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1.
Adv Exp Med Biol ; 1293: 189-206, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33398814

RESUMEN

Three classes of flavoprotein photoreceptors, cryptochromes (CRYs), light-oxygen-voltage (LOV)-domain proteins, and blue light using FAD (BLUF)-domain proteins, have been identified that control various physiological processes in multiple organisms. Accordingly, signaling activities of photoreceptors have been intensively studied and the related mechanisms have been exploited in numerous optogenetic tools. Herein, we summarize the current understanding of photoactivation mechanisms of the flavoprotein photoreceptors and review their applications.


Asunto(s)
Flavoproteínas/metabolismo , Flavoproteínas/efectos de la radiación , Fototransducción/efectos de la radiación , Luz , Optogenética , Criptocromos/genética , Criptocromos/metabolismo , Criptocromos/efectos de la radiación , Flavoproteínas/genética
2.
J Am Chem Soc ; 143(1): 97-102, 2021 01 13.
Artículo en Inglés | MEDLINE | ID: mdl-33369395

RESUMEN

Intermolecular C-C bond-forming reactions are underdeveloped transformations in the field of biocatalysis. Here we report a photoenzymatic intermolecular hydroalkylation of olefins catalyzed by flavin-dependent 'ene'-reductases. Radical initiation occurs via photoexcitation of a rare high-order enzyme-templated charge-transfer complex that forms between an alkene, α-chloroamide, and flavin hydroquinone. This unique mechanism ensures that radical formation only occurs when both substrates are present within the protein active site. This active site can control the radical terminating hydrogen atom transfer, enabling the synthesis of enantioenriched γ-stereogenic amides. This work highlights the potential for photoenzymatic catalysis to enable new biocatalytic transformations via previously unknown electron transfer mechanisms.


Asunto(s)
Alquenos/química , Amidas/síntesis química , Flavoproteínas/química , Oxidorreductasas/química , Alquilación/efectos de la radiación , Biocatálisis/efectos de la radiación , Dominio Catalítico , Dinitrocresoles/química , Dinitrocresoles/efectos de la radiación , Flavoproteínas/efectos de la radiación , Luz , Modelos Químicos , Oxidorreductasas/efectos de la radiación
3.
Sci Rep ; 10(1): 2061, 2020 02 06.
Artículo en Inglés | MEDLINE | ID: mdl-32029866

RESUMEN

Blue Light Using Flavin (BLUF) domains are increasingly being adopted for use in optogenetic constructs. Despite this, much remains to be resolved on the mechanism of their activation. The advent of unnatural amino acid mutagenesis opens up a new toolbox for the study of protein structural dynamics. The tryptophan analogue, 7-aza-Trp (7AW) was incorporated in the BLUF domain of the Activation of Photopigment and pucA (AppA) photoreceptor in order to investigate the functional dynamics of the crucial W104 residue during photoactivation of the protein. The 7-aza modification to Trp makes selective excitation possible using 310 nm excitation and 380 nm emission, separating the signals of interest from other Trp and Tyr residues. We used Förster energy transfer (FRET) between 7AW and the flavin to estimate the distance between Trp and flavin in both the light- and dark-adapted states in solution. Nanosecond fluorescence anisotropy decay and picosecond fluorescence lifetime measurements for the flavin revealed a rather dynamic picture for the tryptophan residue. In the dark-adapted state, the major population of W104 is pointing away from the flavin and can move freely, in contrast to previous results reported in the literature. Upon blue-light excitation, the dominant tryptophan population is reorganized, moves closer to the flavin occupying a rigidly bound state participating in the hydrogen-bond network around the flavin molecule.


Asunto(s)
Proteínas Bacterianas/metabolismo , Flavinas/metabolismo , Flavoproteínas/metabolismo , Luz , Fotorreceptores Microbianos/metabolismo , Triptófano/análogos & derivados , Proteínas Bacterianas/química , Proteínas Bacterianas/efectos de la radiación , Flavinas/química , Flavinas/efectos de la radiación , Flavoproteínas/química , Flavoproteínas/efectos de la radiación , Transferencia Resonante de Energía de Fluorescencia , Enlace de Hidrógeno/efectos de la radiación , Conformación Molecular , Simulación de Dinámica Molecular , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/efectos de la radiación , Triptófano/química , Triptófano/metabolismo , Triptófano/efectos de la radiación
4.
J Phys Chem B ; 123(45): 9592-9597, 2019 11 14.
Artículo en Inglés | MEDLINE | ID: mdl-31596584

