Phototropins (phots) are blue light sensors found in a variety of higher plants and algae. The photochemical reactions of this family of proteins have attracted much attention since their discovery. Phots have two light sensor domains called light-oxygen-voltage 1 (LOV1) and LOV2. After the formation of the characteristic adduct of the LOV domain, a conformational change of the C-terminal region of the LOV2 domain occurs, and characterizing this change is important for understanding biological function, that is, kinase activation. Here, the reaction dynamics of the Jα-helix and the extended region adjacent to the Jα-helix (connector) have been investigated. The conformation of the connector part and the Jα-helix were found to alter significantly in a two-state manner. Furthermore, the conformational change of the kinase domain was also successfully detected as a change in translational diffusion, although the CD intensity due to the kinase domain movement was almost silent. These observations indicate that the tertiary structure of the kinase domain changes. The rate of the kinase domain change is almost the same as that of the change for the LOV2-linker, suggesting that the conformational change of the linker is the rate-determining step for kinase activation.

AbstractCryptochrome (CRY), a blue light sensor protein, possesses a similar domain structure to photolyase (PHR) that, upon absorption of light, repairs DNA damage. In this review, we compare the reaction dynamics of these systems by monitoring the reaction kinetics of conformational change and intermolecular interaction change based on time-dependent diffusion coefficient measurements obtained by using the pulsed laser-induced transient grating technique. Using this method, time-dependent biomolecular interactions, such as transient dissociation reactions in solution, have been successfully detected in real time. Conformational change in (6-4) PHR has not been detected after the photoexcitation by monitoring the diffusion coefficient. However, the repaired DNA dissociates from PHR with a time constant of 50 μs, which must relate to a minor conformational change. However, CRY exhibits a considerable diffusion change with a time constant of 400 ms, which indicates that the protein–solvent interaction is changed by the conformational change. The C-terminal domain of CRY is shown to be responsible for this change.

EL222 is a blue light sensor protein consisting of a light-oxygen-voltage (LOV) domain (EL-LOV domain) at the N-terminus and a helix-turn-helix DNA-binding domain at the C-terminus. EL222 acts as a light dependent transcriptional factor. The photochemical reactions of EL222 and the light sensing properties of the LOV domain were investigated. Concentration dependent experiments revealed that the EL-LOV domain is in equilibrium between the dimer and the monomer in the dark state, and the main photoreaction is the dimerization reaction between a monomer in the ground state and that in the excited state. The equilibrium constant and the intrinsic rate constants of dimerization were determined. EL222 was found to also exhibit photoinduced dimerization even in the absence of target DNA, although the yield of the reaction was low (∼0.08 compared with that of the EL-LOV domain). This observation suggests that there are inhomogeneous conformations, open and closed types, of EL222 in solution.

Photoresponsive DNA modified with azobenzene is an attractive design molecule for efficient photoregulation of DNA hybridization, which may be used for controlling DNA functions. Although the essential step of photocontrolling DNA is the initial isomerization of the azobenzene, the dissociation/association kinetics remain unknown. Here, the time-resolved diffusion method was used to trace the dissociation/association processes of photoresponsive DNA. Although the isomerization of azobenzene occurs in picoseconds, the dissociation of the double-stranded DNA to single-stranded DNA triggered by the trans to cis isomerization takes place ∼107 times slower, with a time constant of 670 μs at 200 μM. From the concentration dependence, the dissociation and association rates were determined. Furthermore, the reaction rate from the single- to double-stranded DNA after the cis to trans isomerization was measured to be 3.6 ms at 200 μM. The difference in the melting temperatures of DNA between tethered trans- and cis-azobenzene is explained by the different rate of dissociation of the double-stranded form.

Although the relationship between structural fluctuations and reactions is important for elucidating reaction mechanisms, experimental data describing such fluctuations of reaction intermediates are sparse. In order to investigate structural fluctuations during a protein reaction, the compressibilities of intermediate species after photoexcitation of a phot1LOV2-linker, which is a typical LOV domain protein with the C-terminal linker including the J-α helix and used recently for optogenetics, were measured in the time-domain by the transient grating and transient lens methods with a high pressure optical cell. The yield of covalent bond formation between the chromophore and a Cys residue (S state formation) relative to that at 0.1 MPa decreased very slightly with increasing pressure. The fraction of the reactive species that yields the T state (linker-unfolded state) decreased almost proportionally with pressure (0.1–200 MPa) to about 65%. Interestingly, the volume change associated with the reaction was much more pressure sensitive. By combining these data, the compressibility changes for the short lived intermediate (S state) and the final product (T state) formation were determined. The compressibility of the S state was found to increase compared with the dark (D) state, and the compressibility decreased during the transition from the S state to the T state. The compressibility change is discussed in terms of cavities inside the protein. By comparing the crystal structures of the phot1LOV2-linker at dark and light states, we concluded that the cavity volumes between the LOV domain and the linker domain increase in the S state, which explains the enhanced compressibility.

