Hydrogen Out-of-plane Research Articles

Protonation of Asp116 and distortion of the all-trans retinal chromophore in Krokinobacter eikastus rhodopsin 2 causes a redshift in absorption maximum upon dehydration.

Water is usually indispensable for protein function. For ion-pumping rhodopsins, water molecules inside the proteins play an important role in ion transportation. In addition to amino acid residues, water molecules regulate the colors of retinal proteins. It was reported that a sodium-pumping rhodopsin, Krokinobacter eikastus rhodopsin 2 (KR2), showed a color change from red to purple upon dehydration under crystalline conditions. Here, we applied comprehensive visible and IR absorption spectroscopy and resonance Raman spectroscopy to KR2 in liposomes under hydration-controlled conditions. A large increase in the hydrogen-out-of-plane (HOOP) vibration at 947 (H-C11=C12-H Au mode) and moderate increases at 893 (C7-H and C10-H) and 808 (C14-H) cm-1 were observed under dehydrated conditions, which were assigned by using systematically deuterated retinal. Moreover, the Asn variant at Asp116, which functions as a counter ion for the protonated retinal Schiff base (PRSB), caused a large redshift in the absorption maximum and constitutive increase in the HOOP modes under hydrated and dehydrated conditions. The protonation of a counter ion at Asp116 clearly causes a redshift in the absorption maximum as the all-trans retinal chromophore twists upon dehydration. Namely, the results strongly suggested that water molecules are important for maintaining the hydrogen-bonding network at the PRSB and deprotonation state of Asp116 in KR2.

Tracking twisted intramolecular charge transfer and isomerization dynamics in 9-(2,2-dicyanovinyl) julolidine using femtosecond stimulated Raman spectroscopy

Understanding the mechanism of how micro-environments affect molecular rotors helps the design and development of molecular sensors. Here, we utilized femtosecond stimulated Raman spectroscopy, helped by quantum chemical calculation, to study the structural dynamics of 9- (2,2-dicyanovinyl) julolidine in cyclohexane, THF, and DMSO solvents. The obtained hydrogen out-of-plane (HOOP) mode and symmetric/anti-symmetric stretching of two nitriles (C≡N) indicate the rotation of the C7=C8 double bond and C4–C7 single bond in the excited-state which provide two non-radiative decay channels to effectively quench the excited-state population on local excited (LE) state via isomerization and twisted intramolecular charge transfer (TICT). In nonpolar solvent, the excited molecule in the LE state radiatively relaxes to the ground state or performs rotation motions via isomerization and TICT to deactivate fluorescence in the LE state. In the polar solvent, the isomerization plays a role to quench the LE state population; simultaneously, an ultrafast intramolecular charge transfer (ICT) from LE state to emissive ICT state was followed by an TICT between ICT state and dark ICT’ state.

Tracking of the molecular geometrical changes in the primary event of photoinduced ring-opening reactions of a spiropyran model in gas phase

Spiropyran (SP) is one of the photochromic molecules that have been extensively studied because of their huge commercial applications. When exposed to the UV light, SP undergoes a molecular arrangement to generate an open-form merocyanine (MC), which can revert to the closed-form photochemically or thermally. A sequential mechanism of the photoisomerisation involved in the photochromic process still remains controversial. The current work aimed at providing a detailed and comprehensive picture of the ring-opening process of the spiropyran by the combination of the CASPT2/CASSCF static electronic structure calculations and on-the-fly surface hopping dynamic simulations. Four different reaction pathways were observed in our results. The internal conversion process (IC) driven by C–O bond rapture can generate both the ring-opening and closed products on the S0 state, while generating only closed forms when driven by a hydrogen-out-of-plane (HOOP) mode. In addition, the ring-opening process of the spiropyran can also occur on the excited state. The C–N bond dissociation was not observed in our simulations results. The current investigation shed light on the complex mechanism of the SP-MC ring-opening process, which would provide helpful information for further design or improvement of SP-MC-based photochromic devices.

