Time-resolved Transient Grating Method Research Articles

Photoreaction of photoactivated adenylate cyclase from cyanobacterium Microcoleus chthonoplastes.

The photochemical reaction of photoactivated adenylate cyclase from cyanobacterium Microcoleus chthonoplastes PCC 7420 (mPAC), which consists of a Per-Arnt-Sim (PAS), a light‑oxygene-voltage (LOV), and an adenylate cyclase (AC) domain, was investigated mainly using the time-resolved transient grating method. An absorption spectral change associated with an adduct formation between its chromophore (flavin mononucleotide) and a cysteine residue was observed with a time constant of 0.66μs. After this reaction, a significant diffusion coefficient (D)-change was observed with a time constant of 38ms. The determined D-value was concentration-dependent indicating a rapid equilibrium between the dimer and tetramer. Combining the results of size exclusion chromatography and CD spectroscopy, we concluded that the photoinduced D-change was mainly attributed to the equilibrium shift from the dimer rich to the tetramer rich states upon light exposure. Since the reaction rate does not depend on concentration, the rate determining step of the tetramer formation is not the collision of proteins by diffusion, but a conformation change. The roles of the PAS and AC domains as well as the N- and C-terminal flanking helices of the LOV domain (A'α- and Jα-helices) were investigated using various truncated mutants. The PAS domain was found to be a strong dimerization site and is related to efficient signal transduction. It was found that simultaneous existence of the A'α- and Jα-helices in mPAC is important for the light-induced conformation change to lead the conformation change which induces the tetramer formation. The results suggest that the angle changes of the coiled-coil structures in the A'α and Jα-helices are essential for this conformation change. The reaction scheme of mPAC is proposed.

Sequential DNA Binding and Dimerization Processes of the Photosensory Protein EL222.

EL222 is a blue light sensor protein, which consists of a light-oxygen-voltage domain as a light sensor and a LuxR-type helix-turn-helix DNA-binding domain. The reaction dynamics of the protein-DNA binding were observed for the first time using the time-resolved transient grating method. The reaction scheme was determined, showing that photoexcited EL222 first binds DNA and the ground state EL222 monomer is subsequently associated with the complex. Rate constants on the millisecond scale were determined for these processes. In addition, binding rates for EL222 with three DNA sequences, with different binding affinities, were measured. Although EL222 binds nonspecific DNA sequences with affinities at least 5-fold lower than the target sequence affinity, the binding rates were almost the same as that for the target DNA. This observation indicates that the specific and nonspecific binding affinities are mainly controlled by differences in the dissociation of DNA binding.

Photoreaction of BlrP1: the role of a nonlinear photo-intensity sensor.

Blue-light-regulated phosphodiesterase 1 (BlrP1) is a blue light sensor protein that controls the hydrolysis of cyclic dimeric guanosine monophosphate, which regulates cellular motility, virulence, and formation of biofilms. In this report, the photoreaction dynamics of BlrP1 and its blue light sensor using a flavin adenine dinucleotide (BLUF) domain were investigated by the time-resolved transient grating method. Only a minor conformational change of the BlrP1-BLUF domain was observed. In contrast, a significant conformational change of the full-length BlrP1 was detected as a diffusion change with a time constant of 21 ms. Interestingly, the extent of the conformational change was concentration-dependent and the dimer form of BlrP1 was found to be the species that exhibited the conformational change. In combination with circular dichroic measurements, a quaternary structural change was determined to be the main origin of the diffusion change. Surprisingly, this conformational change was found to depend strongly on the excitation light intensity. This light-intensity-dependence indicates that the conformational change is induced by the photoexcitation of two monomer units of the dimer. The results suggest that BlrP1 is not only a photosensor but also a light intensity sensor possessing a nonlinear response.

