Time-resolved study on signaling pathway of photoactivated adenylate cyclase and its nonlinear optical response.

Photoactivated adenylate cyclases (PACs) are multidomain BLUF proteins that regulate the cellular levels of cAMP in a light-dependent manner. The signaling route and dynamics of PAC from Oscillatoria acuminata (OaPAC), which consists of a light sensor BLUF domain, an adenylate cyclase domain, and a connector helix (α3-helix), were studied by detecting conformational changes in the protein moiety. Although circular dichroism and small-angle X-ray scattering measurements did not show significant changes upon light illumination, the transient grating method successfully detected light-induced changes in the diffusion coefficient (diffusion-sensitive conformational change (DSCC)) of full-length OaPAC and the BLUF domain with the α3-helix. DSCC of full-length OaPAC was observed only when both protomers in a dimer were photoconverted. This light intensity dependence suggests that OaPAC is a cyclase with a nonlinear light intensity response. The enzymatic activity indeed nonlinearly depends on light intensity, that is, OaPAC is activated under strong light conditions. It was also found that both DSCC and enzymatic activity were suppressed by a mutation in the W90 residue, indicating the importance of the highly conserved Trp in many BLUF domains for the function. Based on these findings, a reaction scheme was proposed together with the reaction dynamics.

Conformational change in an engineered biliverdin-binding cyanobacteriochrome during the photoconversion process.

Cyanobacteriochromes (CBCRs) derived from cyanobacteria are linear-tetrapyrrole-binding photoreceptors related to the canonical red/far-red reversible phytochrome photoreceptors. CBCRs contain chromophore-binding cGMP-specific phosphodiesterase/adenylate cyclase/FhlA (GAF) domains that are highly diverse in their primary sequences and are categorized into many subfamilies. Among this repertoire, the biliverdin (BV)-binding CBCR GAF domains receive considerable attention for their in vivo optogenetic and bioimaging applications because BV is a mammalian intrinsic chromophore and can absorb far-red light that penetrates deep into the mammalian body. The typical BV-binding CBCR GAF domain exhibits reversible photoconversion between far-red-absorbing dark-adapted and orange-absorbing photoproduct states. Herein, we applied various biochemical and spectral studies to identify the details of the conformational change during this photoconversion process. No oligomeric state change was observed, whereas the surface charge would change with a modification of the α-helix structures during the photoconversion process. Combinatorial analysis using partial protease digestion and mass spectrometry identified the region where the conformational change occurred. These results provide clues for the future development of optogenetic tools.

Time-resolved detection of light-induced conformational changes of heliorhodopsin.

Heliorhodopsins (HeRs) are a new category of rhodopsins. They exist as a dimer and exhibit a characteristic inverted topology. HeRs bind all-trans-retinal as a chromophore in the dark, and its isomerization to the 13-cis form by light illumination leads to a photocyclic reaction involving several photo-intermediates: K, L, M, and O. In this study, the kinetics of conformational changes of HeR from Thermoplasmatales archaeon SG8-52-1 (TaHeR) were studied by the transient grating (TG) and circular dichroism (CD) methods. The TG method reveals that the diffusion coefficient (D) does not change until the O formation suggesting no significant conformation change at the surface of the protein during the early steps of the reaction. Subsequently, D decreases upon the O formation. Although two time constants (202 µs and 2.6 ms) are observed for the conversion from the M to O by the absorption detection, D decreases only at the first step (202 µs). Light-induced unfolding of helical structure is detected by the CD method. To examine the contribution of a characteristic helix in the intracellular loop 1 (ICL1 helix), Tyr93 on the ICL1 helix was replaced by Gly (Y93G), and the reaction of this mutant was also investigated. It was found that this replacement partially suppresses the D-change, although the CD-change is almost the same as that of the wild type. These results are interpreted in terms of different sensitivities of TG and CD methods, that is, D is sensitive to the structure of the solvent-exposed surface and selectively observes the conformational change in the ICL1 region. It is suggested that the structure of hydrophilic residues in the ICL1 helix is changed during this process.

Slow Conformational Changes of Blue Light Sensor BLUF Proteins in Milliseconds.

