RESUMO
Strong excitonic interactions are a key design strategy in photosynthetic light harvesting, expanding the spectral cross-section for light absorption and creating considerably faster and more robust excitation energy transfer. These molecular excitons are a direct result of exceptionally densely packed pigments in photosynthetic proteins. The main light-harvesting complexes of diatoms, known as fucoxanthin-chlorophyll proteins (FCPs), are an exception, displaying surprisingly weak excitonic coupling between their chlorophyll (Chl) a's, despite a high pigment density. Here, we show, using single-molecule spectroscopy, that the FCP complexes of Cyclotella meneghiniana switch frequently into stable, strongly emissive states shifted 4-10 nm toward the red. A few percent of isolated FCPa complexes and â¼20% of isolated FCPb complexes, on average, were observed to populate these previously unobserved states, percentages that agree with the steady-state fluorescence spectra of FCP ensembles. Thus, the complexes use their enhanced sensitivity to static disorder to increase their light-harvesting capability in a number of ways. A disordered exciton model based on the structure of the main plant light-harvesting complex explains the red-shifted emission by strong localization of the excitation energy on a single Chl a pigment in the terminal emitter domain due to very specific pigment orientations. We suggest that the specific construction of FCP gives the complex a unique strategy to ensure that its light-harvesting function remains robust in the fluctuating protein environment despite limited excitonic interactions.
Assuntos
Diatomáceas/química , Complexos de Proteínas Captadores de Luz/química , Fotossíntese , Diatomáceas/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismoRESUMO
Photosystem II (PSII) is a huge pigment-protein supercomplex responsible for the primary steps of photosynthesis in green plants. Its light-harvesting antenna exhibits efficient transfer of the absorbed excitation energy to the reaction center and also contains a well-regulated protection mechanism against over-excitation in strong light conditions. The latter is based on conformational changes in antenna complexes that open up excitation decay channels resulting in considerable fluorescence quenching. Meanwhile, fluorescence blinking, observed in single antennas, is likely caused by a similar mechanism. Thus the question arises whether this effect is also present in and relevant to the native supramolecular organization of a fully assembled PSII. To further investigate energy transfer and quenching in single PSII, we performed single-molecule experiments on PSII supercomplexes at 5 °C. Analysis of the fluorescence intensity and mean lifetime allowed us to distinguish detached antennas and specifically analyze PSII supercomplexes. The average fluorescence lifetime in PSII of about 100-150 ps, measured under our extreme excitation conditions, is surprisingly similar to published ensemble lifetime data of photochemical quenching in PSII of a similar size. In our case, this lifetime is nevertheless caused by either one or multiple quenched antennas or by a quencher in the reaction center. The observed reversible light-induced changes in fluorescence intensity on a millisecond timescale are reminiscent of blinking subunits. Our results therefore directly illustrate how environmental control over a fluctuating antenna can regulate light-harvesting in plant photosynthesis.
Assuntos
Complexos de Proteínas Captadores de Luz/química , Complexo de Proteína do Fotossistema II/química , Clorofila/química , Transferência de Energia , Fluorescência , Cinética , Simulação de Dinâmica Molecular , Fotossíntese , Conformação Proteica , Multimerização Proteica , Imagem Individual de MoléculaRESUMO
Most cyanobacteria, under high light conditions, decrease the amount of energy arriving at the reaction centers by increasing thermal energy dissipation at the level of the phycobilisome, the extramembranous antenna. This mechanism is induced by photoactivation of the Orange Carotenoid Protein (OCP). To identify how the activated OCP interacts with phycobilisomes (PBs), several OCP mutants were constructed, and the influence of mutations on photoactivity, stability, and binding to PBs was characterized. The disruption of the salt bridge between Arg155 and Glu244, which stabilizes the interaction between the N- and C-terminal domains, increased the rate of photoactivity and the stability of the photoactivated OCP, suggesting that the activated OCP has an open structure with decreased interdomain interaction. Changing Glu244 to leucine had no effect on OCP binding to PBs. By contrast, substitution of Arg155 with a neutral or a negatively charged amino acid largely decreased OCP binding to the PBs, whereas substitution with a lysine slightly perturbed the interaction. These results strongly suggest that the surface of the N-terminal domain, containing the Arg155, interacts with the PB and that the positive charge of Arg155 plays a key role in photoprotection.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cianobactérias/metabolismo , Luz , Ficobilissomas/farmacologia , Cianobactérias/efeitos dos fármacos , Ligação Proteica , Estrutura Secundária de ProteínaRESUMO
In this study, we demonstrate the selective in vivo detection of diadinoxanthin (DD) and diatoxanthin (DT) in intact Cyclotella cells using resonance Raman spectroscopy. In these cells, we were able to assess both the content of DD and DT carotenoids relative to chlorophyll and their conformation. In addition, the sensitivity and selectivity of the technique allow us to discriminate between different pools of DD on a structural basis, and to follow their fate as a function of the illumination conditions. We report that the additional DD observed when cells are grown in high-light conditions adopts a more twisted conformation than the lower levels of DD present when the cells are grown in low-light (LL) conditions. Thus, we conclude that this pool of DD is more tightly bound to a protein-binding site, which must differ from the one occupied by the DD present in LL conditions.