YtvA is a blue light sensor protein composed of an N-terminal LOV (light–oxygen–voltage) domain, a linker helix, and the C-terminal sulfate transporter and anti-σ factor antagonist domain. YtvA is believed to act as a positive regulator for light and salt stress responses by regulating the σB transcription factor. Although its biological function has been studied, the reaction dynamics and molecular mechanism underlying the function are not well understood. To improve our understanding of the signaling mechanism, we studied the reaction of the LOV domain (YLOV, amino acids 26–127), the LOV domain with its N-terminal extension (N-YLOV, amino acids 1–127), the LOV domain with its C-terminal linker helix (YLOV-linker, amino acids 26–147), and the YLOV domain with the N-terminal extension and the C-terminal linker helix (N-YLOV-linker, amino acids 1–147) using the transient grating method. The signals of all constructs showed adduct formation, thermal diffusion, and molecular diffusion. YLOV showed no change in the diffusion coefficient (D), while the other three constructs showed a significant decrease in D within ∼70 μs of photoexcitation. This indicates that conformational changes in both the N- and C-terminal helices of the YLOV domain indeed do occur. The time constant in the YtvA derivatives was much faster than the corresponding dynamics of phototropins. Interestingly, an additional reaction was observed as a volume expansion as well as a slight increase in D only when both helices were included. These findings suggest that although the rearrangement of the N- and C-terminal helices occurs independently on the fast time scale, this change induces an additional conformational change only when both helices are present.

Aureochrome (Aureo) is a recently discovered blue light sensor protein initially from Vaucheria frigida, in which it controls blue light-dependent branch formation and/or development of a sex organ by a light-dependent change in the affinity for DNA. Although photochemical reactions of Aureo-LOV (LOV is a C-terminal light-oxygen-voltage domain) and the N-terminal truncated construct containing a bZIP (N-terminal basic leucine zipper domain) and a LOV domain have previously been reported, the reaction kinetics of the change in affinity for DNA have never been elucidated. The reactions of Aureo where the cysteines are replaced by serines (AureoCS) as well as the kinetics of the change in affinity for a target DNA are investigated in the time-domain. The dimerization rate constant is obtained as 2.8 × 104 M–1 s–1, which suggests that the photoinduced dimerization occurs in the LOV domain and the bZIP domain dimerizes using the interaction with DNA. Surprisingly, binding with the target DNA is completed very quickly, 7.7 × 104 M–1 s–1, which is faster than the protein dimerization rate. It is proposed that the nonspecific electrostatic interaction, which is observed as a weak binding with DNA, may play a role in the efficient searching for the target sequence within the DNA.

UVR8 is a recently discovered ultraviolet-B (UV-B) photoreceptor protein identified in plants and algae. In the dark state, UVR8 exists as a homodimer, whereas UV-B irradiation induces UVR8 monomerization and initiation of signaling. Although the biological functions of UVR8 have been studied, the fundamental reaction mechanism and associated kinetics have not yet been fully elucidated. Here, we used the transient grating method to determine the reaction dynamics of UVR8 monomerization based on its diffusion coefficient. We found that the UVR8 photodissociation reaction proceeds in three stages: (i) photoexcitation of cross-dimer tryptophan (Trp) pyramids; (ii) an initial conformational change with a time constant of 50 ms; and (iii) dimer dissociation with a time constant of 200 ms. We identified W285 as the key Trp residue responsible for initiating this photoreaction. Although the C-terminus of UVR8 is essential for biological interactions and signaling via downstream components such as COP1, no obvious differences were detected between the photoreactions of wild-type UVR8 (amino acids 1–440) and a mutant lacking the C-terminus (amino acids 1–383). This similarity indicates that the conformational change associated with stage ii cannot primarily be attributed to this region. A UV-B-driven conformational change with a time constant of 50 ms was also detected in the monomeric mutants of UVR8. Dimer recovery following monomerization, as measured by circular dichroism spectroscopy, was decreased under oxygen-purged conditions, suggesting that redox reactivity is a key factor contributing to the UVR8 oligomeric state.

