cm(-1) region with high sensitivity. Key optical components for the best spectroscopic performance are discussed. The TR-ISRS measurements for the excited states of diphenylacetylene in cyclohexane are demonstrated, highlighting the capability of our setup to track femto-second dynamics of all the Raman-active fundamental molecular vibrations. (C) 2016 AIP Publishing LLC.

Green fluorescent protein (GFP) from jellyfish Aequorea victoria, an essential bioimaging tool, luminesces via excited-state proton transfer (ESPT) in which the phenolic proton of the p-hydroxybenzylideneimidazolinone chromophore is transferred to G1u222 through a hydrogen-bond network. In this process, the ESPT mediated by the low-frequency motion of the chromophore has been proposed. We address this issue using femtosecond time-resolved impulsive stimulated Raman spectroscopy. After coherently exciting low frequency modes (<300 cm(-1)) in the excited state of GFP, we examined the excited-state structural evolution and the ESPT dynamics within the dephasing time of the low-frequency vibration. A clear anharmonic vibrational coupling is found between one high-frequency mode of the chromophore (phenolic CH bend) and a low frequency mode at similar to 104 cm(-1). However, the data show that this low-frequency motion does not substantially affect the ESPT dynamics.

Au-Au bond strengthening in photoexcited dimers of an Au(I) complex is captured in solution as oscillations of femtosecond absorption signals. The subsequent dynamics, when compared to the trimer's data, confirm that the bent-to-linear structural change of the trimer occurs in the first few picoseconds.

We report the first femtosecond time-resolved absorption study on ultrafast photoreaction dynamics of a recently discovered retinal protein, KR2, which functions as a light-driven sodium-ion pump. The obtained data show that the excited-state absorption around 460 nm and the stimulated emission around 720 nm decay concomitantly with a time constant of 180 Is. This demonstrates that the deactivation of the S-1 state of KR2, which involves isomerization of the retinal chromophore, takes place three times faster than that of bacteriorhodopsin. In accordance with this rapid electronic relaxation, the photoproduct band assignable to the J intermediate grows up at similar to 620 nm, indicating that the J intermediate is directly formed with the S-1 -> S-0 internal conversion. The photoproduct band subsequently exhibits a similar to 30 nm blue shift with a 500 fs time constant, corresponding to the conversion to the K intermediate. On the basis of the femtosecond absorption data obtained, we discuss the mechanism for the rapid photoreaction of KR2 and its relevance to the unique function of the sodium-ion pump.

Unlike previous coordinative host-guest systems, highly emissive host-guest complexes (up to Phi(F) = 0.5) were successfully prepared upon encapsulation of various fluorescent dyes (e.g., BODIPY and coumarin derivatives) by a Pt(II)-linked coordination capsule in water. Picosecond time-resolved spectroscopy elucidates the photophysical behaviors of the obtained complexes. Notably, the emission color of the fluorescent guest within the capsule can be readily modulated upon pairwise encapsulation with planar aromatic molecules.

Two-photon absorption spectra of 4'-hydroxybenzylidene-2,3-dimethylimidazolinone, a model chromophore of enhanced green fluorescent protein (eGFP), were measured in various solvents. The two-photon absorption band of its anionic form is markedly blue-shifted from the corresponding one-photon absorption band in all solvents. Moreover, the magnitude of the blue shift varies largely depending on the solvent, which does not accord with the assignment of the two-photon absorption band to the transitions to the vibrationally excited S-1 state. Our finding is readily rationalized by considering overlapping contributions of the S-1 <- S-0 and S-2 <- S-0 transitions, suggesting the involvement of the S-2 state also in two-photon fluorescence of eGFP. (C) 2015 Elsevier B.V. All rights reserved.