Photochemistry of Arabidopsis phototropin 1 LOV1: transient tetramerization

The photochemical reaction of the LOV1 (light-oxygen-voltage 1) domain of phototropin 1 from Arabidopsis thaliana was investigated by the time-resolved transient grating method. As with other LOV domains, an absorption spectral change associated with an adduct formation between its chromophore (flavin mononucleotide) and a cysteine residue was observed with a time constant of 1.1 μs. After this reaction, a significant diffusion coefficient (D) change (D of the reactant = 8.2 × 10(-11) m(2) s(-1), and D of the photoproduct = 6.4 × 10(-11) m(2) s(-1)) was observed with a time constant of 14 ms at a protein concentration of 270 μM. From the D value of the ground state and the peak position in size exclusion chromatography, we have confirmed that the phot1LOV1 domain exists as a dimer in the dark. The D-value and the concentration dependence of the rate indicated that the phot1LOV1 domain associates to form a tetramer (dimerization of the dimer) upon photoexcitation. We also found that the chromophore is released from the binding pocket of the LOV domain when it absorbs two photons within a pulse duration, which occurs in addition to the normal photocycle reaction. On the basis of these results, we discuss the molecular mechanism of the light dependent role of the phot1LOV1 domain.

Light-Induced Conformational Change and Transient Dissociation Reaction of the BLUF Photoreceptor Synechocystis PixD (Slr1694)

The light-induced reaction of the BLUF ( blue light photoreceptor using flavin adenine dinucleotide) photoreceptor PixD from Synechocystis sp. PCC6803 (Slr1694) was investigated using the time-resolved transient grating method. A conformational change coupled with a volume contraction of 13 mL mol − 1 was observed with a time constant of 45 ms following photoexcitation. At a weak excitation light intensity, there were no further changes in volume and diffusion coefficient ( D). The determined D-value (3.7 × 10 − 11 m 2 s − 1 ) suggests that PixD exists as a decamer in solution, and this oligomeric state was confirmed by size-exclusion chromatography and blue native polyacrylamide gel electrophoresis. Surprisingly, by increasing the excitation laser power, we observed a large increase in D with a time constant of 350 ms following the volume contraction reaction. The D-value of this photoproduct species (7.5 × 10 − 11 m 2 s − 1 ) is close to that of the PixD dimer. Combined with transient grating and size-exclusion chromatography measurements under light-illuminated conditions, the light-induced increase in D was attributed to a transient dissociation reaction of the PixD decamer to a dimer. For the M93A-mutated PixD, no volume or D-change was observed. Furthermore, we showed that the M93A mutant did not form the decamer but only the dimer in the dark state. These results indicate that the formation of the decamer and the conformational change around the Met residue are important factors that control the regulation of the downstream signal transduction by the PixD photoreceptor.

Temperature-Sensitive Reaction of a Photosensor Protein YcgF: Possibility of a Role of Temperature Sensor

The spectrally silent photoreaction of a blue light sensor protein YcgF, composed of the N-terminal BLUF domain and the C-terminal EAL domain, was investigated by the time-resolved transient grating method. Comparing photoinduced reactions of full-length YcgF with that of the BLUF-linker construct, it was found that a major conformation change after photoinduced dimerization is predominantly localized on the EAL domain. Furthermore, the photoinduced conformational change displayed significant temperature dependence. This result is explained by an equilibrium of reactive and nonreactive YcgF species, with the population of photoreactive species decreasing as the temperature is lowered in the dark state. We consider that the dimer form is the nonreactive species and it is the dominant species at lower temperatures. The temperature sensitivity of the photoreaction of YcgF suggests that this protein could have a biological function as a temperature sensor as well as behaving as a light sensor.