Blue light sensor using flavin (BLUF) proteins consist of flavin-binding BLUF domains and functional domains. Upon blue light excitation, the hydrogen bond network around the flavin chromophore changes, and the absorption spectrum in the visible region exhibits a red shift. Ultimately, the light information received in the BLUF domain is transmitted to the functional region. It has been believed that this red shift is complete within nanoseconds. In this study, slow reaction kinetics were discovered in milliseconds (τ1- and τ2-phase) for all the BLUF proteins examined (AppA, OaPAC, BlrP1, YcgF, PapB, SyPixD, and TePixD). Despite extensive reports on BLUF, this is the first clear observation of the BLUF protein absorption change with the duration in the millisecond time region. From the measurements of some domain-deleted mutants of OaPAC and two chimeric mutants of PixD proteins, it was found that the slower dynamics (τ2-phase) are strongly affected by the size and nature of the C-terminal region adjacent to the BLUF domain. Hence, this millisecond reaction is a significant indicator of conformational changes in the C-terminal region, which is essential for the biological functions. On the other hand, the τ1-phase commonly exists in all BLUF proteins, including any mutants. The origin of the slow dynamics was studied using site-specific mutants. These results clearly show the importance of Trp in the BLUF domain. Based on this, a reaction scheme for the BLUF reaction is proposed.

Time-resolved diffusion reveals photoreactions of BLUF proteins with similar functional domains.

BLUF (blue light sensor using flavin) proteins are the blue light receptors that consist of flavin-binding BLUF domains and functional domains. Upon blue light excitation, the hydrogen bond network around the flavin chromophore changes, and the absorption spectrum in the visible region shifts to red. Light signal received in the BLUF domain is intramolecularly or intermolecularly transmitted to the functional region. In this review, the reactions of three BLUF proteins with similar EAL functional groups within the protein (BlrP1, and YcgF), or with a separated target protein (PapB) are described using time-resolved diffusion technique. The diffusion coefficients (D) of the BLUF domains did not significantly change upon photoexcitation, whereas those of the full-length proteins BlrP1 and YcgF and the PapB-PapA system significantly decreased. The changes in D should be due to diffusion-sensitive conformational changes (DSCC) that alter the friction of diffusion. The time constants of the major D changes of BlrP1 and PapB-PapA were similar (~ 20 ms), although the magnitude of the friction change depended on the proteins. Similarities and differences among the reactions of these proteins were clarified from the viewpoint of DSCC.

A unique photochromic UV-A sensor protein, Rc-PYP, interacting with the PYP-binding protein.

Photoactive yellow protein (PYP) is one of the typical light sensor proteins. Although its photoreaction has been extensively studied, no downstream partner protein has been identified to date. In this study, the intermolecular interaction dynamics observed between PYP from Rhodobacter capsulatus (Rc-PYP) and a possible downstream protein, PYP-binding protein (PBP), were investigated. It was found that UV light induced a long-lived product (pUV*), which interacts with PBP to form a stable hetero-hexamer (Complex-2). The reaction scheme for this interaction was revealed using transient absorption and transient grating methods. Time-resolved diffusion detection showed that a hetero-trimer (Complex-1) is formed transiently, which produced Complex-2 via a second-order reaction. Any other intermediates, including those from pBL, do not interact with PBP. The reaction scheme and kinetics are determined. Interestingly, long-lived Complex-2 dissociates upon excitation with blue light. These results demonstrate that Rc-PYP is a photochromic and new type of UV sensor to sense the relative intensities of UV-A and blue light.

Effect of Hydrated Ionic Liquid on Photocycle and Dynamics of Photoactive Yellow Protein.

The mechanism by which proteins are solvated in hydrated ionic liquids remains an open question. Herein, the photoexcitation dynamics of photoactive yellow protein dissolved in hydrated choline dihydrogen phosphate (Hy[ch][dhp]) were studied by transient absorption and transient grating spectroscopy. The photocyclic reaction of the protein in Hy[ch][dhp] was similar to that observed in the buffer solution, as confirmed by transient absorption spectroscopy. However, the structural change of the protein during the photocycle in Hy[ch][dhp] was found to be different from that observed in the buffer solution. The known change in the diffusion coefficient of the protein was apparently suppressed in high concentrations of [ch][dhp], plausibly due to stabilization of the secondary structure.

Photoreaction Dynamics of a Full-Length Protein YtvA and Intermolecular Interaction with RsbRA.

YtvA from Bacillus subtilis is a sensor protein that responds to blue light stress and regulates the activity of transcription factor σB. It is composed of the N-terminal LOV (light-oxygen-voltage) domain, the C-terminal STAS (sulfate transporter and anti-sigma factor antagonist) domain, and a linker region connecting them. In this study, the photoreaction and kinetics of full-length YtvA and the intermolecular interaction with a downstream protein, RsbRA, were revealed by the transient grating method. Although N-YLOV-linker, which is composed of the LOV domain of YtvA with helices A'α and Jα, exhibits a diffusion change due to the rotational motion of the helices, the YtvA dimer does not show the diffusion change. This result suggests that the STAS domain inhibits the rotational movement of helices A'α and Jα. We found that the YtvA dimer formed a heterotetramer with the RsbRA dimer probably via the interaction between the STAS domains, and we showed the diffusion change upon blue light illumination with a time constant faster than 70 µs. This result suggests a conformational change of the STAS domains; i.e., the interface between the STAS domains of the proteins changes to enhance the friction with water by the rotation structural change of helices A'α and Jα of YtvA.