The effect of pressure on the dissociation reaction of the TePixD decamer was investigated by high-pressure transient grating (TG). The TG signal intensity representing the dissociation reaction of the TePixD decamer significantly decreased by applying a relatively small pressure. On the other hand, the reaction rate increased with increasing pressure. The equilibrium between the pentamer and the decamer was investigated by high-pressure dynamic light scattering. The results indicated that the fraction of the decamer slightly increased in the high-pressure region. From these measurements, it was concluded that the pressure-dependent signal intensity originated from the decrease of the quantum yield of the dissociation reaction of the decamer, indicating that this reaction efficiency is very sensitive to pressure. Using densimetry at high pressures, the compressibility was found to be pressure dependent even in a relatively low pressure range. We attributed the origin of the pressure-sensitive reaction yield to the decrease of compressibility at high pressure. Because the compressibility is related to the volume fluctuation, this observation suggests that the driving force for this reaction is fluctuation of the protein. The relationship between the cavities at the interfaces of the monomer units and the reactivity is also discussed.

Knowledge of the dynamical behavior of proteins, and in particular their conformational fluctuations, is essential to understanding the mechanisms underlying their reactions. Here, transient enhancement of the isothermal partial molar compressibility, which is directly related to the conformational fluctuation, during a chemical reaction of a blue light sensor protein from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (TePixD, Tll0078) was investigated in a time-resolved manner. The UV-Vis absorption spectrum of TePixD did not change with the application of high pressure. Conversely, the transient grating signal intensities representing the volume change depended significantly on the pressure. This result implies that the compressibility changes during the reaction. From the pressure dependence of the amplitude, the compressibility change of two short-lived intermediate (I1 and I2) states were determined to be +(5.6 ± 0.6) × 10−2 cm3⋅mol−1⋅MPa−1 for I1 and +(6.6 ± 0.7)×10−2 cm3⋅mol−1⋅MPa−1 for I2. This result showed that the structural fluctuation of intermediates was enhanced during the reaction. To clarify the relationship between the fluctuation and the reaction, the compressibility of multiply excited TePixD was investigated. The isothermal compressibility of I1 and I2 intermediates of TePixD showed a monotonic decrease with increasing excitation laser power, and this tendency correlated with the reactivity of the protein. This result indicates that the TePixD decamer cannot react when its structural fluctuation is small. We concluded that the enhanced compressibility is an important factor for triggering the reaction of TePixD. To our knowledge, this is the first report showing enhanced fluctuations of intermediate species during a protein reaction, supporting the importance of fluctuations.

Phototropins are blue-light-sensitive photoreceptor proteins in plants. Phototropins consist of two LOV (light, oxygen, and voltage sensor) domains (LOV1 and LOV2) that undergo photochemical reactions. Although the photochemical reaction of the LOV2 domain has been investigated extensively, the reaction of the LOV1 domain remains unresolved. In this study, the reactions of the Arabidopsis phototropin 2 LOV1 (phot2LOV1) domain were revealed by the transient grating (TG) method. The TG signal showed a significant diffusion coefficient (D) change upon photoexcitation. This change was sensitive to the protein concentration and the observation time range. These observations were explained by assuming that there are reactive and nonreactive forms, and the fraction of these species is concentration dependent. From the concentration dependence of the dynamics, the monomer was found to form a dimer; however, the dimer does not exhibit an observable reaction. In the dark state, both species were in equilibrium and were not distinguishable spectroscopically. For the LOV1 domain with the hinge domain, the reaction scheme was the same as the LOV1 domain sample, but the D change was affected by the presence of the hinge region. This observation suggested that the hinge region undergoes a conformational change during the photoreaction.

TePixD is a blue-light sensor protein from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (TePixD Tll0078). Although the photochemistry has been examined, so far the photoproduct remains unknown. We have measured the diffusion coefficient (D) of TePixD in the dark by dynamic light scattering and have discovered a very peculiar diffusion property; the decamer oligomer has a larger D than that of the pentamer. Furthermore, D of the pentamer was found to be very close to that of the TePixD decamer photoreaction product. In order to investigate this reaction further, elution profiles of size-exclusion chromatography were measured under dark and illuminated conditions at low (40 μM) and high (1.1 mM) TePixD concentrations. On the basis of these results, we have concluded that the main photoreaction of the TePixD decamer is the dissociation into the pentamer. The secondary structure change associated with this reaction was found to be minor according to circular dichroism analysis.

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 m2 s−1, and D of the photoproduct = 6.4 × 10−11 m2 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.

Recently, conformational changes of the amino-terminal helix (A′α helix), in addition to the reported conformational changes of the carboxyl-terminal helix (Jα helix), have been proposed to be important for the regulatory function of the light-oxygen-voltage 2 domain (LOV2) of phototropin 1 from Arabidopsis. However, the reaction dynamics of the A′α helix have not been examined. Here, the unfolding reactions of the A′α and Jα helices of the LOV2 domain of phototropin 1 from Arabidopsis thaliana were investigated by the time-resolved transient grating (TG) method. A mutant (T469I mutant) that renders the A′α helix unfolded in the dark state showed unfolding of the Jα helix with a time constant of 1 ms, which is very similar to the time constant reported for the wild-type LOV2-linker sample. Furthermore, a mutant (I608E mutant) that renders the Jα helix unfolded in the dark state exhibited an unfolding process of the A′α helix with a time constant of 12 ms. On the basis of these experimental results, it is suggested that the unfolding reactions of these helices occurs independently.

