Norio Hamada

Ultrafast photoreactions in protein nanospaces as revealed by fs fluorescence dynamics measurements on photoactive yellow protein and related systems

We have investigated primary processes of ultrafast photoreactions of various photoresponsive proteins by means of fs fluorescence dynamics measurements. Based on these studies, the effects of the protein nanospace (PNS), containing the chromophore, on the dynamics and mechanisms of the ultrafast and highly efficient reactions of these proteins have been elucidated. In this article, we discuss mainly the results of our studies on ultrafast photoisomerization of photoactive yellow protein (PYP), its mutants and analogues. The chromophore of the PYP, deprotonated coumaric acid thioester (O−-phenyl-CHCH–CO–S–CH2–), fixed in PNS by hydrogen (H) bonding interactions at the head part, O−-phenyl-, and by covalent bonding at the tail part, –CO–S–, undergoes ultrafast twisting by flipping the thioester bond, owing to the intrachromophore head to tail charge transfer caused by the photoexcitation. In the site-directed mutants where the PNS structure is looser and more disordered, the photoinduced twisting reaction becomes slowed compared with the wild-type PYP and moreover, the twisting becomes much slower in the denatured PYP, showing the supreme importance of more regulated PNS for the fast twisting. We found also coherent vibrations in the fluorescence decay curves which might be coupled with the twisting. Furthermore, we found for a PYP analogue with replaced chromophore ultrafast dynamic Stokes shift of fluorescence rather than the quenching due to twisting, indicating the importance of chromophore-PNS fine adjustment for the ultrafast twisting.

Coherent oscillations in ultrafast fluorescence of photoactive yellow protein.

The ultrafast photoinduced dynamics of photoactive yellow protein in aqueous solution were studied at room temperature by femtosecond fluorescence spectroscopy using an optical Kerr-gate technique. Coherent oscillations of the wave packet were directly observed in the two-dimensional time-energy map of ultrafast fluorescence with 180 fs time resolution and 5 nm spectral resolution. The two-dimensional map revealed that four or more oscillatory components exist within the broad bandwidth of the fluorescence spectrum, each of which is restricted in the respective narrow spectral region. Typical frequencies of the oscillatory modes are 50 and 120 cm(-1). In the landscape on the map, the oscillatory components were recognized as the ridges which were winding and descending with time. The amplitude of the oscillatory and winding behaviors is a few hundred cm(-1), which is the same order as the frequencies of the oscillations. The mean spectral positions of the oscillatory components in the two-dimensional map are well explained by considering the vibrational energies of intramolecular modes in the electronic ground state of the chromophore. The entire view of the wave packet oscillations and broadening in the electronic excited state, accompanied by fluorescence transitions to the vibrational sublevels belonging to the electronic ground state, was obtained.

Ultrafast Hydrogen-Bonding Dynamics in the Electronic Excited State of Photoactive Yellow Protein Revealed by Femtosecond Stimulated Raman Spectroscopy

The ultrafast structural dynamics in the electronic excited state of photoactive yellow protein (PYP) is studied by femtosecond stimulated Raman spectroscopy. Stimulated Raman spectra in the electronic excited state, S(1), can be obtained by using a Raman pump pulse in resonance with the S(1)-S(0) transition. This is confirmed by comparing the experimental results with numerical calculations based on the density matrix treatment. We also investigate the hydrogen-bonding network surrounding the wild-type (WT)-PYP chromophore in the ground and excited states by comparing its stimulated Raman spectra with those of the E46Q-PYP mutant. We focus on the relative intensity of the Raman band at 1555 cm(-1), which includes both vinyl bond C═C stretching and ring vibrations and is sensitive to the hydrogen-bonding network around the phenolic oxygen of the chromophore. The relative intensity for the WT-PYP decreases after actinic excitation within the 150 fs time resolution and reaches a similar intensity to that for E46Q-PYP. These observations indicate that the WT-PYP hydrogen-bonding network is immediately rearranged in the electronic excited state to form a structure similar to that of E46Q-PYP.

Selective Photoinduced Energy Transfer from a Thiophene Rotaxane to Acceptor

An energy transfer process was investigated using cyclodextrin-oligothiophene rotaxanes (2T-[2]rotaxane). The excited energy of 2T-[2]rotaxane is transferred to the sexithiophene derivative which is included in the cavity of β-CD stoppers of 2T-[2]rotaxane.

Structure of photoactive yellow protein (PYP) E46Q mutant at 1.2 A resolution suggests how Glu46 controls the spectroscopic and kinetic characteristics of PYP.

