RESUMEN
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.
Asunto(s)
Adenilil Ciclasas , Proteínas Bacterianas , Luz , Transducción de Señal , Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , Adenilil Ciclasas/efectos de la radiación , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/efectos de la radiación , Subunidades de Proteína , Activación Enzimática/efectos de la radiación , MutaciónRESUMEN
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.
Asunto(s)
Cianobacterias , Fotorreceptores Microbianos , Biliverdina/química , Fotorreceptores Microbianos/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , LuzRESUMEN
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.
Asunto(s)
Rodopsina , Rodopsinas Microbianas , Rodopsinas Microbianas/química , Dicroismo Circular , Retinaldehído/química , Conformación ProteicaRESUMEN
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.
Asunto(s)
Fotorreceptores Microbianos , Synechocystis , Proteínas Bacterianas/química , Luz , Synechocystis/química , Fotorreceptores Microbianos/química , Aminoácidos/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Bacterianas , Flavinas , Proteínas Bacterianas/química , Difusión , Flavinas/química , Enlace de Hidrógeno , LuzRESUMEN
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.
Asunto(s)
Proteínas Bacterianas , Flavoproteínas , Proteínas Bacterianas/química , Dinitrocresoles , Flavoproteínas/química , Luz , Estructura Terciaria de ProteínaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas , Proteínas Bacterianas/química , Dimerización , Estructura Secundaria de ProteínaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/química , Líquidos Iónicos/química , Fosforilcolina/química , Fotorreceptores Microbianos/química , Agua/química , Tampones (Química) , Difusión , Luz , Solubilidad , Análisis Espectral/métodosRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/química , Fotorreceptores Microbianos/química , Proteínas Bacterianas/aislamiento & purificación , Fotorreceptores Microbianos/aislamiento & purificación , Rhodobacter capsulatus/química , Espectrofotometría Ultravioleta , Rayos UltravioletaRESUMEN
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.
RESUMEN
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.
Asunto(s)
Adenilil Ciclasas/metabolismo , Proteínas Bacterianas/metabolismo , Cianobacterias/metabolismo , Adenilil Ciclasas/química , Adenilil Ciclasas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Cromatografía en Gel , Dicroismo Circular , Luz , Mutagénesis , Dominios Proteicos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Biological functions essentially consist of a series of chemical reactions, including intermolecular interactions, and also involve the cooperation of a number of biological molecules performing these reactions. To understand this function at the molecular level, all steps of the reactions must be elucidated. However, since the biosystems including the surrounding environment are notably large, the reactions have to be elucidated from several different approaches. A variety of techniques have been developed to obtain structural information, and the knowledge of the three-dimensional structure of biomolecules has increased dramatically. Contrarily, the current information on reaction dynamics, which is essential for understanding reactions, is still not enough. Although frequently used techniques, such as spectroscopy, have revealed several important processes of reactions, there are various hidden dynamics that are not detected by these methods (silent dynamics). For example, although water molecules are essential for bioreactions, dynamics of the protein-water interaction are very difficult to trace and spectrally silent. Transient association/dissociations of proteins with partner proteins are difficult to observe. Another important property to understand the reaction of proteins is fluctuations, which are random movements that do not change the average structure and energy. The importance of fluctuations has been pointed out in order to explain enzymatic activity; however, it is extremely difficult to detect changes in fluctuation during a reaction. In this Account, unique time-resolved methods, time-resolved thermodynamics, and time-resolved diffusion methods, both of which are able to detect silent dynamics in solution at physiological temperature, are described.Thermodynamic properties are important for characterizing materials, in particular, macromolecules such as biomolecules. Therefore, the data available regarding these properties, for several stable proteins, is abundant. However, it is almost impossible to characterize short-lived intermediate species in irreversible reactions using traditional thermodynamic techniques. Similarly, although the translational diffusion coefficient is a useful property to determine the protein size and intermolecular interactions, there have been no reports revealing reaction dynamics. The transient grating (TG) method enables us to measure these quantities in a time-resolved manner for a variety of irreversible reactions. With this method, it is now possible to study biomolecule reactions from the viewpoint of thermodynamic properties and diffusion, and to elucidate reaction dynamics that cannot be detected by other spectroscopic methods.Here, the principles of the methodologies used, their characteristic advantages, and their applications to protein reactions are described. The TG measurements of octopus rhodopsin revealed a spectrally hidden intermediate and determined an energetic profile along the reaction coordinate. This emphasizes that the measurement in solution, not for trapped intermediates, is important to characterize the reaction intermediates. The application of these methods to a blue light sensor PixD revealed many spectrally silent dynamics as well as the importance of fluctuation for the reaction. As an example of the time-resolved heat capacity change and transient thermal expansion measurements, the reaction of PYP was briefly described. The reaction scheme of another blue light sensor protein, phototropins, and a spectrally silent DNA binding process of EL222 were fully elucidated by the time-resolved diffusion method.