PixD (Slr1694) is a blue light receptor that contains a BLUF (blue light sensors using a flavin chromophore) domain. A protein–protein interaction between PixD and a response regulator PixE (Slr1693) is essential to achieve light signal transduction for phototaxis of the species. Although the initial photochemical reaction of PixD, the red shift of the flavin absorption spectrum, has been investigated, the subsequent reaction dynamics remain largely unresolved. Only the disassembly of the PixD10–PixE5 dark complex has been characterized by static size exclusion chromatography. In this report, interprotein reaction dynamics were examined using time-resolved transient grating spectroscopy. The dissociation process was clearly observed as the light-induced diffusion coefficient change in the time domain, and the kinetics was determined. More strikingly, disassembly was found to take place only after photoactivation of two PixD subunits in the complex. This result suggests that the biological response of PixD does not follow a linear correlation with the light intensity but appears to be light-intensity-dependent.

Proteins of the cryptochrome/photolyase family share high sequence similarities, common folds, and the flavin adenine dinucleotide (FAD) cofactor, but exhibit diverse physiological functions. Mammalian cryptochromes are essential regulatory components of the 24 h circadian clock, whereas (6−4) photolyases recognize and repair UV-induced DNA damage by using light energy absorbed by FAD. Despite increasing knowledge about physiological functions from genetic analyses, the molecular mechanisms and conformational dynamics involved in clock signaling and DNA repair remain poorly understood. The (6−4) photolyase, which has strikingly high similarity to human clock cryptochromes, is a prototypic biological system to study conformational dynamics of cryptochrome/photolyase family proteins. The entire light-dependent DNA repair process for (6−4) photolyase can be reproduced in a simple in vitro system. To decipher pivotal reactions of the common FAD cofactor, we accomplished time-resolved measurements of radical formation, diffusion, and protein conformational changes during light-dependent repair by full-length (6−4) photolyase on DNA carrying a single UV-induced damage. The (6−4) photolyase by itself showed significant volume changes after blue-light activation, indicating protein conformational changes distant from the flavin cofactor. A drastic diffusion change was observed only in the presence of both (6−4) photolyase and damaged DNA, and not for (6−4) photolyase alone or with undamaged DNA. Thus, we propose that this diffusion change reflects the rapid (50 μs time constant) dissociation of the protein from the repaired DNA product. Conformational changes with such fast turnover would likely enable DNA repair photolyases to access the entire genome in cells.

Anabaena sensory rhodopsin (ASR), a microbial rhodopsin in the cyanobacterium sp. PCC7120, has been suggested to regulate cell processes in a light-quality-dependent manner (color-discrimination) through interaction with a water-soluble transducer protein (Tr). However, light-dependent ASR–Tr interaction changes have yet to be demonstrated. We applied the transient grating (TG) method to investigate protein–protein interaction between ASR with Tr. The molecular diffusion component of the TG signal upon photostimulation of ASRAT (ASR with an all-trans retinylidene chromophore) revealed that Tr dissociates from ASR upon formation of the M-intermediate and rebinds to ASR during the decay of M; that is, light induces transient dissociation of ASR and Tr during the photocycle. Further correlating the dissociation of the ASR–Tr pair with the M-intermediate, no transient dissociation was observed after the photoexcitation of the blue-shifted ASR13C (ASR with 13-cis, 15-syn chromophore), which does not produce M. This distinction between ASRAT and ASR13C, the two isomeric forms in a color-sensitive equilibrium in ASR, provides a potential mechanism for color-sensitive signaling by ASR.

A recently developed method to monitor reaction kinetics of intermolecular interaction is presented in this perspective. This method is based on time-dependent diffusion coefficient measurements using the pulsed laser induced transient grating technique. Using this method, time dependent biomolecular interactions, such as transient association and dissociation reactions in solution, have been successfully detected in real time. The principles and particular applications are described. In particular, unique features of this time-dependent diffusion coefficient method are emphasized by comparison with other techniques.

Following the disruption of the covalent bond between the cysteine and flavin of Phot1LOV2−linker, the unfolded conformation of the linker folds with a time constant of 13 ms, which is considerably (∼104 times) slower than the helix formation rate measured for an α-helical polypeptide in solution.

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.