Wavelength-Dependent Photoreaction of PYP from Rhodobacter capsulatus.

PYPs (photoactive yellow proteins) are blue light sensor proteins found in more than 100 species. Compared with the extensive and intensive studies of the reactions of PYP from Halorhodospira halophila (Hh-PYP), studies of the reactions of other PYPs are scarce. Here, the photoreaction of PYP from Rhodobacter capsulatus (Rc-PYP) was studied in detail using ultraviolet-visible absorption and transient grating methods. Rc-PYP exhibits two absorption peaks at 375 and 438 nm. By using the transient absorption and the temperature-dependent absorption spectrum, the absorption spectra of two forms, pUV and pBL, were determined. Upon photoexcitation of pBL, two intermediates are observed before returning back to the dark state, with a time constant of 1.2 ms, which is 3 orders of magnitude faster than the dark recovery of Hh-PYP. Upon photoexcitation of pUV, two intermediates are observed to produce a long-lived final product, although one of the processes is spectrally silent. The diffusion coefficients decreased transiently for both pBL and pUV reactions, suggesting a relatively large conformational change during the reactions. It is particularly interesting to observe that the blue light irradiation of the long-lived product of pUV returns the product to the dark state. This result suggests different opposing responses of the biological function due to photoexcitation by ultraviolet and blue lights.

Time-Resolved Diffusion Detection with Microstopped Flow System.

The transient grating (TG) method is a powerful technique for monitoring the time dependence of the diffusion coefficient during photochemical reactions. However, the applications of this technique have been limited to photochemical reactions. Here, a microstopped flow (µ-SF) system is developed to expand the technique's applicability. The constructed µ-SF system can be used for a solution with a total volume as small as 3 µL, and mixing times for absorption and diffusion measurements were determined to be 400 µs and 100 ms, respectively. To demonstrate this system with the TG method, an acid-induced denaturation of a photosensor protein, phototropin LOV2 domain with a linker, was studied from the viewpoint of the reactivity. This system can be used not only for time-resolved diffusion measurement but also for conventional absorption or fluorescence detection methods. In particular, this system has a great advantage for a target solution in that only a very small amount is needed.

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.

Photoinduced dimerization of a photosensory DNA-binding protein EL222 and its LOV domain.

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.

Photochemical Reactions of the LOV and LOV-Linker Domains of the Blue Light Sensor Protein YtvA.

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.

Dynamics of Inter-DNA Chain Interaction of Photoresponsive DNA.

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 ∼10(7) 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.

Time-resolved fluctuation during the photochemical reaction of a photoreceptor protein: phototropin1LOV2-linker.

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.

Anomalous pressure effects on the photoreaction of a light-sensor protein from Synechocystis, PixD (Slr1694), and the compressibility change of its intermediates.

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.

Reaction dynamics of the UV-B photosensor UVR8.

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.

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.

Effects of N- and C-terminal regions on oligomeric formation of blue light sensor protein SyPixD.

A sensor of blue-light using flavin adenine dinucleotide (BLUF) is a typical blue light photoreceptor domain that is found in many photosensor proteins in bacteria and some eukaryotic algae. SyPixD in Synechocystis is one of the well-studied BLUF proteins. In the dark state, it forms a decamer and, upon photoexcitation, a dissociation reaction takes place to yield dimers. Such change in the intermolecular interactions of the protomers is important for the biological function. The effect of the N- and C-terminal sequences on the stability of SyPixD oligomeric states and photoreactions of SyPixD were studied to understand how the oligomeric form is maintained with weak interaction. It was found that a few residues that frequently persist at the N-terminus after removing a tag for purification are sensitive to the stability of the decamer structure. Even two or three residues at the N-terminus considerably reduces decamer stability, whereas four or more residues completely prevent decamer formation. Unexpectedly, truncating C-terminal sequences, which locate far from any protomer interface and of which structure is undetermined in crystal structure, also destabilizes the decamer structure. This destabilization is also apparent from the dissociation reaction dynamics detected by the transient grating and transient absorption measurements. The dissociation reaction is faster and the yield increases when the C-terminus does not contain seven amino acid residues. Photoexcitation induces a conformational change in the C-terminus of the decamer but not the dimer.