Enthalpy and Volume Changes on the pH Jump Process Studied by the Transient Grating Technique

-nitrobenzaldehyde is studied by using the time-resolved transient grating method. For low pH solutions, the grating signal due to the proton is observed clearly without any pH indicator. The origin of the signal is attributed to a pure volume grating, from which the partial molar volume of the proton can be determined accurately. In an alkali solution, the hydroxyl ion is observed as the main species of the grating signal. The enthalpy changes of the reaction in pH=6.5 and 8.2 are determined to be –131.2 and -133.6 kJ/mol, respectively, indicating that the reaction is exothermic. The negative volume change at the lower pH solutions is due to the electrostriction by the creation of the ionic species. At the higher pH, the reaction volume changes to positive, which is mainly due to the water formation. This pH jump reaction was used to study the unfolding process of apomyoglobin.

Dynamics of Conformational Changes of Arabidopsis Phototropin 1 LOV2 with the Linker Domain

Conformational changes of Arabidopsis phot1-LOV2 with the linker (phot1-LOV2-linker) were investigated from the viewpoint of the changes in molecular volume and molecular diffusion coefficient (D) by time-resolved transient grating (TG) and transient lens (TrL) methods. Although the absorption spectrum change completes within a few microseconds, the D-value detected by the TG method decreased drastically with a time constant of 1.0 ms from 9.2(±0.4)×10−11 m2/s to 5.0(±0.3)×10−11 m2/s. This time-dependent D was interpreted in terms of the unfolding of α-helices in the linker region. The change of the α-helices was confirmed by observing the recovery of the circular dichroism intensity. The TrL signal showed that the molecular volume decreases with two time constants; 300 μs and 1.0 ms. The former time constant is close to the previously observed photo-dissociation reaction rate of the phot1-LOV2 (without the linker) dimer, and the latter one agrees well with the rate of the D-change. Considering a similar time constant of the dissociation reaction of the LOV2 dimer, we interpreted these kinetics in terms of the dissociation step of the linker region from the LOV2 domain (T390pre state). After this step, the protein volume and D are decreased significantly with the lifetime of 1.0 ms. The D decrease indicates the increase of the intermolecular interaction between the protein and water molecules. On the basis of these observations, a two-step mechanism of the linker unfolding is proposed.

Dynamics of Water-Soluble Stilbene Dendrimers upon Photoisomerization

The energy-releasing process and the conformational dynamics of water-soluble stilbene dendrimers upon photoexcitation are studied by the time-resolved transient grating method. While the energy relaxation and the conformational change are completed within 30 ns for the first generation (W-G1), slower dynamics are observed for the second (W-G2) and the third (W-G3) generations. The enthalpy change by the photoisomerization increases and the dynamics of the conformational change becomes slower with an increase in the generation. The diffusion coefficients (D) of the trans- and cis-forms of W-G2 and W-G3 are almost the same, whereas a slight difference is observed for W-G1. These characteristic features are consistently explained in terms of the electric charges on the dendron surface.

Energy Releasing and Conformational Dynamics of Stilbene Dendrimers upon Photoisomerization

The energy-releasing process and the conformational dynamics of stilbene dendrimers (tetramethoxystilbene (generation (G) 0, G1, G2, and G4) upon photoexcitation are studied by the time-resolved transient grating method. For all dendrimers, the excited triplet (T1) state formation was observed. From the time-resolved studies on energy and the molecular volumes changes, it is found that the conformational change completes with the decay of the T1 state for G0−G2. However, the dynamics is slightly slower for G4, which is attributed to the conformational change of the dendron part. The molecular volume change due to the conformational rearrangement of the dendron upon the trans to cis isomerization is slightly positive (molecular expansion). The expansion is explained in terms of the creation of the void volume surrounded by the dendron side chains. Although the diffusion coefficient (D) of the trans and cis forms of G0 is almost the same, the difference becomes larger with increasing generation. This is clear evidence for the large conformational change upon isomerization. The larger D of the trans form compared with that of the cis form is explained in terms of the surface roughness of the dendrimers.

Photoreaction of caged ATP studied by the time-resolved transient grating method.