Photoactive yellow protein from Ectothiorhodospira halophila is a photoreceptor protein involved in the negative phototaxis of this bacterium. Its chromophore (p-coumaric acid) is deprotonated in the ground state, which is stabilized by a hydrogen-bond network between Tyr42, Glu46 and Thr50. Glu46 is a key residue as it has been suggested that the proton at Glu46 is transferred to the chromophore during its photoconversion from the dark state to the signalling state. The structure of E46Q mutant protein was determined at 1.2 A resolution, revealing that the phenolic O atom of p-coumaric acid is hydrogen bonded to NH(2) of Gln46 in E46Q with a longer distance (2.86 +/- 0.02 A) than its distance (2.51 A) to Glu46 OH in the wild type. This and the decreased thermal stability of E46Q relative to the wild type show that this hydrogen bond is weakened in the E46Q mutant compared with the corresponding bond in the wild type. Several characteristic features of E46Q such as an alkali shift in the pK(a) and the rapid photocycle can be explained by this weakened hydrogen bond. Furthermore, the red shift in the absorption maximum in E46Q can be explained by the delocalization of the electron on the phenolic oxygen of p-coumaric acid owing to the weakening of this hydrogen bond.

Photo-induced protein dynamics measured by femtosecond time-resolved luminescence

Abstract The early stage in the photo-induced dynamics of photoactive yellow protein in aqueous solution has been investigated at room temperature by the femtosecond luminescence spectroscopy using an optical Kerr-gate technique. Remarkable oscillatory components have been directly observed with the time resolution of 180 fs and the spectral resolution of 5 nm . Mapping the phase of oscillations in time at each wavelength reveals that each oscillation is restricted in the respective narrow spectral region, the period and amplitude of which are 500– 800 fs and 100– 200 cm −1 , respectively. Since the mean spectral position of each component seems to be related to the molecular vibrational energy, the oscillatory components are possibly attributed to the combination of the intramolecular vibrations of the chromophore and the surrounding protein motions.

Transient vibronic structure in ultrafast fluorescence spectra of photoactive yellow protein.

The ultrafast photo‐induced dynamics of wild‐type photoactive yellow protein and its site‐directed mutant of E46Q in aqueous solution was studied at room temperature by femtosecond fluorescence spectroscopy using the optical Kerr‐gate method. The vibronic structure appears, depending on the excitation photon energy, in the time‐resolved fluorescence spectra just after photoexcitation, which winds with time and disappears on a time scale of sub‐picoseconds. This result indicates that the wavepacket is localized in the electronic excited state followed by dumped oscillations and broadening, and also that the initial condition of the wavepacket prepared depending on the excitation photon energy affects much the following ultrafast dynamics in the electronic excited state.

Vibrational Energy Flow in Photoactive Yellow Protein Revealed by Infrared Pump–Visible Probe Spectroscopy

Vibrational energy flow in the electronic ground state of photoactive yellow protein (PYP) is studied by ultrafast infrared (IR) pump-visible probe spectroscopy. Vibrational modes of the chromophore and the surrounding protein are excited with a femtosecond IR pump pulse, and the subsequent vibrational dynamics in the chromophore are selectively probed with a visible probe pulse through changes in the absorption spectrum of the chromophore. We thus obtain the vibrational energy flow with four characteristic time constants. The vibrational excitation with an IR pulse at 1340, 1420, 1500, or 1670 cm(-1) results in ultrafast intramolecular vibrational redistribution (IVR) with a time constant of 0.2 ps. The vibrational modes excited through the IVR process relax to the initial ground state with a time constant of 6-8 ps in parallel with vibrational cooling with a time constant of 14 ps. In addition, upon excitation with an IR pulse at 1670 cm(-1), we observe the energy flow from the protein backbone to the chromophore that occurs with a time constant of 4.2 ps.

Ultrafast Dynamics of Photoactive Yellow Protein via the Photoexcitation and Emission Processes

Pump‐dump fluorescence spectroscopy was performed for photoactive yellow protein (PYP) at room temperature. The effect of the dump pulse on the population of the potential energy surface of the electronic excited state was examined as depletion in the stationary fluorescence intensity. The dynamic behavior of the population in the electronic excited state was successfully probed in the various combinations of the pump‐dump delay, the dump‐pulse wavelength, the dump‐pulse energy and the observation wavelength. The experimental results were compared with the results obtained by the femtosecond time‐resolved fluorescence spectroscopy.

Wide-bandgap nonlinear crystal LiGaS 2 for femtosecond mid-infrared spectroscopy with chirped-pulse upconversion.

Femtosecond time-resolved mid-infrared (MIR) spectroscopy based on chirped-pulse upconversion is a promising method for observing molecular vibrational dynamics. A quantitative study on nonlinear media for upconversion is still essential for wide applications, particularly at the frequencies below 2000  cm-1. We evaluate wide-bandgap nonlinear crystals of Li-containing ternary chalcogenides based on their performance as the upconversion medium for femtosecond MIR spectroscopy. The upconversion efficiency is measured as a function of the MIR pulse frequency and the chirped pulse energy. LiGaS2 is found to be an efficient crystal for the upconversion of MIR pulses in a wide frequency range of 1100-2700  cm-1, especially below 2000  cm-1. By using LiGaS2 as an efficient upconversion crystal, we develop a MIR pump-probe spectroscopy system with a spectral resolution of 2.5  cm-1, a time resolution of 0.2 ps, and a probe window of 120  cm-1. Vibrational relaxation dynamics of CO stretching modes of Mn2(CO)10 in cyclohexane and bovine serum albumin in D2O are demonstrated with a high signal-to-noise ratio.