CONSPECTUS: Bis-diimine Cu(I) complexes exhibit strong absorption in the visible region owing to the metal-to-ligand charge transfer (MLCT) transitions, and the triplet MLCT ((MLCT)-M-3) states have long lifetimes. Because these characteristics are highly suitable for photosensitizers and photocatalysts, Cu(I) complexes have been attracting much interest. An intriguing feature of the Cu(I) complexes is the photoinduced structural change called "flattening". Bis-diimine Cu(I) complexes usually have tetrahedron-like D-2d structures in the ground (S-0) state, in which two ligands are perpendicularly attached to the Cu(I) ion. With MLCT excitation, the central Cu(I) ion is formally oxidized to Cu(II), which induces the structural change to the "flattened" square-planar-like structure that is seen for usual Cu(II) complexes. In this Account, we review our recent studies on ultrafast excited-state dynamics of bis-diimine Cu(I) complexes carried out using femtosecond time-resolved optical spectroscopy. Focusing on three prototypical bis-diimine Cu(I) complexes that have 1,10-phenanthroline ligands with different substituents at the 2,9-positions, i.e., [Cu(phen)(2)](+) (phen = 1,10-phenanthroline), [Cu(dmphen)(2)](+) (dmphen = 2,9-dimethyl-1,10-phenanthroline), and [Cu(dpphen)(2)](+) (dpphen = 2,9-diphenyl-1,10-phenanthroline), we examined their excited-state dynamics by time-resolved emission and absorption spectroscopies with 200 fs time resolution, observed the excited-state coherent nuclear motion with 30 fs time resolution and performed complementary theoretical calculations. This combined approach vividly visualizes excited-state processes in the MLCT state of bis-diimine Cu(I) complexes. It was demonstrated that flattening distortion, internal conversion, and intersystem crossing occur on the femtosecond early picosecond time scale, and their dynamics is clearly identified separately. The flattening distortion predominantly occurs in the S-1, state on the subpicosecond time-scale, and the precursor S-1 state retaining the initial undistorted structure appears as a metastable state before the structural change. This observation indicates that the traditional understanding based on the Jahn- Teller effect appears irrelevant for realistically discussing the photoinduced structural change of bis-diimine Cu(I) complexes. The lifetime of the precursor S-1 state significantly depends on the substituents in the three complexes, indicating that the flattering distortion requires a longer time as the substituents at 2,9-positions of the ligands become builder. It is suggested that the substituents are rotated to avoid steric repulsions to achieve the flattened structure at the global minimum of the S-1 state, implying the necessity of discussion based on a multidimensional potential energy surface to properly consider this excited-state structural change. After the flattening distortion, the S-1 states of [Cu(dmphen)(2)](+) and [Cu(dpphen)(2)](+), which have bulky substituents, relax to the T-1 state by intersystem crossing on the similar to 10 ps time scale, while the flattened S, state of [Cu(phen)(2)](+) relaxes directly to the So state on the similar to 2 ps time scale. This difference is rationalized in terms of the different magnitude of the flattening distortion and relevant changes in the potential energy surfaces. Clear understanding of the ultrafast excited-state process provides a solid basis for designing and using Cu(I) complexes, such as controlling the structural change to efficiently utilize the energy of the MLCT state in solar energy conversion.

Time-resolved impulsive-Raman with narrowband photoexcitation was utilized to study structural dynamics of bis-diimine copper complex in solution. A copper-ligand symmetric stretch band showed up with frequency oscillation, demonstrating its anharmonic coupling with large-amplitude distortional motions.

Ultrafast dynamics of photoactive yellow protein was investigated by time-resolved impulsive stimulated-Raman spectroscopy. Time-Domain vibrational data revealed rapid change of the hydrogen-bonding structure in the excited state and vibrational structure of the first ground-state intermediate.

Structural dynamics of green fluorescent protein was studied by femtosecond time-resolved impulsive Raman spectroscopy. The excited-state vibrational spectra of the protein with three different chromophore forms were obtained and they revealed their structural differences and excited-state deprotonation.