RESUMEN

Real-time observation of structure changes associated with protein function remains a major challenge. Ultrafast pump-probe methods record dynamics in light activated proteins, but the assignment of spectroscopic observables to specific structure changes can be difficult. The BLUF (blue light using flavin) domain proteins are an important class of light sensing flavoprotein. Here, we incorporate the unnatural amino acid (UAA) azidophenylalanine (AzPhe) at key positions in the H-bonding environment of the isoalloxazine chromophore of two BLUF domains, namely, PixD and AppABLUF; both proteins retain the red-shift on irradiation characteristic of photoactivity. Steady state and ultrafast time resolved infrared difference measurements of the azido mode reveal site-specific information on the nature and dynamics of light driven structure change. AzPhe dynamics are thus shown to be an effective probe of BLUF domain photoactivation, revealing significant differences between the two proteins and a differential response of the two sites to chromophore excitation.


Asunto(s)
Azidas/química , Flavoproteínas/química , Sondas Moleculares/química , Fenilalanina/análogos & derivados , Sustitución de Aminoácidos , Aminoácidos/química , Flavinas/química , Flavoproteínas/genética , Flavoproteínas/efectos de la radiación , Enlace de Hidrógeno , Luz , Mutación , Fenilalanina/química , Conformación Proteica/efectos de la radiación , Dominios Proteicos/efectos de la radiación , Estructura Terciaria de Proteína/efectos de la radiación , Espectrofotometría Infrarroja
5.
J Phys Chem B ; 121(40): 9366-9371, 2017 10 12.
Artículo en Inglés | MEDLINE | ID: mdl-28892628

RESUMEN

Optogenetics has been, and will continue to be, a boon to mechanistic studies of cellular processes. Genetically encodable proteins that sensitize the production of reactive oxygen species (ROS) are expected to play an increasingly important role, particularly in elucidating mechanisms of temporally and spatially dependent cell signaling. However, a substantial challenge in developing such photosensitizing proteins has been to funnel the optical excitation energy into the initial selective production of only one ROS. Singlet molecular oxygen, O2(a1Δg), is a ROS known to have a wide range of effects on cell function. Nevertheless, mechanistic details of singlet oxygen's behavior in a cell are lacking. On the basis of the rational optimization of a LOV-derived flavoprotein, we now report the development and photophysical characterization of a protein-encased photosensitizer that efficiently and selectively produces singlet oxygen at the expense of other ROS, especially ROS that derive from photoinduced electron transfer reactions. These results set the stage for a plethora of new experiments to elucidate ROS-mediated events in cells.


Asunto(s)
Flavoproteínas/efectos de la radiación , Oxígeno/metabolismo , Oxígeno Singlete/química , Mononucleótido de Flavina/química , Mononucleótido de Flavina/efectos de la radiación , Flavoproteínas/química , Flavoproteínas/genética , Flavoproteínas/metabolismo , Cinética , Mutagénesis Sitio-Dirigida , Procesos Fotoquímicos , Fotones , Temperatura
6.
J Am Chem Soc ; 139(41): 14638-14648, 2017 10 18.
Artículo en Inglés | MEDLINE | ID: mdl-28876066

RESUMEN

The flavin chromophore in blue-light-using FAD (BLUF) photoreceptors is surrounded by a hydrogen bond network that senses and responds to changes in the electronic structure of the flavin on the ultrafast time scale. The hydrogen bond network includes a strictly conserved Tyr residue, and previously we explored the role of this residue, Y21, in the photoactivation mechanism of the BLUF protein AppABLUF by the introduction of fluorotyrosine (F-Tyr) analogues that modulated the pKa and reduction potential of Y21 by 3.5 pH units and 200 mV, respectively. Although little impact on the forward (dark- to light-adapted form) photoreaction was observed, the change in Y21 pKa led to a 4000-fold increase in the rate of dark-state recovery. In the present work we have extended these studies to the BLUF protein PixD, where, in contrast to AppABLUF, modulation in the Tyr (Y8) pKa has a profound impact on the forward photoreaction. In particular, a decrease in Y8 pKa by 2 or more pH units prevents formation of a stable light state, consistent with a photoactivation mechanism that involves proton transfer or proton-coupled electron transfer from Y8 to the electronically excited FAD. Conversely, the effect of pKa on the rate of dark recovery is markedly reduced in PixD. These observations highlight very significant differences between the photocycles of PixD and AppABLUF, despite their sharing highly conserved FAD binding architectures.


Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/efectos de la radiación , Flavoproteínas/metabolismo , Flavoproteínas/efectos de la radiación , Flúor/metabolismo , Luz , Fotorreceptores Microbianos/metabolismo , Fotorreceptores Microbianos/efectos de la radiación , Tirosina/metabolismo , Sitios de Unión , Color , Transporte de Electrón , Flavina-Adenina Dinucleótido/metabolismo , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Dominios Proteicos , Protones , Synechocystis/química
7.
Proc Natl Acad Sci U S A ; 114(7): 1480-1485, 2017 02 14.
Artículo en Inglés | MEDLINE | ID: mdl-28137837

RESUMEN

Blue light using flavin adenine dinucleotide (BLUF) proteins are essential for the light regulation of a variety of physiologically important processes and serve as a prototype for photoinduced proton-coupled electron transfer (PCET). Free-energy simulations elucidate the active site conformations in the AppA (activation of photopigment and puc expression) BLUF domain before and following photoexcitation. The free-energy profile for interconversion between conformations with either Trp104 or Met106 closer to the flavin, denoted Trpin/Metout and Trpout/Metin, reveals that both conformations are sampled on the ground state, with the former thermodynamically favorable by ∼3 kcal/mol. These results are consistent with the experimental observation of both conformations. To analyze the proton relay from Tyr21 to the flavin via Gln63, the free-energy profiles for Gln63 rotation were calculated on the ground state, the locally excited state of the flavin, and the charge-transfer state associated with electron transfer from Tyr21 to the flavin. For the Trpin/Metout conformation, the hydrogen-bonding pattern conducive to the proton relay is not thermodynamically favorable on the ground state but becomes more favorable, corresponding to approximately half of the configurations sampled, on the locally excited state. The calculated energy gaps between the locally excited and charge-transfer states suggest that electron transfer from Tyr21 to the flavin is more facile for configurations conducive to proton transfer. When the active site conformation is not conducive to PCET from Tyr21, Trp104 can directly compete with Tyr21 for electron transfer to the flavin through a nonproductive pathway, impeding the signaling efficiency.


Asunto(s)
Proteínas Bacterianas/química , Simulación por Computador , Flavoproteínas/química , Fotorreceptores Microbianos/química , Rhodobacter sphaeroides/metabolismo , Proteínas Bacterianas/efectos de la radiación , Dominio Catalítico , Transporte de Electrón , Mononucleótido de Flavina/química , Flavoproteínas/efectos de la radiación , Glutamina/química , Enlace de Hidrógeno , Luz , Metionina/química , Modelos Moleculares , Fotorreceptores Microbianos/efectos de la radiación , Conformación Proteica/efectos de la radiación , Dominios Proteicos , Rhodobacter sphaeroides/efectos de la radiación , Triptófano/química , Tirosina/química , Tirosina/efectos de la radiación
8.
Nat Struct Mol Biol ; 20(7): 859-67, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23728293

RESUMEN

The anoxygenic phototrophic bacterium Rhodobacter sphaeroides uses different energy sources, depending on environmental conditions including aerobic respiration or, in the absence of oxygen, photosynthesis. Photosynthetic genes are repressed at high oxygen tension, but at intermediate levels their partial expression prepares the bacterium for using light energy. Illumination, however, enhances repression under semiaerobic conditions. Here, we describe molecular details of two proteins mediating oxygen and light control of photosynthesis-gene expression: the light-sensing antirepressor AppA and the transcriptional repressor PpsR. Our crystal structures of both proteins and their complex and hydrogen/deuterium-exchange data show that light activation of AppA-PpsR2 affects the PpsR effector region within the complex. DNA binding studies demonstrate the formation of a light-sensitive ternary AppA-PpsR-DNA complex. We discuss implications of these results for regulation by light and oxygen, highlighting new insights into blue light-mediated signal transduction.