A new numerical analysis method for experimental single-pair fluorescence resonance energy transfer (sp-FRET) data is proposed. In this method, every single data point was plotted in a style of a cumulative distribution function and dedicated to curve-fitting analysis, so that the analysis does not depend on bin size. A series of numerical simulations showed that this analysis has a more efficient and accurate resolvability of components than a fitting method based on Gaussian functions to a histogram plot. A simulated data based on experimental FRET distributions were also used to discuss the fitting errors of this method. The proposed method was applied to sp-FRET experiments of doubly dye-labeled double-strand DNA with a short sequence. Mixtures of up to three species were analyzed, and the contributions up to four subpopulations were successfully resolved.

Enthalpy and volume changes of irreversible chemical reactions were properties that were difficult to access by use of traditional thermodynamical techniques. However, recent developments of several types of spectroscopic methods enable us to measure these quantities for a variety of irreversible reactions. The principles of three experimental methods, specifically, the photoacoustic (PA), transient grating (TG) and transient lens (TrL) methods are explained, and their characteristic points are compared in detail. Enthalpy and volume changes associated with various photochemical reactions (photodissociation reactions, photoisomerization reactions, photoinduced proton-releasing reactions, electron transfer processes) and creation of the excited states of organic molecules in solution phase are reviewed, and these properties are discussed.

TePixD, a cyanobacterial sensor of blue light using flavin adenine dinucleotide (FAD) (BLUF) which exists in a decamer form, was found to exhibit photoreaction sensitive to light intensity. While the number of excited molecules increased monotonically as the laser power increased, the number of decamers exhibiting a global conformational change initially increased, and then decreased with the increase of excitation intensity. This unusual power dependence was analyzed based on a Poisson distribution equation, demonstrating that decamers containing more than one excited monomer subunit do not undergo conformational change. Our results suggest that TePixD functions not only as a photosensor, but also by sensing light intensity.

Aureochrome is a recently discovered blue light photosensor that controls a light-dependent morphology change. As a photosensor, it has a unique DNA binding domain (bZIP). Although the biological functions of aureochrome have been revealed, the fundamental photochemistry of this protein has not been elucidated. The photochemical reaction dynamics of the LOV (light, oxygen, or voltage) domain of aureochrome-1 (AUREO1-LOV) and the LOV domain with the bZIP domain (AUREO1-ZL) were studied by employing the transient-grating (TG) technique, using size-exclusion chromatography to verify results. For both samples, adduct formation takes place with a time constant of 2.8 μs. Although significant diffusion changes were observed for both AUREO1-LOV and AUREO1-ZL after adduct formation, the origins of these changes were significantly different. The TG signal of AUREO1-LOV was strongly concentration-dependent. From analysis of the signal, it was concluded that AUREO1-LOV exists in equilibrium between the monomer and dimer, and dimerization of the monomer is the main reaction, i.e., irradiation with blue light enhances the strength of the interdomain interaction. On the other hand, the reaction of AUREO1-ZL is independent of concentration, suggesting that an intraprotein conformational change occurs in the bZIP domain with a time constant of 160 ms. These results revealed the different reactions and roles of the two domains; the LOV domain acts as a photosensor, leading to a subsequent conformational change in the bZIP domain, which should change its ability to bind to DNA. A model is proposed that demonstrates how aureochrome uses blue light to control its affinity for DNA.

Reaction dynamics of a chloride ion pump protein, halorhodopsin (HR), from Natronomonas pharaonis (N. pharaonis) (NpHR) was studied by the pulsed-laser-induced transient grating (TG) method. A detailed investigation of the TG signal revealed that there is a spectrally silent diffusion process besides the absorption-observable reaction dynamics. We interpreted these dynamics in terms of release, diffusion, and uptake of the Cl− ion. From a quantitative global analysis of the signals at various grating wavenumbers, it was concluded that the release of the Cl− ion is associated with the L2 → (L2 (or N) ⇆ O) process, and uptake of Cl− occurs with the (L2 (or N) ⇆ O) →NpHR′ process. The diffusion coefficient of NpHR solubilized in a detergent did not change during the cyclic reaction. This result contrasts the behavior of many photosensor proteins and implies that the change in the H-bond network from intra- to intermolecular is not significant for the activity of this protein pump.

The photochemical reactions of Arabidopsis phototropin 2 light- oxygen-voltage domain 2 (LOV2) with the linker region (LOV2-linker), without the linker (LOV2), and LOV1 were studied using the time-resolved transient grating (TG) and transient lens (TrL) methods. Although the absorption spectra did not change after the formation of the adduct species, a small volume expansion process with a time constant of 9 ms was observed for LOV2. For the LOV2-linker, at 293 K, a volume contraction process with a time constant of 140 μs was observed in addition to a volume expansion process with 9 ms and the diffusion coefficient change with 2 ms. The reaction intermediate species were characterized on the basis of their thermodynamic properties, such as changes in enthalpy, thermal expansion, and heat capacity. For the first intermediate (S390), the values of these properties were similar to those of the ground state for both LOV2 and LOV2-linker. A relatively large thermal expansion volume (0.09 cm3mol− 1K− 1) and a positive heat capacity change (4.7 kJ mol− 1K− 1) were detected for the intermediates of LOV2-linker. These characteristic features were interpreted in terms of structural fluctuation and exposure of hydrophobic residues in the linker domain, respectively. The enthalpy change of S390 of the LOV1 domain was significantly greater than changes for the LOV2 or LOV2-linker samples. Data from this study support a major conformational change of the linker region in the photochemical reaction of phototropin.