Modulation of Light Energy Transfer from Chromophore to Protein in the Channelrhodopsin ReaChR

The function of photoreceptors relies on efficient transfer of absorbed light energy from the chromophore to the protein to drive conformational changes that ultimately generate an output signal. In retinal-binding proteins, mainly two mechanisms exist to store the photon energy after photoisomerization: 1) conformational distortion of the prosthetic group retinal, and 2) charge separation between the protonated retinal Schiff base (RSBH+) and its counterion complex. Accordingly, energy transfer to the protein is achieved by chromophore relaxation and/or reduction of the charge separation in the RSBH+-counterion complex. Combining FTIR and UV-Vis spectroscopy along with molecular dynamics simulations, we show here for the widely used, red-activatable Volvox carteri channelrhodopsin-1 derivate ReaChR that energy storage and transfer into the protein depends on the protonation state of glutamic acid E163 (Ci1), one of the counterions of the RSBH+. Ci1 retains a pKa of 7.6 so that both its protonated and deprotonated forms equilibrate at physiological conditions. Protonation of Ci1 leads to a rigid hydrogen-bonding network in the active-site region. This stabilizes the distorted conformation of the retinal after photoactivation and decelerates energy transfer into the protein by impairing the release of the strain energy. In contrast, with deprotonated Ci1 or removal of the Ci1 glutamate side chain, the hydrogen-bonded system is less rigid, and energy transfer by chromophore relaxation is accelerated. Based on the hydrogen out-of-plane (HOOP) band decay kinetics, we determined the activation energy for these processes in dependence of the Ci1 protonation state.

Nonadiabatic Dynamics Simulations on Early-Time Photochemistry of Spirobenzopyran.

Photoinduced ring-opening, decay, and isomerization of spirobenzopyran have been explored by the OM2/MRCI nonadiabatic dynamics simulations based on Tully's fewest-switches surface hopping scheme. The efficient S1 to S0 internal conversion as observed in experiments is attributed to the existence of two efficient excited-state decay pathways. The first one is related to the C-N dissociation, and the second one is done to the C-O dissociation. The C-O dissociation pathway is dominant, and more than 90% trajectories decay to the S0 state via the C-O bond-fission related S1/S0 conical intersections. Near these regions in the S0 state, trajectories can either return to spirobenzopyran or proceed to various intermediates including merocyanine via a series of bond rotations. Our nonadiabatic dynamics simulations also demonstrate that the hydrogen-out-of-plane (HOOP) motion is important for efficient and ultrafast excited-state deactivation. On the other hand, we have also found that the replacement of methyl groups by hydrogen atoms in spirobenzopyran can artificially introduce different intramolecular hydrogen transfers leading to hydrogen-transferred intermediates. This finding is important for the community and demonstrates that such a kind of structural truncation, sometimes, could be problematic, leading to incorrect photodynamics. Our present work provides valuable insights into the photodynamics of spirobenzopyran, which could be helpful for the design of spiropyran-based photochromic materials.

The origin of absorptive features in the two-dimensional electronic spectra of rhodopsin.

In rhodopsin, the absorption of a photon causes the isomerization of the 11-cis isomer of the retinal chromophore to its all-trans isomer. This isomerization is known to occur through a conical intersection (CI) and the internal conversion through the CI is known to be vibrationally coherent. Recently measured two-dimensional electronic spectra (2DES) showed dramatic absorptive spectral features at early waiting times associated with the transition through the CI. The common two-state two-mode model Hamiltonian was unable to elucidate the origin of these features. To rationalize the source of these features, we employ a three-state three-mode model Hamiltonian where the hydrogen out-of plane (HOOP) mode and a higher-lying electronic state are included. The 2DES of the retinal chromophore in rhodopsin are calculated and compared with the experiment. Our analysis shows that the source of the observed features in the measured 2DES is the excited state absorption to a higher-lying electronic state and not the HOOP mode.

FTIR Analysis of a Light-driven Inward Proton-pumping Rhodopsin at 77 K.

Parvularcula oceani xenorhodopsin (PoXeR) is a light-driven inward proton pump that was discovered from deep ocean marine bacteria. PoXeR is categorized into the same family of Anabaena sensory rhodopsin (ASR) that functions as a photochromic sensor. In this study, we applied light-induced difference Fourier-transform infrared (FTIR) spectroscopy to PoXeR at 77 K, and the obtained spectra were compared with those of ASR. The structure and structural changes in the primary processes of PoXeR are common to all microbial rhodopsins. The red-shifted K formation (PoXeRK ) was accompanied by retinal photoisomerization from the all-trans to 13-cis form resulting in a distorted structure of the retinal. The observed hydrogen out-of-plane (HOOP) vibrations were sensitive to H/D exchange, indicating that the chromophore distortion by retinal isomerization is located near the Schiff base region in PoXeR. The hydrogen-bonding strength of the protonated Schiff base is similar between PoXeR and ASR, whose acceptor is presumably a water molecule. Unlike ASR, however, PoXeR contains strongly hydrogen-bonded water (O-D stretch at 2277 cm-1 in D2 O), which is also the case for outward proton pumps. The detailed structure, structural changes upon retinal photoisomerization, as well as functional correlation in PoXeR are discussed based on the present FTIR study.