Assuntos
Carotenoides/análise , Diatomáceas/metabolismo , Análise Espectral Raman/métodos , Sítios de Ligação , Carotenoides/metabolismo , Cromatografia Líquida de Alta Pressão , Diatomáceas/crescimento & desenvolvimento , Luz , Xantofilas/análise , Xantofilas/metabolismoRESUMO
The role of conformational changes in explaining the huge catalytic power of enzymes is currently one of the most challenging questions in biology. Although it is now widely regarded that enzymes modulate reaction rates by means of short- and long-range protein motions, it is almost impossible to distinguish between conformational changes and catalysis. We have solved this problem using the chlorophyll biosynthetic enzyme NADPH:protochlorophyllide (Pchlide) oxidoreductase, which catalyses a unique light-driven reaction involving hydride and proton transfers. Here we report that prior excitation of the enzyme-substrate complex with a laser pulse induces a more favourable conformation of the active site, enabling the coupled hydride and proton transfer reactions to occur. This effect, which is triggered during the Pchlide excited-state lifetime and persists on a long timescale, switches the enzyme into an active state characterized by a high rate and quantum yield of formation of a catalytic intermediate. The corresponding spectral changes in the mid-infrared following the absorption of one photon reveal significant conformational changes in the enzyme, illustrating the importance of flexibility and dynamics in the structure of enzymes for their function.
Assuntos
Luz , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/efeitos da radiação , Synechocystis/enzimologia , Biocatálise/efeitos da radiação , Domínio Catalítico/efeitos da radiação , Modelos Moleculares , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Conformação Proteica/efeitos da radiação , Prótons , Relação Estrutura-Atividade , Fatores de TempoRESUMO
In high light, the antenna system in oxygenic photosynthetic organisms switches to a photoprotective mode, dissipating excess energy in a process called non-photochemical quenching (NPQ). Diatoms exhibit very efficient NPQ, accompanied by a xanthophyll cycle in which diadinoxanthin is de-epoxidized into diatoxanthin. Diatoms accumulate pigments from this cycle in high light, and exhibit faster and more pronounced NPQ. The mechanisms underlying NPQ in diatoms remain unclear, but it can be mimicked by aggregation of their isolated light-harvesting complexes, FCP (fucoxanthin chlorophyll-a/c protein). We assess this model system by resonance Raman measurements of two peripheral FCPs, trimeric FCPa and nonameric FCPb, isolated from high- and low-light-adapted cells (LL,HL). Quenching is associated with a reorganisation of these proteins, affecting the conformation of their bound carotenoids, and in a manner which is highly dependent on the protein considered. FCPa from LL diatoms exhibits significant changes in diadinoxanthin structure, together with a smaller conformational change of at least one fucoxanthin. For these LL-FCPa, quenching is associated with consecutive events, displaying distinct spectral signatures, and its amplitude correlates with the planarity of the diadinoxanthin structure. HL-FCPa aggregation is associated with a change in planarity of a 515-nm-absorbing fucoxanthin, and, to a lesser extent, of diadinoxanthin. Finally, in FCPb, a blue-absorbing fucoxanthin is primarily affected. FCPs thus possess a plastic structure, undergoing several conformational changes upon aggregation, dependent upon their precise composition and structure. NPQ in diatoms may therefore arise from a combination of structural changes, dependent on the environment the cells are adapted to.
RESUMO
The photosynthetic light-harvesting systems of purple bacteria and plants both utilize specific carotenoids as quenchers of the harmful (bacterio)chlorophyll triplet states via triplet-triplet energy transfer. Here, we explore how the binding of carotenoids to the different types of light-harvesting proteins found in plants and purple bacteria provides adaptation in this vital photoprotective function. We show that the creation of the carotenoid triplet states in the light-harvesting complexes may occur without detectable conformational changes, in contrast to that found for carotenoids in solution. However, in plant light-harvesting complexes, the triplet wavefunction is shared between the carotenoids and their adjacent chlorophylls. This is not observed for the antenna proteins of purple bacteria, where the triplet is virtually fully located on the carotenoid molecule. These results explain the faster triplet-triplet transfer times in plant light-harvesting complexes. We show that this molecular mechanism, which spreads the location of the triplet wavefunction through the pigments of plant light-harvesting complexes, results in the absence of any detectable chlorophyll triplet in these complexes upon excitation, and we propose that it emerged as a photoprotective adaptation during the evolution of oxygenic photosynthesis.