Asunto(s)
Proteínas Bacterianas/efectos de la radiación , ADN Bacteriano/química , Flavoproteínas/química , Regulación Bacteriana de la Expresión Génica/efectos de la radiación , Simulación del Acoplamiento Molecular , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Rhodobacter sphaeroides/efectos de la radiación , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cromatografía en Gel , Secuencia Conservada , ADN Bacteriano/metabolismo , Flavoproteínas/metabolismo , Flavoproteínas/efectos de la radiación , Cinética , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Oxígeno/metabolismo , Fotosíntesis , Proteínas del Complejo del Centro de Reacción Fotosintética/efectos de la radiación , Unión Proteica , Conformación Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Rhodobacter sphaeroides/genética , Rhodobacter sphaeroides/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido
9.
Plant J ; 67(5): 869-84, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21575090

RESUMEN

The time-resolved response of Arabidopsis thaliana towards changing light and/or temperature at the transcriptome and metabolome level is presented. Plants grown at 21°C with a light intensity of 150 µE m⁻² sec⁻¹ were either kept at this condition or transferred into seven different environments (4°C, darkness; 21°C, darkness; 32°C, darkness; 4°C, 85 µE m⁻² sec⁻¹; 21 °C, 75 µE m⁻² sec⁻¹; 21°C, 300 µE m⁻² sec⁻¹ ; 32°C, 150 µE m⁻² sec⁻¹). Samples were taken before (0 min) and at 22 time points after transfer resulting in (8×) 22 time points covering both a linear and a logarithmic time series totaling 177 states. Hierarchical cluster analysis shows that individual conditions (defined by temperature and light) diverge into distinct trajectories at condition-dependent times and that the metabolome follows different kinetics from the transcriptome. The metabolic responses are initially relatively faster when compared with the transcriptional responses. Gene Ontology over-representation analysis identifies a common response for all changed conditions at the transcriptome level during the early response phase (5-60 min). Metabolic networks reconstructed via metabolite-metabolite correlations reveal extensive environment-specific rewiring. Detailed analysis identifies conditional connections between amino acids and intermediates of the tricarboxylic acid cycle. Parallel analysis of transcriptional changes strongly support a model where in the absence of photosynthesis at normal/high temperatures protein degradation occurs rapidly and subsequent amino acid catabolism serves as the main cellular energy supply. These results thus demonstrate the engagement of the electron transfer flavoprotein system under short-term environmental perturbations.


Asunto(s)
Arabidopsis/fisiología , Flavoproteínas/metabolismo , Regulación de la Expresión Génica de las Plantas/fisiología , Metaboloma/fisiología , Transcriptoma/fisiología , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/efectos de la radiación , Análisis por Conglomerados , Oscuridad , Flavoproteínas/efectos de la radiación , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Cinética , Luz , Redes y Vías Metabólicas/fisiología , Redes y Vías Metabólicas/efectos de la radiación , Metaboloma/efectos de la radiación , Metabolómica , Análisis de Secuencia por Matrices de Oligonucleótidos , Fotosíntesis/fisiología , Fotosíntesis/efectos de la radiación , Proteolisis/efectos de la radiación , Temperatura , Factores de Tiempo , Transcriptoma/efectos de la radiación
10.
Neurochem Int ; 56(3): 379-86, 2010 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-20036704

RESUMEN

Synaptic stimulation in brain slices is accompanied by changes in tissue autofluorescence, which are a consequence of changes in tissue metabolism. Autofluorescence excited by ultraviolet light has been most extensively studied, and is due to reduced pyridine nucleotides (NADH and NADPH, collectively termed NAD(P)H). Stimulation generates a characteristic compound NAD(P)H response, comprising an initial fluorescence decrease and then an overshooting increase that slowly recovers to baseline levels. Evoked NAD(P)H transients are relatively easy to record, do not require the addition of exogenous indicators and have good signal-noise ratios. These characteristics make NAD(P)H imaging methods very useful for tracking the spread of neuronal activity in complex brain tissues, however the cellular basis of synaptically-evoked autofluorescence transients has been the subject of recent debate. Of particular importance is the question of whether signals are due primarily to changes in neuronal mitochondrial function, and/or whether astrocyte metabolism triggered by glutamate uptake may be a significant contributor to the overshooting NAD(P)H fluorescence increases. This mini-review addresses the subcellular origins of NAD(P)H autofluorescence and the evidence for mitochondrial and glycolytic contributions to compound transients. It is concluded that there is no direct evidence for a contribution to NAD(P)H signals from glycolysis in astrocytes following synaptic glutamate uptake. In contrast, multiple lines of evidence, including from complimentary flavoprotein autofluorescence signals, imply that mitochondrial NADH dynamics in neurons dominate compound evoked NAD(P)H transients. These signals are thus appropriate for studies of mitochondrial function and dysfunction in brain slices, in addition to providing robust maps of postsynaptic neuronal activation following physiological activation.


