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.

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.

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.

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.

Selective Photoinduced Dimerization and Slow Recovery of a BLUF Domain of EB1.

The EAL-BLUF fragment from Magnetococcus marinus BldP1 (EB1) light-dependently hydrolyzes c-di-GMP. Herein, the photoreaction of the BLUF domain of EB1 (eBLUF) is studied. It is found for the first time that a monomeric BLUF domain forms a dimer upon illumination and its dark recovery is very slow. The dimer of light- and dark-state protomers (LD-dimer) is much more stable than that of two light-state protomers (LL-dimer), and the dark recovery of the LD-dimer is approximately 20 times slower than that of the LL-dimer, which is suitable for optogenetic tools. The secondary structure of the L-monomer is different from those of the D-monomer and the LD-dimer. The transient grating measurements reveal that this conformational change occurs simultaneously with dimerization. Although the W91A mutant exhibits a spectral red shift, it forms a heterodimer with the L-monomer of wild-type eBLUF with similar stability to the LD-dimer. This suggests that the conformation of the dimerization site of W91A is similar to that of the dark state (dark-mimic mutant); that is, the light-induced structural changes in the chromophore cavity are not transferred to the other part of the protein. The selective photoinduced dimerization of eBLUF is potentially useful to control interprotein interactions between two different effector domains bound to these proteins.

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.

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.

A Time-Resolved Diffusion Technique for Detection of the Conformational Changes and Molecular Assembly/Disassembly Processes of Biomolecules.

Biological liquid-liquid phase separation (LLPS) is driven by dynamic and multivalent interactions, which involves conformational changes and intermolecular assembly/disassembly processes of various biomolecules. To understand the molecular mechanisms of LLPS, kinetic measurements of the intra- and intermolecular reactions are essential. In this review, a time-resolved diffusion technique which has a potential to detect molecular events associated with LLPS is presented. This technique can detect changes in protein conformation and intermolecular interaction (oligomer formation, protein-DNA interaction, and protein-lipid interaction) in time domain, which are difficult to obtain by other methods. After the principle and methods for signal analyses are described in detail, studies on photoreactive molecules (intermolecular interaction between light sensor proteins and its target DNA) and a non-photoreactive molecule (binding and folding reaction of α-synuclein upon mixing with SDS micelle) are presented as typical examples of applications of this unique technique.

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 light­oxygene-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 0.66 µs. After this reaction, a significant diffusion coefficient (D)-change was observed with a time constant of 38 ms. 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.

Enzymatic activity of the blue light-regulated phosphodiesterase BlrP1 from Klebsiella pneumoniae shows a nonlinear dependence on light intensity.

The blue light-regulated phosphodiesterase BlrP1 from Klebsiella pneumoniae hydrolyzes cyclic dimeric guanosine monophosphate (GMP) in a blue light-dependent manner. It contains a photosensing BLUF domain and a functional EAL domain. Previously, it was reported that conformational changes in the dimer upon light illumination occurred only when both protomers of the dimer were excited. Based on this observation, it was proposed that BlrP1 might be a nonlinear light intensity sensor. To test this, here, the correlation between the turnover number of the hydrolysis reaction (kcat ) and the fraction of the excited protein (fred ) was measured by simultaneously monitoring the reaction rate and fred . Our results show that kcat is proportional to fred2 . Thus, BlrP1 works as a nonlinear light intensity sensor to sense a strong light environment.

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 detection of SDS-induced conformational changes in α-synuclein by a micro-stopped-flow system.

An intrinsically disordered protein, α-synuclein (αSyn), binds to negatively charged phospholipid membranes and adopts an α-helical structure. This conformational change is also induced by interaction with sodium dodecyl sulfate (SDS), which is an anionic surfactant used in previous studies to mimic membrane binding. However, while the structure of the αSyn and SDS complex has been studied widely by various static measurements, the process of structural change from the denatured state to the folded state remains unclear. In this study, the interaction dynamics between αSyn and SDS micelles was investigated using time-resolved measurements with a micro-stopped-flow system, which has been recently developed. In particular, the time-resolved diffusion based on the transient grating technique in combination with a micro-stopped-flow system revealed the gradual change in diffusion triggered by the presence of SDS micelles. This change is induced not only by binding to SDS micelles, but also by an intramolecular conformational change. It was interesting to find that the diffusion coefficient decreased in an intermediate state and then increased to the final state in the binding reaction. We also carried out stopped-flow-kinetic measurements of circular dichroism and intramolecular fluorescence resonance energy transfer, and the D change was assigned to the formation of a compact structure derived from the helix bending on the micelle.

Photoreaction Dynamics of Full-Length Phototropin from Chlamydomonas reinhardtii.

Phototropin (phot) is a blue light sensor involved in the light responses of several species from green algae to higher plants. Phot consists of two photoreceptive domains (LOV1 and LOV2) and a Ser/Thr kinase domain. These domains are connected by a hinge and a linker domain. So far, studies on the photochemical reaction dynamics of phot have been limited to short fragments, and the reactions of intact phot have not been well elucidated. Here, the photoreactions of full-length phot and of several mutants from Chlamydomonas reinhardtii (Cr) were investigated by the transient grating and circular dichroism (CD) methods. Full-length Cr phot is in monomeric form in both dark and light states and shows conformational changes upon photoexcitation. When LOV1 is excited, the hinge helix unfolds with a time constant of 77 ms. Upon excitation of LOV2, the linker helix unfolds initially followed by a tertiary structural change of the kinase domain with a time constant of 91 ms. The quantum yield of conformational change after adduct formation of LOV2 is much smaller than that of LOV1, indicating that reactive and nonreactive forms exist. The conformational changes associated with the excitations of LOV1 and LOV2 occur independently and additively, even when they are excited simultaneously. Hence, the role of LOV1 is not to enhance the kinase activity in addition to LOV2 function; we suggest LOV1 has different functions such as regulation of intermolecular interactions.

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.

Time-Resolved Study of Interprotein Signaling Process of a Blue Light Sensor PapB-PapA Complex.

PapB is a short BLUF protein from the purple bacterium Rhodopseudomonas palustris, and it has the function of regulating the phosphodiesterase activity of its partner protein PapA. Because PapA is an enzymatic protein containing an EAL domain, which hydrolyzes a cyclic-dimeric-GMP, the PapB-PapA complex regulates, for example, biofilm formation in a light-dependent manner. The reaction dynamics of PapB, as well as dynamics of signal transduction in the PapB-PapA complex, were studied mostly by time-resolved transient grating and CD methods. It was found that although PapB exists as a monomer in both dark and light states, the diffusion coefficient slightly increases and partial molar volume changes upon light irradiation with time constants of 80 µs, 3.2 ms, and 24 ms. The last conformational change leads to a signaling state that is attributed to the α-helix unfolding in the C-terminal region of the BLUF domain. Interestingly, when PapA is added to a PapB solution to form a PapB-PapA complex, the diffusion coefficient decreases significantly, with a time constant of 26 ms, which is similar to the rate of the last conformational change of PapB. It was found that this change is due to the conformational change of PapA, and the light signal from PapB is transmitted through the conformational change of PapB. Compared with another BLUF protein, BlrP1, which possesses the EAL effector domain and requires photoexcitation of two monomer units of the dimer for a major conformational change, the conformational change of the PapB-PapA complex is induced by excitation of one monomer PapB among the heterotetramer.

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.

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.