A conformational change of the transducer HtrII upon photoexcitation of the associated photoreceptor sensory rhodopsin II (SRII) was investigated by monitoring the kinetics of volume changes and the diffusion coefficient (D) of the complex during the photochemical reaction cycle. To localize the region of the transducer responsible, we truncated it at various positions in the cytoplasmic HAMP (histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) domain. The truncations do not alter receptor binding, which is dependent primarily on membrane-embedded domain interactions. We found that the light-induced reduction in D occurs in transducers of lengths 120 and 157 residues (Tr120 and Tr157), which are both predicted to contain a HAMP domain consisting of two amphipathic α-helices (AS-1 and AS-2). In contrast, the change in D was abolished in a transducer of 114 amino acid residues (Tr114), which lacks a distal portion of the second α-helix AS-2. The volume changes in SRII–Tr114 are comparable in amplitude and kinetics with those in SRII–Tr120 and SRII–Tr157, confirming the integrity of the complex, which was previously concluded from the similar SRII binding affinity and similar blocking of SRII proton transport by full-length HtrII and Tr114. Our results indicate that a substantial conformational change occurs in the HAMP domain during SRII–HtrII signaling. The data presented here are the first demonstration of stimulus-induced conformational changes of a HAMP domain and provide evidence that the presence of AS-2 is crucial for the conformational alterations. The reduction in diffusion coefficient is likely to due to structural changes in the AS-1 and AS-2 helices such that hydrogen bonding with the surrounding water molecules is increased, thereby increasing friction with the solvent. Similar structural changes may be a general feature in HAMP domain switching, which occurs in diverse signaling proteins, including sensor kinases, taxis receptors/transducers, adenylyl cyclases, and phosphatases.

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 m2 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 m2 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.

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.

Transient grating signals after photoexcitation of Arabidopsis phototropin 1 light–oxygen–voltage 2 (phot1LOV2) domain without the linker were found to be very sensitive to temperature. In particular, the diffusion signal drastically increased with rising temperature. The signal was consistently explained by the superposition of the photo-induced dissociation and association reactions. This observation indicated the presence of an equilibrium between the monomer and dimer forms of the phot1LOV2 domain in the dark. The equilibrium was confirmed by a gel chromatographic technique. The equilibrium constants at various temperatures were calculated from the fraction of the dimer, and the stabilization enthalpy and entropy were determined. Interestingly, the transient grating signal of phot1LOV2 with the linker (phot1LOV2-linker), which exists as the monomer form, was also temperature dependent; the diffusion signal intensity decreased with increasing temperature. Because the diffusion signal reflects a conformation change of the linker upon photoexcitation, this temperature dependence indicated that there were two forms of the phot1LOV2-linker. One form exhibited a conformational change upon photoexcitation whereas the other form showed no change. These two forms are not distinguishable spectroscopically. The fraction of these species depended on the temperature. Considering the monomer–dimer equilibrium of the phot1LOV2 domain, we suggest that the nonreactive form possesses the linker region that is dissociated from the LOV2 domain. Because the dissociation of the linker region from the LOV2 domain is a key step for the conformation change of the phot1LOV2-linker to induce biological activity, we proposed that the phototropins could have a role as a temperature sensor.

Photoreverse reactions of octopus rhodopsin (Rh) from acid-metarhodopsin (Acid-Meta), which is the final product of the photoreaction of Rh, to Rh were studied by the time-resolved transient absorption and transient grating methods. The time course of the absorption signal showed a rapid change within 500 ns followed by one phase with a time constant of ∼470 μs, whereas the transient grating signal indicates three phases with time constants of <500 ns, ∼490 μs, and 2.6 ms. The faster two phases indicate the conformational change in the vicinity of the chromophore, and the slowest one represents conformational change far from the chromophore. The absorption spectrum of the first intermediate created just after the laser excitation (<500 ns) is already very similar to the final product, Rh. This behavior is quite different from that of the forward reaction from Rh to Acid-Meta, in which several intermediates with different absorption spectra are involved within 50 ns–500 μs. This result indicates that the conformation around the chromophore is easily adjusted from all-trans to 11-cis forms compared with that from 11-cis to all-trans forms. Furthermore, it was found that the protein energy is quickly relaxed after the excitation. One of the significantly different properties between Rh and Acid-Meta is the diffusion coefficient (D). D is reduced by about half the transformation from Rh to Acid-Meta. This large reduction was interpreted in terms of the helix opening of the Rh structure.