The substituent effect on the excited-state dynamics of bis-diimine Cu(I) complexes was investigated by femtosecond time-resolved absorption spectroscopy with the S-1 <-- S-0 metal-to-ligand charge transfer (MLCT) photoexcitation. The time-resolved absorption of [Cu(phen)(2)](+) (phen = 1,10-phenanthroline) showed a slight intensity increase of the S-1 absorption with a time-constant of 0.1-0.2 ps, reflecting the flattening distortion occurring in the S-1 state. The transient absorption of the 'flattened' S-1 state was clearly observed, although its fluorescence was not observed in the previous fluorescence up-conversion study in the visible region. The flattened S-1 state decayed with a time constant of similar to 2 ps, and the S-0 bleaching recovered accordingly. This clarifies that the S-1 state of [Cu(phen)(2)](+) is predominantly relaxed to the S-0 state by internal conversion. The time-resolved absorption of [Cu(dpphen)(2)](+) (dpphen = 2,9-diphenyl-1,10-phenanthroline) showed a 0.9 ps intensity increase of the S1 absorption due to the flattening distortion, and then exhibited a 11 ps spectral change due to the intersystem crossing. This excited-state dynamics of [Cu(dpphen)(2)](+) is very similar to that of [Cu(dmphen)(2)](+) (dmphen = 2,9-dimethyl-1,10-phenanthroline). In the ultrafast pump-probe measurements with 35 fs time resolution, [Cu(phen)(2)](+) and [Cu(dpphen)(2)](+) exhibited oscillation due to the nuclear wavepacket motions of the initial S-1 state, and the oscillation was damped as the structural change took place. This indicates that the initial S-1 states have well-defined vibrational structures and that the vibrational coherence is retained in their short lifetimes. The present time-resolved absorption study, together with the previous time-resolved fluorescence study, provides a unified view for the ultrafast dynamics of the MLCT excited state of the Cu(I) complexes.

We studied the signaling-state formation of a BLUF (blue light using FAD) protein, PapB, from the purple bacterium Rhodopseudomonas palustris, using femtosecond time-resolved absorption spectroscopy. Upon photoexcitation of the dark state, FADH(center dot) (neutral flavin semiquinone FADH radical) was observed as the intermediate before the formation of the signaling state. The kinetic analysis based on singular value decomposition showed that FADH(center dot) mediates the signaling-state formation, showing that PapB is the second example of FADH(center dot)-mediated formation of the signaling state after Slr1694 (M. Gauden et al. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 10895-10900). The mechanism of the signaling-state formation is discussed on the basis of the comparison between femtosecond time-resolved absorption spectra of the dark state and those obtained by exciting the signaling state. FADH(center dot) was observed also with excitation of the signaling state, and surprisingly, the kinetics of FADH(center dot) was indistinguishable from the case of exciting the dark state. This result suggests that the hydrogen bond environment in the signaling state is realized before the formation of FADH(center dot) in the photocycle of PapB.

Light absorption by the photoreceptor microbial rhodopsin triggers trans cis isomerization of the retinal chromophore surrounded by seven transmembrane alpha-helices. Sensory rhodopsin I (SRI) is a dual functional photosensory rhodopsin both for positive and negative phototaxis in microbes. By making use of the highly stable SRI protein from Salinibacter ruber (SrSRI), the early steps in the photocycle were studied by time-resolved spectroscopic techniques. All of the temporal behaviors of the S-n <- S-1 absorption, ground-state bleaching, K intermediate absorption, and stimulated emission were observed in the femto- to picosecond time region by absorption spectroscopy. The primary process exhibited four dynamics similar to other microbial rhodopsins. The first dynamics (tau(1) similar to 54 fs) corresponds to the population branching process from the Franck-Condon region to the reactive (S-1(r)) and nonreactive (S-1(nr)) S-1 states. The second dynamics (tau(2) = 0.64 ps) is the isomerization process of the Sir state to generate the ground-state 13-cis form, and the third dynamics (tau(3) = 1.8 ps) corresponds to the internal conversion of the S-1(nr) state. The fourth component (tau(3)' = 2.5 ps) is assignable to the J-decay (K-formation). This reaction scheme was further supported by the results of fluorescence spectroscopy. To investigate the protein response(s), the spectral changes of the tryptophan bands were monitored by ultraviolet resonance Raman spectroscopy. The intensity change following the K formation in the chromophore structure (tau similar to 17 ps) was significantly small in SrSRI as compared with other microbial rhodopsins. We also analyzed the effect(s) of Cl- binding on the ultrafast dynamics of SrSRI. Compared with a chloride pump Halorhodopsin, Cl- binding to SrSRI was less effective for the excited-state dynamics, whereas the binding altered the structural changes of tryptophan following the K-formation, which was the characteristic feature for SrSRI. On the basis of these results, a primary photoreaction scheme of SrSRI together with the role of chloride binding is proposed.