Asunto(s)
Astrocitos/metabolismo , Encéfalo/metabolismo , NADP/metabolismo , NADP/efectos de la radiación , Neuronas/metabolismo , Transmisión Sináptica/fisiología , Animales , Astrocitos/citología , Encéfalo/citología , Flavoproteínas/metabolismo , Flavoproteínas/efectos de la radiación , Colorantes Fluorescentes/metabolismo , Colorantes Fluorescentes/efectos de la radiación , Humanos , Redes y Vías Metabólicas/fisiología , Mitocondrias/metabolismo , Neuroquímica/métodos , Neuronas/citología
11.
Biochemistry ; 48(36): 8585-93, 2009 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-19663499

RESUMEN

Cryptochromes (CRYs) are blue-light photoreceptors with known or presumed functions in light-dependent and light-independent gene regulation in plants and animals. Although the photochemistry of plant CRYs has been studied in some detail, the photochemical behavior of animal cryptochromes remains poorly defined in part because it has been difficult to purify animal CRYs with their flavin cofactors. Here we describe the purification of type 4 CRYs of zebrafish and chicken as recombinant proteins with full flavin complement and compare the spectroscopic properties of type 4 and type 1 CRYs. In addition, we analyzed photoinduced proteolytic degradation of both types of CRYs in vivo in heterologous systems. We find that even though both types of CRYs contain stoichiometric flavin, type 1 CRY is proteolytically degraded by a light-initiated reaction in Drosophila S2, zebrafish Z3, and human HEK293T cell lines, but zebrafish CRY4 (type 4) is not. In vivo degradation of type 1 CRYs does not require continuous illumination, and a single light flash of 1 ms duration leads to degradation of about 80% of Drosophila CRY in 60 min. Finally, we demonstrate that in contrast to animal type 2 CRYs and Arabidopsis CRY1 neither insect type 1 nor type 4 CRYs have autokinase activities.


Asunto(s)
Proteínas Aviares/química , Flavinas/química , Flavoproteínas/química , Fotoquímica , Proteínas de Pez Cebra/química , Animales , Anopheles , Proteínas Aviares/metabolismo , Línea Celular , Pollos , Criptocromos , Desoxirribodipirimidina Fotoliasa/química , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/efectos de la radiación , Escherichia coli/enzimología , Flavina-Adenina Dinucleótido/química , Flavinas/metabolismo , Flavinas/efectos de la radiación , Flavoproteínas/metabolismo , Flavoproteínas/efectos de la radiación , Humanos , Luz , Fotoquímica/métodos , Ratas , Proteínas de Pez Cebra/metabolismo
12.
Biochem Soc Trans ; 37(Pt 2): 382-6, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19290867

RESUMEN

(13)C-NMR experiments were performed on photo-excited fully and partially (13)C-labelled LOV2 domains of the blue-light receptor phototropin. In the present paper, we report on nuclear-spin polarized tryptophan resonances that are generated by light-induced intraprotein electron transfer to the FMN cofactor. The spectra are discussed with respect to earlier data obtained from (13)C-NMR experiments on unlabelled LOV2 domains that have been reconstituted with FMN (13)C isotopologues.


Asunto(s)
Flavoproteínas/química , Triptófano/química , Isótopos de Carbono , Criptocromos , Transporte de Electrón , Mononucleótido de Flavina/química , Mononucleótido de Flavina/efectos de la radiación , Flavoproteínas/efectos de la radiación , Luz , Espectroscopía de Resonancia Magnética , Estructura Terciaria de Proteína
13.
Plant Cell ; 21(1): 118-30, 2009 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-19141709