The photoreaction of caged ATP, P3-[1-(2-nitrophenyl)ethyl]adenosine-5'-triphosphate, has been investigated using the time-resolved transient grating (TG) method. We found that a feature of the TG signal time profile depends sensitively on the grating wavenumber (q) after the photoexcitation of caged ATP. This q-dependent feature of the TG signal was interpreted based on a model where the ATP release rate is comparable to the molecular diffusion process. We found that the TG signals at various q can be consistently analyzed based on this model and the ATP release rate determined. The enthalpy and volume changes of the reaction have been determined by quantitative measurement of the grating and photoacoustic signals.

Energy and volume changes induced by photoinitiated proton releasing reaction with apomyoglobin

The photodissociation reaction of o-nitrobenzaldehyde (NBA) aqueous solution and of the NBA solution with a protein, apomyoglobin (ApoMb), were studied by the time-resolved transient grating (TG) technique. The amount of released thermal energy and the volume change accompanied with this reaction were determined by the TG and photoacoustic method. Without the protein, the photoproduct of NBA releases a proton in the solution (pH jump reaction). The time profile of the grating signal of NBA and ApoMb shows two diffusing species with diffusion coefficients of 1.39±0.15 and 0.14±0.02×10−9 m2 s−1, respectively. From the diffusion coefficients, we suggest that the photoproduct of NBA induces a molecular recombination reaction between ApoMb and another small molecule or ion in the solution.

3D1515 ファラオニスセンサリーロドプシンII及びトランスデューサー複合体の過渡回折格子法測定(18.光生物(A)視覚,一般講演,日本生物物理学会第40回年会)

3D1515 ファラオニスセンサリーロドプシンII及びトランスデューサー複合体の過渡回折格子法測定(18.光生物(A)視覚,一般講演,日本生物物理学会第40回年会)

A Spectrally Silent Transformation in the Photolysis of Octopus Rhodopsin: A Protein Conformational Change Without Any Accompanying Change of the Chromophore's Absorption

A spectrally silent transformation in the photolysis of octopus rhodopsin was detected by the time-resolved transient grating method. Our results showed that at least two photointermediates, which share the same chromophore absorption spectrum, exist after the final absorption changes. Previously, mesorhodopsin was thought to decay to the final photoproduct, acid metarhodopsin with a lifetime of 38 μs at 15°C, but the present results show that there is at least one intermediate species (called transient acid metarhodopsin) with a lifetime of 180 μs at 15°C, before forming acid metarhodopsin. This indicates that the parts of the protein distant from the chromophore are still changing even after the changes in microenvironment around the chromophore are over. From the signal intensity detected by the transient grating method, the volume change of the spectrally silent transformation was found to be Δ V = 13 ml/mol. The activation energy of the spectrally silent transformation is much lower than those of other transformations of octopus rhodopsin. Since stable acid metarhodopsin has not been shown to activate the G protein, this transient acid metarhodopsin may be responsible for G protein activation.

A pH-Jump Reaction Studied by the Transient Grating Method: Photodissociation ofo-Nitrobenzaldehyde

A proton-releasing photodissociation reaction of o-nitrobenzaldehyde is investigated by using the time-resolved transient grating method. The grating signal is very sensitive to the pH of the solution. The chemical species existing in the solution are identified from the diffusion constant of the species. For low-pH solutions, the grating signal due to the proton is observed clearly without any pH indicator. The origin of the signal is attributed to a pure volume grating, from which the partial molar volume of the proton can be determined accurately. The diffusion constant of the proton decreases with increasing the pH. This change is explained by the electrostatic interaction between the proton and the anion product, o-nitrosobenzoic anion. In an alkali solution, the hydroxyl ion is observed as the main species of the grating signal. By the quantitative measurement of the grating signal and the photoacoustic signal, the enthalpy change and the volume change of the reaction and the partial molar volumes o...