A Nonadiabatic Excited State Molecular Mechanics/Extended Hückel Ehrenfest Method

In this work we study intramolecular vibronic relaxation effects and photoisomerization dynamics in atomistic molecular systems by means of a mixed self-consistent quantum–classical Ehrenfest formalism for nonadiabatic molecular dynamics (NA-MD) in the excited state. The quantum mechanical part of the method is based on the extended Hückel formalism, whereas the nuclei are treated with the molecular mechanics method. The self-consistent coupling between quantum and classical degrees of freedom is achieved by nonadiabatic Hellmann–Feynman–Pulay forces that conserve the total (quantum–classical) energy. Moreover, this work demonstrates the capabilities of a method that combines two simple though efficient computational schemes to describe complex excited state effects such as photoisomerization dynamics of stilbene and azobenzene molecules. In particular, for stilbene we point out the key importance of hydrogen out-of-plane (HOOP) modes to trigger the isomerization of the phenyl rings. For azobenzene, we identify the inversion-assisted rotation and the associated pedal-cycle motion of the CNNC dihedral as the driving isomerization mechanism. The method can also be applied for NA-MD studies of molecules adsorbed on extended solid surfaces and charge transfer processes.

Detailed Molecular Dynamics of the Photochromic Reaction of Spiropyran: A Semiclassical Dynamics Study

A realistic semiclassical dynamics simulation study is reported for the photoinduced ring-opening reaction of spiropyran. The main simulation results show that one pathway involves hydrogen out-of-plane (HOOP) torsion of phenyl ring nearby N atom in 254 fs on the excited state and the isomerization from cis- to trans-SP that is complete in about 10 ps on the ground state after the electron transitionπσ*; the other dominate pathway corresponds to the ring-opening reaction of trans-SP to form the most stable merocyanine (MC) product. Unlike the previous theoretical finding, one C−C bond cleavage on the real molecule rather than the C−N dissociation of the model one is more probable than the ring-opening reaction after the photoexcitation of SP. The simulation findings provide more important complementarity for interpreting experimental observations, confirming the previously theoretical studies of photochromic ring-opening process and even supplying other possible reaction mechanisms.

Mapping the Excited State Potential Energy Surface of a Retinal Chromophore Model with Multireference and Equation-of-Motion Coupled-Cluster Methods.

The photoisomerization of the retinal chromophore of visual pigments proceeds along a complex reaction coordinate on a multidimensional surface that comprises a hydrogen-out-of-plane (HOOP) coordinate, a bond length alternation (BLA) coordinate, a single bond torsion and, finally, the reactive double bond torsion. These degrees of freedom are coupled with changes in the electronic structure of the chromophore and, therefore, the computational investigation of the photochemistry of such systems requires the use of a methodology capable of describing electronic structure changes along all those coordinates. Here, we employ the penta-2,4-dieniminium (PSB3) cation as a minimal model of the retinal chromophore of visual pigments and compare its excited state isomerization paths at the CASSCF and CASPT2 levels of theory. These paths connect the cis isomer and the trans isomer of PSB3 with two structurally and energetically distinct conical intersections (CIs) that belong to the same intersection space. MRCISD+Q energy profiles along these paths provide benchmark values against which other ab initio methods are validated. Accordingly, we compare the energy profiles of MRPT2 methods (CASPT2, QD-NEVPT2, and XMCQDPT2) and EOM-SF-CC methods (EOM-SF-CCSD and EOM-SF-CCSD(dT)) to the MRCISD+Q reference profiles. We find that the paths produced with CASSCF and CASPT2 are topologically and energetically different, partially due to the existence of a "locally excited" region on the CASPT2 excited state near the Franck-Condon point that is absent in CASSCF and that involves a single bond, rather than double bond, torsion. We also find that MRPT2 methods as well as EOM-SF-CCSD(dT) are capable of quantitatively describing the processes involved in the photoisomerization of systems like PSB3.