Asunto(s)
Proteínas/metabolismo , Termodinámica , Difusión , Proteínas/química , Factores de TiempoRESUMEN
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.
Asunto(s)
Klebsiella pneumoniae/enzimología , Luz , Dinámicas no Lineales , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/efectos de la radiación , Color , Activación Enzimática/efectos de los fármacos , Hidrólisis/efectos de la radiación , Cinética , Dominios ProteicosRESUMEN
Photosensor proteins are important not only because of their biological functions but also because of their applications in optogenetics. To understand the molecular mechanism behind their biological functions and consequently seek possible applications to optogenetics, the dynamics of their intermolecular interaction (for example, association/dissociation reaction and conformational changes) upon photoexcitation need to be elucidated. Although it has been difficult to trace such reactions in the time domain using traditional spectroscopic techniques, the time-resolved diffusion method based on the transient grating technique has been demonstrated to possess a significant advantage in detecting such spectrally silent dynamics in a time-resolved manner. In this paper, the principle and studies on blue light sensor proteins, phototropins, are described. Reaction kinetics of dimerization, dissociation reactions, and conformational changes were measured, and reaction schemes were determined. This method can be employed to study protein reactions from the viewpoint of diffusion and to elucidate the reaction schemes and kinetics that cannot be detected by other spectroscopic methods.
RESUMEN
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.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/efectos de la radiación , Fosfoproteínas/química , Fosfoproteínas/efectos de la radiación , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/efectos de la radiación , Bacillus subtilis/química , Bacillus subtilis/metabolismo , Bacillus subtilis/efectos de la radiación , Proteínas Bacterianas/metabolismo , Dispersión Dinámica de Luz , Luz , Modelos Moleculares , Fosfoproteínas/metabolismo , Procesos Fotoquímicos , Fotorreceptores Microbianos/metabolismo , Dominios y Motivos de Interacción de Proteínas/efectos de la radiación , Estructura Cuaternaria de Proteína/efectos de la radiaciónRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/efectos de la radiación , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/efectos de la radiación , Rhodobacter capsulatus/química , Conformación Proteica/efectos de la radiación , Rayos UltravioletaRESUMEN
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.
RESUMEN
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.
Asunto(s)
Proteínas Algáceas/química , Chlamydomonas reinhardtii/química , Fototropinas/química , Proteínas Algáceas/genética , Dominio Catalítico , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/efectos de la radiación , Cromatografía en Gel , Dicroismo Circular , Criptocromos/química , Criptocromos/genética , Mononucleótido de Flavina/química , Mononucleótido de Flavina/genética , Luz , Modelos Moleculares , Mutación , Procesos Fotoquímicos , Fototropinas/genética , Conformación Proteica , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
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.
Asunto(s)
Mediciones Luminiscentes , Fototropinas/análisis , Difusión , Factores de TiempoRESUMEN
Phytochromes (Phys) are photoreceptor proteins that sense red/far-red light in plants, fungi, and bacteria. The proteins consist of a light-sensing photosensory module and a signaling output module, which is typically a histidine kinase (HK) domain in bacteriophytochromes. Although the time-resolved detection of the HK domain is essential for obtaining insights into the reaction mechanism of photoactivation, it has been very difficult to detect the change. Here, the reaction of Cph1, one of the Phys found in the cyanobacterium Synechocystis sp. PCC6803, was studied using time-resolved translational diffusion detection. It was found that the kinetics of the HK domain movement of the Cph1 dimer could be monitored successfully. The diffusion coefficient of the Cph1 dimer decreases significantly with a time constant similar to that of the final step of the reaction monitored by the transient absorption method (780 ms), whereas the monomer does not exhibit this change. We attribute this change to the closed-to-open type of conformational change in the HK domain of the Cph1 dimer without the secondary structure change. The fact that the rate is similar to that from the transient absorption method suggests that the proton uptake at His260 is the rate-determining step of the conformational change.