Assuntos
Complexos de Proteínas Captadores de Luz/química , Luz , Modelos Moleculares , Bacterioclorofilas/química , Carotenoides/química , Carotenoides/metabolismo , Elétrons , Complexos de Proteínas Captadores de Luz/metabolismo , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/metabolismo , Ligação Proteica , Conformação Proteica , Proteobactérias/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral Raman , Spinacia oleracea/metabolismo , VibraçãoRESUMO
The light-dependent reduction of protochlorophyllide, a key step in the synthesis of chlorophyll, is catalyzed by the enzyme protochlorophyllide oxidoreductase (POR) and requires two photons (O. A. Sytina et al., Nature, 2008, 456, 1001-1008). The first photon activates the enzyme-substrate complex, a subsequent second photon initiates the photochemistry by triggering the formation of a catalytic intermediate. These two events are characterized by different spectral changes in the infra-red spectral region. Here, we investigate the vibrational frequencies of the POR-bound and unbound substrate, and product, and thus provide a detailed assignment of the spectral changes in the 1800-1250 cm(-1) region associated with the catalytic conversion of PChlide:NADPH:TyrOH into Chlide:NADP(+):TyrO(-). Fluorescence line narrowed spectra of the POR-bound Pchlide reveal a C=O keto group downshifted by more than 20 cm(-1) to a relatively low vibrational frequency of 1653 cm(-1), as compared to the unbound Pchlide, indicating that binding of the chromophore to the protein occurs via strong hydrogen bond(s). The frequencies of the C=C vibrational modes are consistent with a six-coordinated state of the POR-bound Pchlide, suggesting that there are two coordination interactions between the central Mg atom of the chromophore and protein residues, and/or a water molecule. The frequencies of the C=C vibrational modes of Chlide are consistent with a five-coordinated state, indicating a single interaction between the central Mg atom of the chromophore and a water molecule. Rapid-scan FTIR measurements on the Pchlide:POR:NADPH complex at 4 cm(-1) spectral resolution reveal a new band in the 1670 cm(-1) region. The FTIR spectra of the enzyme activation phase indicate involvement of a nucleotide-binding structural motif, and an increased exposure of the protein to solvent after activation.
Assuntos
NADP/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Fotoquímica , Catálise , Ativação Enzimática , Cinética , Luz , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Intense sunlight is dangerous for photosynthetic organisms. Cyanobacteria, like plants, protect themselves from light-induced stress by dissipating excess absorbed energy as heat. Recently, it was discovered that a soluble orange carotenoid protein, the OCP, is essential for this photoprotective mechanism. Here we show that the OCP is also a member of the family of photoactive proteins; it is a unique example of a photoactive protein containing a carotenoid as the photoresponsive chromophore. Upon illumination with blue-green light, the OCP undergoes a reversible transformation from its dark stable orange form to a red "active" form. The red form is essential for the induction of the photoprotective mechanism. The illumination induces structural changes affecting both the carotenoid and the protein. Thus, the OCP is a photoactive protein that senses light intensity and triggers photoprotection.
Assuntos
Proteínas de Bactérias/metabolismo , Carotenoides/metabolismo , Luz , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cor , Modelos Moleculares , Fotoquímica , Estrutura Terciária de Proteína , Synechocystis/química , Synechocystis/genética , Synechocystis/metabolismo , Synechocystis/efeitos da radiaçãoRESUMO
The bacterium Caulobacter crescentus encodes a two-component signaling protein, LovK, that contains an N-terminal photosensory LOV domain coupled to a C-terminal histidine kinase. LovK binds a flavin cofactor, undergoes a reversible photocycle, and displays regulated ATPase and autophosphorylation activity in response to visible light. Femtosecond to nanosecond visible absorption spectroscopy demonstrates congruence between full-length LovK and isolated LOV domains in the mechanism and kinetics of light-dependent cysteinyl-C4(a) adduct formation and rupture, while steady-state absorption and fluorescence line narrowing (FLN) spectroscopies reveal unique features in the electronic structure of the LovK flavin cofactor. In agreement with other sensor histidine kinases, ATP binds specifically to LovK with micromolar affinity. However, ATP binding to the histidine kinase domain of LovK has no apparent effect on global protein structure as assessed by differential Fourier transform infrared (FTIR) spectroscopy. Cysteinyl adduct formation results in only minor changes in the structure of LovK as determined by differential FTIR. This study provides insight into the structural underpinnings of LOV-mediated signal transduction in the context of a full-length histidine kinase. In particular, the data provide evidence for a model in which small changes in the tertiary/quaternary structure of LovK, as triggered by photon detection in the N-terminal LOV sensory domain, are sufficient to regulate histidine kinase activity.