The kinetics of conformational change in the N-terminal region of photoactive yellow protein (PYP) was studied by the time-resolved diffusion measurement. The transient grating signal that represented the protein diffusion of the ground state and pB state depended on the observation time range. An analysis of the signal based on the time-dependent diffusion coefficient clearly showed that protein diffusion changed with a time constant of 170 μs, corresponding to the pR2 → pB′ transition. Since a previous diffusion study of N-terminal truncated PYPs had revealed that the change in the diffusion coefficient reflected the unfolding of the α-helices in the N-terminal region of PYP, the results indicate that this unfolding took place at the same rate as the pR2 → pB′ transition. This demonstrates that the response of the conformational change in the N-terminal region was quite fast, probably due to changes in a specific hydrogen-bonding network of this domain.

The interaction between sensory rhodopsin II (SRII) and its transducer HtrII was studied by the time-resolved laser-induced transient grating method using the D75N mutant of SRII, which exhibits minimal visible light absorption changes during its photocycle, but mediates normal phototaxis responses. Flash-induced transient absorption spectra of transducer-free D75N and D75N joined to 120 amino-acid residues of the N-terminal part of the SRII transducer protein HtrII (ΔHtrII) showed only one spectrally distinct K-like intermediate in their photocycles, but the transient grating method resolved four intermediates (K1–K4) distinct in their volumes. D75N bound to HtrII exhibited one additional slower kinetic species, which persists after complete recovery of the initial state as assessed by absorption changes in the UV-visible region. The kinetics indicate a conformationally changed form of the transducer portion (designated Tr*), which persists after the photoreceptor returns to the unphotolyzed state. The largest conformational change in the ΔHtrII portion was found to cause a ΔHtrII-dependent increase in volume rising in 8 μs in the K4 state and a drastic decrease in the diffusion coefficient (D) of K4 relatively to those of the unphotolyzed state and Tr*. The magnitude of the decrease in D indicates a large structural change, presumably in the solvent-exposed HAMP domain of ΔHtrII, where rearrangement of interacting molecules in the solvent would substantially change friction between the protein and the solvent.

Cryptochromes (CRYs) are widespread flavoproteins with homology to photolyases (PHRs), a class of blue-light-activated DNA repair enzymes. Unlike PHRs, both plant and animal CRYs have a C-terminal domain. This cryptochrome C-terminal (CCT) domain mediates interactions with other proteins, while the PHR-like domain converts light energy into a signal via reduction and radical formation of the flavin adenine dinucleotide cofactor. However, the mechanism by which the PHR-like domain regulates the CCT domain is not known. Here, we applied the pulsed-laser-induced transient grating method to detect conformational changes induced by blue-light excitation of full-length Arabidopsis thaliana cryptochrome 1 (AtCRY1). A significant reduction in the diffusion coefficient of AtCRY1 was observed upon photoexcitation, indicating that a large conformational change occurs in this monomeric protein. AtCRY1 containing a single mutation (W324F) that abolishes an intra-protein electron transfer cascade did not exhibit this conformational change. Moreover, the conformational change was much reduced in protein lacking the CCT domain. Thus, we conclude that the observed large conformational changes triggered by light excitation of the PHR-like domain result from C-terminal domain rearrangement. This inter-domain modulation would be critical for CRYs' ability to transduce a blue-light signal into altered protein–protein interactions for biological activity. Lastly, we demonstrate that the transient grating technique provides a powerful method for the direct observation and understanding of photoreceptor dynamics.Download high-res image (135KB)Download full-size imageResearch Highlights► We examined conformation change in full-length AtCRY1. ► A large conformational change was successfully detected, and the rate was determined. ► Intra-protein electron transfer cascade is important for the conformational changes. ► The changes result from the C-terminal domain rearrangement. ► The light signal alters protein–protein interactions for bioactivity.