Relaxation of nitrobenzene from the excited singlet state

The lifetime of the lowest excited singlet (S1) state of nitrobenzene in ethanol was measured using a picosecond time-resolved transient grating method. The lifetime was found to be 6 ps and the rate constant of the intersystem crossing relaxation process from the S1 state was determined to be 1.3–1.6×1011 s−1. The relaxation mechanism from the S1 state is also discussed.

Photothermal Expansion of Droplets in Emulsions Detected by the Transient Grating Method

Abstract A nonlinear optical effect due to the thermal energy after the photoexcitation of a light-absorbing chromophore or a metal ion contained in micro droplets (oil in water (O/W) and water in oil (W/O)) was investigated by the time-resolved transient grating method. The temporal profiles of the diffusive components of the thermal grating signals are very different from that observed in a homogeneous solution, and also depend on the emulsion systems. The profiles were consistently analyzed in terms of the droplet heating, thermal expansion, and subsequent cooling process. The size of the droplets determined from this analysis agrees with that from the dynamic light scattering method.

Nonradiative Relaxation Processes and Electronically Excited States of Nitrobenzene Studied by Picosecond Time-Resolved Transient Grating Method

The exothermic nonradiative relaxation processes from photoexcited nitrobenzene (NB) are studied by the picosecond time-resolved transient grating method. The decay rate constants and energy of the excited NB are determined. The lifetime of the lowest excited singlet state is found to be very short ( e10 ps) and a surprisingly short lifetime of the lowest excited triplet state (480 ps) is detected. From quantitative measurements of the thermal energies released from the excited states of NB, the lowest excited triplet state is determined to be located at (22-27) 10 3 cm -1 . The triplet formation is very efficient and its quantum yield is found to be grater than 0.80. From the temperature and solvent dependence of the triplet lifetime, it is suggested that motions of the nitro group may cause the surprisingly rapid relaxation from the lowest excited triplet state to the ground state. The photophysical properties and excited states of monosubstituted nitrobenzenes are also discussed.

Picosecond Time-Resolved Transient Grating Method for Heat Detection: Excited-State Dynamics of FeCl3 and o-Hydroxybenzophenone in Aqueous Solution

Transient grating signals after photoexcitation to electronically excited states of FeCl3 and o-hydroxybenzophenone (o-HBP) in aqueous solutions were investigated at various temperatures with short-pulsed laser systems. The observed signals can be expressed by a superposition of the density grating (Dens.G) signal and the temperature grating (Temp.G) signal. Above 4 °C, an acoustic oscillation is predominant in the signal, while the acoustic oscillation disappears and a pure Temp.G signal is observed clearly at 4 °C. The rise profile of the pure Temp.G signal was analyzed in terms of the deactivation from the photoexcited state, and it is found that a very fast thermalization process takes place among the solvent molecules. The lifetime of the charge-transfer state of [Fe(OH)(H2O)5]2+ is determined to be 55 ps from the rise profile of the Temp.G signal. Using a subpicosecond laser system and the population grating signal, two time constants (400 fs and 7.5 ps) are obtained for the dynamics of o-HBP. The dynamics of the photoexcited states of FeCl3 and o-HBP are also discussed. Since the temporal response of the Temp.G component is 10−100 times shorter than that of the Dens.G signal, this signal can be used for studying ultrafast heat-releasing processes.

Dynamics of a liquid crystal molecule at a solid-liquid interface detected by the time-resolved transient grating method

Dynamics of MBBA (N-(4-methoxybenzylidene)-4- n-butylaniline) in ethanol at a liquid-solid interface is selectively detected using the transient grating method with total internal reflection for probe light. The thermal grating signal decays much faster than that in bulk liquid. This faster decay is explained by the thermal conduction from the liquid to solid phase. Species grating signal from the interface also decays faster than that from the bulk liquid. The fringe length dependence of the rate constant reveals that the diffusion constant at the interface region (49 nm) is similar to that in the bulk liquid. The faster decay of the species grating is mainly attributed to the enhanced cis-trans back isomerization of MBBA at the interface.