RESUMEN

Arabidopsis thaliana cryptochrome 2 (CRY2) mediates photoperiodic promotion of floral initiation and blue light inhibition of hypocotyl elongation. It has been hypothesized that photoexcitation derepresses CRY2 by disengaging its C-terminal domain from the N-terminal PHR domain. To test this hypothesis, we analyzed activities of CRY2 fused to green fluorescent protein (GFP) at either the N terminus (GFP-CRY2) or the C terminus (CRY2-GFP). While GFP-CRY2 exerts light-dependent biochemical and physiological activities similar to those of the endogenous CRY2, CRY2-GFP showed constitutive biochemical and physiological activities. CRY2-GFP is constitutively phosphorylated, it promotes deetiolation in both dark and light, and it activates floral initiation in both long-day and short-day photoperiods. These results are consistent with the hypothesis that photoexcited CRY2 disengages its C-terminal domain from the PHR domain to become active. Surprisingly, we found that CRY2-GFP, but not GFP-CRY2, formed distinct nuclear bodies in response to blue light. Compared with GFP-CRY2 or the endogenous CRY2, CRY2-GFP degradation was significantly retarded in response to blue light, suggesting that the nuclear bodies may result from accumulation of photoexcited CRY2-GFP waiting to be degraded. Consistent with this interpretation, we showed that both GFP-CRY2 and endogenous CRY2 formed nuclear bodies in the presence of the 26S-proteasome inhibitors that block blue light-dependent CRY2 degradation.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Flavoproteínas/metabolismo , Cuerpos de Inclusión Intranucleares/metabolismo , Luz , Arabidopsis/citología , Arabidopsis/genética , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/efectos de la radiación , Criptocromos , Flavoproteínas/genética , Flavoproteínas/efectos de la radiación , Regulación de la Expresión Génica de las Plantas , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/efectos de la radiación , Modelos Moleculares , Fosforilación , Fotorreceptores de Plantas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , ARN de Planta/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/efectos de la radiación
15.
Biochemistry ; 47(52): 13842-9, 2008 Dec 30.
Artículo en Inglés | MEDLINE | ID: mdl-19063612

RESUMEN

Light is a key stimulus for plant biological functions, several of which are controlled by light-activated kinases known as phototropins, a group of kinases that contain two light-sensing domains (LOV, light-oxygen-voltage domains) and a C-terminal serine/threonine kinase domain. The second sensory domain, LOV2, plays a key role in regulating kinase enzymatic activity via the photochemical formation of a covalent adduct between a LOV2 cysteine residue and an internally bound flavin mononucleotide (FMN) chromophore. Subsequent conformational changes in LOV2 lead to the unfolding of a peripheral Jalpha helix and, ultimately, phototropin kinase activation. To date, the mechanism coupling bond formation and helix dissociation has remained unclear. Previous studies found that a conserved glutamine residue [Q513 in the Avena sativa phototropin 1 LOV2 (AsLOV2) domain] switches its hydrogen bonding pattern with FMN upon light stimulation. Located in the immediate vicinity of the FMN binding site, this Gln residue is provided by the Ibeta strand that interacts with the Jalpha helix, suggesting a route for signal propagation from the core of the LOV domain to its peripheral Jalpha helix. To test whether Q513 plays a key role in tuning the photochemical and transduction properties of AsLOV2, we designed two point mutations, Q513L and Q513N, and monitored the effects on the chromophore and protein using a combination of UV-visible absorbance and circular dichroism spectroscopy, limited proteolysis, and solution NMR. The results show that these mutations significantly dampen the changes between the dark and lit state AsLOV2 structures, leaving the protein in a pseudodark state (Q513L) or a pseudolit state (Q513N). Further, both mutations changed the photochemical properties of this receptor, in particular the lifetime of the photoexcited signaling states. Together, these data establish that this residue plays a central role in both spectral tuning and signal propagation from the core of the LOV domain through the Ibeta strand to the peripheral Jalpha helix.


Asunto(s)
Flavoproteínas/fisiología , Glutamina , Fototransducción , Fosfotransferasas/fisiología , Avena/enzimología , Avena/fisiología , Secuencia Conservada , Criptocromos , Mononucleótido de Flavina , Flavoproteínas/genética , Flavoproteínas/efectos de la radiación , Luz , Mutación Missense , Fosfotransferasas/genética , Fosfotransferasas/efectos de la radiación , Fotoquímica , Proteínas de Plantas , Conformación Proteica
16.
Proc Natl Acad Sci U S A ; 105(50): 20038-43, 2008 Dec 16.
Artículo en Inglés | MEDLINE | ID: mdl-19060199

RESUMEN

Fluorescent proteins (FPs) based on green fluorescent protein (GFP) are widely used throughout cell biology to study protein dynamics, and have extensive use as reporters of virus infection and spread. However, FP-tagging of viruses is limited by the constraints of viral genome size resulting in FP loss through recombination events. To overcome this, we have engineered a smaller ( approximately 10 kDa) flavin-based alternative to GFP ( approximately 25 kDa) derived from the light, oxygen or voltage-sensing (LOV) domain of the plant blue light receptor, phototropin. Molecular evolution and Tobacco mosaic virus (TMV)-based expression screening produced LOV variants with improved fluorescence and photostability in planta. One variant in particular, designated iLOV, possessed photophysical properties that made it ideally suited as a reporter of subcellular protein localization in both plant and mammalian cells. Moreover, iLOV fluorescence was found to recover spontaneously after photobleaching and displayed an intrinsic photochemistry conferring advantages over GFP-based FPs. When expressed either as a cytosolic protein or as a viral protein fusion, iLOV functioned as a superior reporter to GFP for monitoring local and systemic infections of plant RNA viruses. iLOV, therefore, offers greater utility in FP-tagging of viral gene products and represents a viable alternative where functional protein expression is limited by steric constraints or genome size.