The photochemical reaction dynamics of a BLUF (sensors of blue light using FAD) protein, PixD, from a thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (TePixD, Tll0078) were studied by pulsed laser-induced transient grating method. After the formation of an intermediate species with a red-shifted absorption spectrum, two new reaction phases reflecting protein conformational changes were discovered; one reaction phase manifested itself as expansion of partial molar volume with a time constant of 40 μs, whereas the other reaction phase represented a change in the diffusion coefficient D [i.e., the diffusion-sensitive conformational change (DSCC)]. D decreased from 4.9 × 10− 11 to 4.4 × 10− 11 m2 s− 1 upon the formation of the first intermediate, and subsequently showed a more pronounced decrease to 3.2 × 10− 11 m2 s− 1 upon formation of the second intermediate. From a global analysis of signals at various grating wavenumbers, the time constant of D-change was determined to be 4 ms. Although the magnitude and rate constant of the faster volume change were independent of protein concentration, the amplitude of the signal that reflects the later DSCC significantly decreased as the protein concentration decreased. This concentration dependence suggests that two species exist in solution: a reactive species exhibiting the DSCC, and a second species that is nonreactive. The fraction of these species was found to be dependent on the concentration. The difference in reactivity was attributed to the different oligomeric states of TePixD (i.e., pentamer and decamer). The equilibrium of these states in the dark was confirmed by size-exclusion chromatography at various concentrations. These results demonstrated that only the decamer state is responsible for the conformational change. The results may suggest that the oligomeric state is functionally important in the signal transduction of this photosensory protein.

The photochemical reaction dynamics of a light-oxygen-voltage (LOV) domain from the blue light sensor protein, FKF1 (flavin-binding Kelch repeat F-box) was studied by means of the pulsed laser-induced transient grating method. The observed absorption spectral changes upon photoexcitation were similar to the spectral changes observed for typical LOV domain proteins (e.g., phototropins). The adduct formation took place with a time constant of 6 μs. After this reaction, a significant conformational change with a time constant of 6 ms was observed as a change in the diffusion coefficient. An FKF1-LOV mutant without the conserved loop connecting helices E and F, which is present only in the FKF1/LOV Kelch protein 2/ZEITLUPE family, did not show these slow phase dynamics. This result indicates that the conformational change in the loop region represents a major change in the FKF1-LOV photoreaction.

SyPixD (Slr1694) is a blue-light receptor that contains a BLUF (blue-light sensor using a flavin chromophore) domain for the function of phototaxis. The key reaction of this protein is a light-induced conformational change and subsequent dissociation reaction from the decamer to the dimer. In this study, anomalous effects of pressure on this reaction were discovered, and changes in the compressibility of its short-lived intermediates were investigated. While the absorption spectra of the dark and light states are not sensitive to pressure, the formation yield of the first intermediate decreases with pressure to about 40% at 150 MPa. Upon blue-light illumination with a sufficiently strong intensity, the transient grating signal, which represents the dissociation of the SyPixD decamer, was observed at 0.1 MPa, and the signal intensity significantly decreased with increasing pressure. This behavior shows that the dissociation of the decamer from the second intermediate state is suppressed by pressure. However, while the decamer undergoes no dissociation upon excitation of one monomer unit at 0.1 MPa, dissociation is gradually induced with increasing pressure. For solving this strange behavior, the compressibility changes of the intermediates were measured as a function of pressure at weak light intensity. Interestingly, the compressibility change was negative at low pressure, but became positive with increasing pressure. Because the compressibility is related to the volume fluctuation, this observation suggests that the driving force for this reaction is fluctuation of the protein. The relationship between the cavities at the interfaces of the monomer units and the reactivity was also discussed.

Although the relationship between structural fluctuations and reactions is important for elucidating reaction mechanisms, experimental data describing such fluctuations of reaction intermediates are sparse. In order to investigate structural fluctuations during a protein reaction, the compressibilities of intermediate species after photoexcitation of a phot1LOV2-linker, which is a typical LOV domain protein with the C-terminal linker including the J-α helix and used recently for optogenetics, were measured in the time-domain by the transient grating and transient lens methods with a high pressure optical cell. The yield of covalent bond formation between the chromophore and a Cys residue (S state formation) relative to that at 0.1 MPa decreased very slightly with increasing pressure. The fraction of the reactive species that yields the T state (linker-unfolded state) decreased almost proportionally with pressure (0.1–200 MPa) to about 65%. Interestingly, the volume change associated with the reaction was much more pressure sensitive. By combining these data, the compressibility changes for the short lived intermediate (S state) and the final product (T state) formation were determined. The compressibility of the S state was found to increase compared with the dark (D) state, and the compressibility decreased during the transition from the S state to the T state. The compressibility change is discussed in terms of cavities inside the protein. By comparing the crystal structures of the phot1LOV2-linker at dark and light states, we concluded that the cavity volumes between the LOV domain and the linker domain increase in the S state, which explains the enhanced compressibility.

A recently developed method to monitor reaction kinetics of intermolecular interaction is presented in this perspective. This method is based on time-dependent diffusion coefficient measurements using the pulsed laser induced transient grating technique. Using this method, time dependent biomolecular interactions, such as transient association and dissociation reactions in solution, have been successfully detected in real time. The principles and particular applications are described. In particular, unique features of this time-dependent diffusion coefficient method are emphasized by comparison with other techniques.