Multiple Pathways for the Primary Step of the Spiropyran Photochromic Reaction: A CASPT2//CASSCF Study

CASSCF and CASPT2 studies on the reaction mechanism of the photochromic ring-opening process of a spiropyran (SP) (1',3',3'-trimethylspiro-[2H-1-benzopyran-2,2'-indoline], also known as BIPS) have been performed and possible excited-state C-O (and C-N) bond cleavage pathways and S1-to-S0 nonadiabatic transition channels have been explored. (1) The C-O bond dissociation in SP does not follow a conical-intersection mechanism that has been proposed in a model study with a simplified benzopyran. The CASSCF-optimized crossing points are actually avoided crossings with a large S1-S0 energy gap at the CASPT2 level; thus, they could not act as efficient S1-to-S0 funnels. (2) C-O bond cleavage paths on S1 leading to both the CCC (cis-cis-cis with respect to the configuration around α, β, γ) and TCC (trans-cis-cis) intermediates of merocyanine (MC) are barrierless, in line with the experimentally observed ultrafast formation of MC. (3) An unexpected low-energy hydrogen-out-of-plane (HOOP) valley on the (π→σ*) surface was located not far from the C-O bond cleavage path and was suggested to be an efficient S1-to-S0 nonadiabatic decay channel. Triggered by the active HOOP mode, the molecule can easily access the S1-HOOP valley and then make a transition to the S0 surface through the narrow S1-S0 gap that exists in an extended region. Nonadiabatic decay through a conical intersection on C-N dissociation path as well as the HOOP funnel is responsible for high internal conversion yields of SP. These findings shedding light on the complex mechanism of SP-MC interconversion provide fundamental information for design spiropyran-based photochromic devices.

Nonlinear optical signatures of ultraviolet light-induced ring opening in α-terpinene

Photoinduced electrocyclic ring opening reactions in conjugated cylcoalkenes are among the most elementary processes in organic chemistry. One prototypical ring opening reaction transforms cyclohexadiene into hexatriene. It is known that a sequence of sub-100 fs internal conversion transitions precedes bond breaking in cyclohexadiene and some of its derivatives. However, these excited state dynamics have never been directly monitored in solution because of insufficient time resolution. Here we aim to uncover the extraordinary photophysics behind related ultrafast internal conversion processes in a derivative of cyclohexadiene, α-terpinene (α-TP), solvated in cyclohexane. Transient absorption anisotropy experiments conducted with 20 fs laser pulses at 267 nm expose non-exponential depopulation kinetics for the ππ* electronic state of α-TP. Our data show that population transfer rapidly accelerates within the first 100 fs after photoexcitation. In addition, recurrences in two-dimensional photon echo (2DPE) line shapes reveal strong vibronic coupling in a normal mode near 523 cm−1, which involves torsions of the C=C bonds and hydrogen out-of-plane (HOOP) wagging on a vinyl group. With the support of several experiments, we hypothesize that the excited state wavepacket inα-TP undergoes several recurrences in the C=C stretching coordinate before displacement along the C=C torsion/vinyl HOOP coordinate finally sets it free from the Franck–Condon region of the potential energy surface. The unconfined wavepacket departs the ππ* electronic state by way of a conical intersection with a lower energy excited state. The present observations are made possible by recent improvements to both the time resolution and detection sensitivity of our experimental setup. This work demonstrates that it is now possible to acquire 2DPE signals in the deep ultraviolet, which are comparable with high-quality measurements in the visible spectral region. These technical developments open the door to studies of many beautiful models for elementary chemical dynamics.

Mixed Potential Energy Surfaces of the Ultrafast Isomerization of Retinal in Bacteriorhodopsin

We observe, using electronic two-dimensional photon echo spectroscopy, that the cis and trans potential energy surfaces of the ultrafast isomerization of retinal in bacteriorhodopsin are mixed via the hydrogen out of plane (HOOP) mode.

Sampling excited state dynamics: influence of HOOP mode excitations in a retinal model

Zero point energy and classical thermal sampling techniques are compared in semi-classical photodynamics of the pentadienyliminium cation, a minimal retinal model. Using both methods, the effects of vibrational hydrogen-out-of-plane (HOOP) excitations on the photo-reactivity are probed at the ab initio CASSCF level. With 2376 individual trajectories the calculations reveal a clear picture of the relation between the excited state reaction coordinate, surface crossing and product generation. The productivity is strongly coupled with hydrogen torsion and the number of hopping attempts before the molecule finally decays.