The Cu(I) complexes having phenanthroline derivatives as ligands are known to exhibit photo-induced 'flattening' structural change in the metal-to-ligand charge transfer (MLCT) excited state. Our recent ultrafast spectroscopic studies of [Cu(dmphen)(2)](+) (dmphen = 2,9-dimethyl-1,10-phenanthroline) showed that the photo-induced structural change predominantly occurs in the S-1 state on a subpicosecond time scale, with the appearance of the 'perpendicular' S-1 state before the structural change. In this work, we carried out femto/picosecond time-resolved emission spectroscopy of [Cu(phen)(2)](+) (phen = 1,10-phenanthroline) and [Cu(dpphen)(2)](+) (dpphen = 2,9-diphenyl-1,10-phenanthroline) in dichloromethane with the S-2 <- S-0 photo-excitation to examine the substituent effect on the ultrafast structural change. The femtosecond time-resolved emission spectra of the two complexes exhibit ultrafast fluorescence changes that are attributed to the structural change in the S-1 state after fast (50-100 fs) S-2 -> S-1 internal conversion. By comparing with the dynamics of [Cu(dmphen)(2)](+), it was found that the time constant of the structural change increases as the substituents at 2- and 9- positions of the ligand become bulkier, i.e., [Cu(phen)(2)](+) (200 fs) < [Cu(dmphen)(2)](+) (660 fs) < [Cu(dpphen)(2)](+) (920 fs). This implies that the complex needs a longer time to flatten with the bulkier substituent. This demonstrates that the dynamics of the photo-induced structural change of Cu(I) complexes is substantially affected by the substituent of the ligand. The dynamics of the ultrafast structural change and the substituent effect are discussed with the multidimensional S-1 potential energy surface of Cu(I) complexes.

Anabaena sensory rhodopsin (ASR) is a microbial rhodopsin found in eubacteria and functions as a photosensor. The photoreaction of ASR is photochromic between all-trans, 15-anti (ASR(AT)), and 13-cis, 15-syn (ASR(13C)) isomers. To understand primary protein dynamics in the photoreaction starting in ASR(AT) and ASR(13C), picosecond time-resolved ultraviolet resonance Raman spectra were obtained. In the intermediate state appearing in the picosecond temporal region, spectral changes of Trp bands were observed. For both ASR(AT) and ASR(13C), the intensities of the Trp bands were bleached within the instrumental response time and recovered with a time constant of 30 ps. This suggests that the rates of structural changes in the Trp residue in the vicinity of the chromophore do not depend on the direction of the isomerization of retinal. A comparison between spectra of the wild-type and Trp mutants indicates that the structures of Trp76 and Trp46 change upon the primary photoreaction of retinal. (C) 2013 Elsevier B.V. All rights reserved.

Newly-developed ultraviolet-resonance femtosecond stimulated-Raman spectroscopy was utilized to study the initial structural evolution of photoactive yellow protein chromophore in solution. The obtained spectra changed drastically within 1 ps, demonstrating rapid in-plane deformations of the chromophore. © Owned by the authors, published by EDP Sciences, 2013.

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 S-1 potential minimum in the femtosecond time region. The S-1 Raman spectra exhibit spectral patterns that are similar to the ground-state spectrum, suggesting that pCA(-) yet retains a planar-trans conformation throughout the S-1 lifetime. We concluded that S-1 pCA(-) undergoes a femtosecond in-plane deformation, rather than a substantial C-et=C-et 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.