Asunto(s)
Flavoproteínas/análisis , Proteínas Luminiscentes/análisis , Virus de Plantas/fisiología , Plantas/virología , Proteínas Virales/análisis , Animales , Criptocromos , Evolución Molecular Dirigida , Flavinas/química , Flavoproteínas/genética , Flavoproteínas/metabolismo , Flavoproteínas/efectos de la radiación , Fluorescencia , Genes Reporteros , Ingeniería Genética , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/efectos de la radiación , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas Luminiscentes/efectos de la radiación , Microscopía Confocal , Microscopía Fluorescente , Oxígeno/metabolismo , Fotoblanqueo , Virus de Plantas/genética , Virus de Plantas/metabolismo , Proteínas Recombinantes de Fusión , Virus del Mosaico del Tabaco/genética , Virus del Mosaico del Tabaco/fisiología , Proteínas Virales/genética , Proteínas Virales/metabolismo , Proteínas Virales/efectos de la radiación
17.
Nat Chem Biol ; 4(8): 491-7, 2008 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-18604202

RESUMEN

Protein photosensors are versatile tools for studying ligand-regulated allostery and signaling. Fundamental to these processes is the amount of energy that can be provided by a photosensor to control downstream signaling events. Such regulation is exemplified by the phototropins--plant serine/threonine kinases that are activated by blue light via conserved LOV (light, oxygen and voltage) domains. The core photosensor of oat phototropin 1 is a LOV domain that interacts in a light-dependent fashion with an adjacent alpha-helix (J alpha) to control kinase activity. We used solution NMR measurements to quantify the free energy of the LOV domain-J alpha-helix binding equilibrium in the dark and lit states. These data indicate that light shifts this equilibrium by approximately 3.8 kcal mol(-1), thus quantifying the energy available through LOV-J alpha for light-driven allosteric regulation. This study provides insight into the energetics of light sensing by phototropins and benchmark values for engineering photoswitchable systems based on the LOV-J alpha interaction.


Asunto(s)
Regulación Alostérica/efectos de la radiación , Flavoproteínas/química , Flavoproteínas/efectos de la radiación , Luz , Termodinámica , Criptocromos , Activación Enzimática/efectos de la radiación , Oxígeno , Proteínas de Plantas , Proteínas Serina-Treonina Quinasas , Estructura Terciaria de Proteína/efectos de la radiación
18.
PLoS Biol ; 6(7): e160, 2008 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-18597555

RESUMEN

Cryptochromes are a class of flavoprotein blue-light signaling receptors found in plants, animals, and humans that control plant development and the entrainment of circadian rhythms. In plant cryptochromes, light activation is proposed to result from photoreduction of a protein-bound flavin chromophore through intramolecular electron transfer. However, although similar in structure to plant cryptochromes, the light-response mechanism of animal cryptochromes remains entirely unknown. To complicate matters further, there is currently a debate on whether mammalian cryptochromes respond to light at all or are instead activated by non-light-dependent mechanisms. To resolve these questions, we have expressed both human and Drosophila cryptochrome proteins to high levels in living Sf21 insect cells using a baculovirus-derived expression system. Intact cells are irradiated with blue light, and the resulting cryptochrome photoconversion is monitored by fluorescence and electron paramagnetic resonance spectroscopic techniques. We demonstrate that light induces a change in the redox state of flavin bound to the receptor in both human and Drosophila cryptochromes. Photoreduction from oxidized flavin and subsequent accumulation of a semiquinone intermediate signaling state occurs by a conserved mechanism that has been previously identified for plant cryptochromes. These results provide the first evidence of how animal-type cryptochromes are activated by light in living cells. Furthermore, human cryptochrome is also shown to undergo this light response. Therefore, human cryptochromes in exposed peripheral and/or visual tissues may have novel light-sensing roles that remain to be elucidated.