Photoinduced Isomerization of the Photoactive Yellow Protein (PYP) Chromophore: Interplay of Two Torsions, a HOOP Mode and Hydrogen Bonding

We report on a detailed theoretical analysis, based on extensive ab initio calculations at the CC2 level, of the S(1) potential energy surface (PES) of the photoactive yellow protein (PYP) chromophore. The chromophore's photoisomerization pathway is shown to be fairly complex, involving an intimate coupling between single-bond and double-bond torsions. Furthermore, these torsional modes are shown to couple to a third coordinate of hydrogen out-of-plane (HOOP) type whose role in the isomerization is here identified for the first time. In addition, it is demonstrated that hydrogen bonding at the phenolate moiety of the chromophore can hinder the single-bond torsion and thus facilitates double-bond isomerization. These results suggest that the interplay between intramolecular factors and H-bonding determines the isomerization in native PYP.

UV resonance Raman study of cation-π interactions in an indole crown ether.

UV resonance Raman (UVRR) spectroscopy is used to probe changes in vibrational structure associated with cation-π interactions for the most prevalent amino acid π -donor, tryptophan. The model compound studied here is a diaza crown ether with two indole substituents. In the presence of sodium or potassium sequestered in the crown ether, or a protonated diaza group on the compound, the indole moieties participate in a cation-π interaction in which the pyrrolo group acts as the primary π-donor. Systematic shifts in relative intensity in the 760-780 cm-1 region are observed upon formation of this cation-π interaction; we propose that these modifications reflect shifts of the delocalized, ring-breathing W18 and hydrogen-out-of-plane (HOOP) vibrational modes in this spectral region. The observed changes are attributed to perturbations of the π-electron density as well as of normal modes that involve large displacement of the hydrogen atom on the C2 position of the pyrrole ring. Modest variations in the UVRR spectra for the three complexes studied here are correlated to differences in cation-π strength. Specifically, the UVRR spectrum of the sodium-bound complex differs from those of the potassium-bound or protonated-diaza complexes, and may reflect the observation that the C2 hydrogen atom in the sodium-bound complex exhibits the greatest perturbation relative to the other species. Normal modes sensitive to hydrogen-bonding, such as the tryptophan W10, W9, and W8 modes, also undergo shifts in the presence of the salts. These shifts reflect the strength of interaction of the indole N-H group with the iodide or hexafluorophosphate counteranion. The current observation that the W18 and HOOP normal mode regions of the indole crown ether compound are sensitive to cation-pyrrolo π interactions suggests that this region may provide reliable spectroscopic evidence of these important interactions in proteins.

Exciton Coherence and Energy Transport in the Light-Harvesting Dimers of Allophycocyanin

Femtosecond transient grating and photon echo spectroscopies with a sub-20 fs time resolution are applied to allophycocyanin (APC), a protein located at the base of the phycobilisome antenna of cyanobacteria. Coupling between pairs of phycocyanobilin pigments with nondegenerate energy levels gives rise to the four-level exciton electronic structure of APC. Spectroscopic signals obtained in multiple experiments (e.g., linear absorption, fluorescence, transient grating, 2D Fourier transform photon echo) are used to constrain the parameters of a Frenkel exciton Hamiltonian. Comparison between experiment and theory yields a robust microscopic understanding of the electronic and nuclear relaxation dynamics. In agreement with previous work, transient absorption anisotropy establishes that internal conversion between the exciton states of the dimer occurs with time constants of 35, 220, and 280 fs. The sub-100 fs dynamics are decomposed into three distinct relaxation processes: electronic population transfer, intramolecular vibrational energy redistribution, and the dephasing of electronic and nuclear coherences. Model calculations show that the sub-100 fs red-shift in the transient absorption signal spectrum reflects interference between stimulated emission (ESE) and excited state absorption (ESA) signal components. It is also established that the pigment fluctuations in the dimer are not well-correlated, although further experiments will be required to precisely quantify the amount of correlation. The findings of this paper suggest that the light harvesting function of APC is enhanced by nondegeneracy of the pigments comprising the dimer and strong vibronic coupling of intramolecular modes on the phycocyanobilins. We find that the exciton states are 96% localized to the individual molecular sites within a particular dimer. Localization of the transition densities, in turn, is suggested to promote significant vibronic coupling which serves to both broaden the absorption line shape and open channels for fast internal conversion. The dominant internal conversion channel is assigned to a promoting mode near 800 cm(-1) involving hydrogen out-of-plane (HOOP) wagging motion similar to that observed in phytochrome and retinal. This rate enhancement ensures that all photoexcitations quickly and efficiently relax to the electronic origin of the lower energy exciton state from which energy transfer to the reaction center occurs.

Acceleration of the Z to E photoisomerization of penta-2,4-dieniminium by hydrogen out-of-plane motion: theoretical study on a model system of retinal protonated Schiff base

We report the result of comparison between two reaction coordinates [on the potential energy surface of the first excited state (S(1))] produced by CASSCF and these energies recalculated by MRMP2 in the Z to E photoisomerization of penta-2,4-dieniminium (PDI) as the minimal model of the retinal protonated Schiff base (RPSB). One coordinate is the S(1) state minimum-energy-path (MEP) in mass-weighted coordinates from the S(1) vertically excited point, where a strong hydrogen-out-of plane (HOOP) motion is not exhibited. The energy profile of the S(1) MEP at the MRMP2//CASSCF level shows a barrier for the rotation around the reactive C-C and hits the S(1)/S(0) degeneracy space where the central C-C-C-C dihedral angle is distorted by 65 degrees . The other coordinate is an S(1) coordinate obtained by the relaxed scan strategy. The relaxed coordinate along the central C-C-C-C dihedral angle, which we call the HOOP coordinate, shows strong HOOP motion. According to the MRMP2//CASSCF calculation, there is no barrier on the HOOP coordinate. Furthermore, the S(1) to S(0) transition may be possible without the large skeletal deformation by HOOP motion because the HOOP coordinate encounters the S(1)/S(0) degeneracy space where the central C-C-C-C dihedral angle is distorted by only 40 degrees . Consequently, if PDI is a suitable model molecule for the RPSB as often assumed, the 11-cis to all-trans photoisomerization is predicted to be accelerated by the HOOP motion.

Structural Changes of Salinibacter Sensory Rhodopsin I upon Formation of the K and M Photointermediates

Sensory rhodopsin I (SRI) is one of the most interesting photosensory receptors in nature because of its ability to mediate opposite signals depending on light color by photochromic one-photon and two-photon reactions. Recently, we characterized SRI from eubacterium Salinibacter ruber (SrSRI). This protein allows more detailed information about the structure and structural changes of SRI during its action to be obtained. In this paper, Fourier transform infrared (FTIR) spectroscopy is applied to SrSRI, and the spectral changes upon formation of the K and M intermediates are compared with those of other archaeal rhodopsins, SRI from Halobacterium salinarum (HsSRI), sensory rhodopsin II (SRII), bacteriorhodopsin (BR), and halorhodopsin (HR). Spectral comparison of the hydrogen out-of-plane (HOOP) vibrations of the retinal chromophore in the K intermediates shows that extended choromophore distortion takes place in SrSRI and HsSRI, as well as in SRII, whereas the distortion is localized in the Schiff base region in BR and HR. It appears that sensor and pump functions are distinguishable from the spectral feature of HOOP modes. The HOOP band at 864 cm(-1) in SRII, important for negative phototaxis, is absent in SrSRI, suggesting differences in signal transfer mechanism between SRI and SRII. The strongly hydrogen-bound water molecule, important for proton pumps, is observed at 2172 cm(-1) in SrSRI, as well as in BR and SRII. The formation of the M intermediate accompanies the appearance of peaks at 1753 (+) and 1743 (-) cm(-1), which can be interpreted as the protonation signal of the counterion (Asp72) and the proton release signal from an unidentified carboxylic acid, respectively. The structure and structural changes of SrSRI are discussed on the basis of the present infrared spectral comparisons with other rhodopsins.

Fs photo-isomerization in bacteriorhodopsin by few-cycle pulses

We have build up non-collinear optical parametric amplifier (NOPA) laser system with adaptive pulse tailoring, which enables to generate sub-4-fs laser pulses. Using the ultrashort laser pulses generated by NOPA, we have performed pump–probe measurement of bacteriorhodopsin (bR) using ultrashort pulses, which can observe real-time frequencies of molecular vibrations. Both of the spectra of pump and probe pulses are nearly the same and cover a spectral range from 500 to 710 nm with a nearly constant phase. The time-gated Fourier transform of the measured absorbance change trace revealed that the frequency of the C C stretching mode was modulated along with the torsion motion around C 13 C 14 during the photoisomerization of bR. It shows the bond length of C 13 C 14 is modulated on the deformation around C 13 C 14 during the photoisomerization. The hydrogen-out-of-plane (HOOP) mode and the in-plane C C–H bending mode coupled with C–C stretching mode are found to be intermixed with each other in the same period of the torsion motion. It also shows us the real-time conformation change of the molecule, which makes both of the modes indistinguishable.