Asunto(s)
Proteínas del Ojo/metabolismo , Flavinas/metabolismo , Flavoproteínas/metabolismo , Fototransducción , Células Fotorreceptoras de Invertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Animales , Relojes Biológicos/fisiología , Línea Celular , Criptocromos , Drosophila melanogaster , Espectroscopía de Resonancia por Spin del Electrón , Proteínas del Ojo/efectos de la radiación , Flavinas/efectos de la radiación , Flavoproteínas/efectos de la radiación , Expresión Génica , Humanos , Organismos Modificados Genéticamente , Oxidación-Reducción , Células Fotorreceptoras de Invertebrados/efectos de la radiación , Células Fotorreceptoras de Vertebrados/efectos de la radiación , Spodoptera , Rayos Ultravioleta
19.
Photochem Photobiol ; 84(4): 1016-23, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-18494763

RESUMEN

Photoreaction of a blue-light photoreceptor Cryptochrome-DASH (Cry-DASH), a new member of the Cryptochrome family, from zebrafish was studied by UV-visible absorption spectroscopy in aqueous solutions at 293 K. Zebrafish Cry-DASH binds two chromophores, a flavin adenine dinucleotide (FAD) and a N5,N10-methenyl-5,6,7,8-tetrahydrofolate (MTHF) noncovalently. The bound FAD exists in the oxidized form (FAD(ox)) in the dark. Blue light converts FAD(ox) to the neutral radical form (FADH*). Formed FADH* is transformed to the fully reduced form FADH(2) (or FADH(-)) by successive light irradiation, or reverts to FAD(ox). FADH(2) (or FADH(-)) reverts to FADH* or possibly to FAD(ox) directly. The effect of dithiothreitol suggests a possible electron transfer between FAD in zebrafish Cry-DASH and reductants in the external medium. This is the first report on the photoreaction pathway and kinetics of a vertebrate Cry-DASH family protein.


Asunto(s)
Transporte de Electrón , Flavoproteínas/metabolismo , Flavoproteínas/efectos de la radiación , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/efectos de la radiación , Pez Cebra/metabolismo , Animales , Criptocromos , Oscuridad , Flavoproteínas/química , Luz , Células Fotorreceptoras/metabolismo , Espectrofotometría , Proteínas de Pez Cebra/química
20.
Biophys J ; 95(1): 312-21, 2008 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-18339766

RESUMEN

The flavoprotein AppA from Rhodobacter sphaeroides contains an N-terminal, FAD-binding BLUF photoreceptor domain. Upon illumination, the AppA BLUF domain forms a signaling state that is characterized by red-shifted absorbance by 10 nm, a state known as AppA(RED). We have applied ultrafast spectroscopy on the photoaccumulated AppA(RED) state to investigate the photoreversible properties of the AppA BLUF domain. On light absorption by AppA(RED), the FAD singlet excited state FAD(RED)* decays monoexponentially in 7 ps to form the neutral semiquinone radical FADH(*), which subsequently decays to the original AppA(RED) molecular ground state in 60 ps. Thus, FAD(RED)* is deactivated rapidly via electron and proton transfer, probably from the conserved tyrosine Tyr-21 to FAD, followed by radical-pair recombination. We conclude that, in contrast to many other photoreceptors, the AppA BLUF domain is not photoreversible and does not enter alternative reaction pathways upon absorption of a second photon. To explain these properties, we propose that a molecular configuration is formed upon excitation of AppA(RED) that corresponds to a forward reaction intermediate previously identified for the dark-state BLUF photoreaction. Upon excitation of AppA(RED), the BLUF domain therefore enters its forward reaction coordinate, readily re-forming the AppA(RED) ground state and suppressing reverse or side reactions. The monoexponential decay of FAD* indicates that the FAD-binding pocket in AppA(RED) is significantly more rigid than in dark-state AppA. Steady-state fluorescence experiments on wild-type, W104F, and W64F mutant BLUF domains show tryptophan fluorescence maxima that correspond with a buried conformation of Trp-104 in dark and light states. We conclude that Trp-104 does not become exposed to solvent during the BLUF photocycle.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Flavoproteínas/química , Flavoproteínas/ultraestructura , Modelos Químicos , Modelos Moleculares , Fotoquímica/métodos , Proteínas Bacterianas/efectos de la radiación , Simulación por Computador , Flavoproteínas/efectos de la radiación , Luz , Conformación Proteica/efectos de la radiación , Estructura Terciaria de Proteína/efectos de la radiación , Dosis de Radiación
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