Assuntos
Proteínas de Bactérias/química , Caulobacter crescentus/enzimologia , Transdução de Sinal Luminoso/fisiologia , Luz , Proteínas Quinases/química , Proteínas de Bactérias/metabolismo , Sequência Conservada , Elétrons , Ativação Enzimática , Mononucleotídeo de Flavina/química , Mononucleotídeo de Flavina/metabolismo , Histidina Quinase , Ligação Proteica , Proteínas Quinases/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de ProteínaRESUMO
The peridinin chlorophyll-a protein (PCP) is a water-soluble, trimeric light harvesting complex found in marine dinoflagellates that binds peridinin and Chl-a in an unusual stoichiometric ratio of 4:1. In this paper, the pathways of excited-state energy transfer and relaxation in PCP were identified by means of femtosecond visible-pump, mid-infrared probe spectroscopy. In addition, excited-state relaxation of peridinin dissolved in organic solvent (CHCl(3) and MeOH) was investigated. For peridinin in solution, the transient IR signatures of the low-lying S(1) and intramolecular charge transfer (ICT) states were similar, in line with a previous ultrafast IR study. In PCP, excitation of the optically allowed S(2) state of peridinin results in ultrafast energy transfer to Chl-a, in competition with internal conversion to low-lying optically forbidden states of peridinin. After vibrational relaxation of the peridinin hot S(1) state in 150 fs, two separate low-lying peridinin singlet excited states are distinguished, assigned to an ICT state and to a slowly transferring, vibrationally relaxed S(1) state. These states exhibit different lactone bleaches, indicating that the ICT and S(1) states localize on distinct peridinins. Energy transfer from the peridinin ICT state to Chl-a constitutes the dominant energy transfer channel and occurs with a time constant of 2 ps. The peridinin S(1) state mainly decays to the ground state through internal conversion, in competition with slow energy transfer to Chl-a. The singlet excited state of Chl-a undergoes intersystem crossing (ISC) to the triplet state on the nanosecond timescale, followed by rapid triplet excitation energy transfer (TEET) from Chl-a to peridinin, whereby no Chl-a triplet is observed but rather a direct rise of the peridinin triplet. The latter contains some Chl-a features due to excitonic coupling of the pigments. The peridinin triplet state shows a lactone bleach mode at 1748 cm(-1), while that of the peridinin ICT state is located at 1745 cm(-1), indicating that the main channels of singlet and triplet energy transfer in PCP proceed through distinct peridinins. Our results are consistent with an energy transfer scheme where the ICT state mainly localizes on Per621/611 and Per623/613, the S(1) state on Per622/612 and the triplet state on Per624/614.
Assuntos
Carotenoides/química , Clorofilídeos/química , Transferência de Energia , Conformação Molecular , Solventes/química , Espectrofotometria Infravermelho , Fatores de TempoRESUMO
Phototropins control phototropism, chloroplast movement, stomatal opening, and leaf expansion in plants. Phototropin 1 (phot1) is composed of a kinase domain linked to two blue light-sensing domains, LOV2 and LOV1, which bind flavin mononucleotide. Disruption of the interaction between the LOV2 domain and a helical segment named Jalpha, joining LOV to the kinase domain, induces the subsequent kinase activity of phototropin 1 and further-downstream signal transduction. Here we study the effects of temperature and hydration on the light-triggered signal propagation in the phot1 LOV2 domain of Avena sativa (AsLOV2/Jalpha), using Fourier transform infrared spectroscopy to unravel part of the molecular mechanism of phototropin 1. We report that AsLOV2/Jalpha shows an intense signal in the amide I and II regions, arising mainly from beta-sheet changes and the unbinding of the Jalpha helix from the Per-ARNT-Sim core and its subsequent partial unfolding. Importantly, these structural changes only occur under conditions of full hydration and at temperatures above 280 K. We characterized a newly isolated low-hydration intermediate that shows a downshift of high-frequency amide I signals and that possibly corresponds to loop tightening, without large beta-sheet or Jalpha structural changes. In addition, we report a heterogeneity in AsLOV2/Jalpha involving two different C(4)=O conformer populations, coexisting in the dark state and characterized by C(4)=O carbonyl frequencies at 1712 cm(-1) and 1694 cm(-1) that are attributable to a single H-bond and two H-bonds at this site, respectively. Such conformers display slightly shifted absorption spectra and cause a splitting of the 475-nm band in the ultraviolet/visible spectra of LOV domains at low temperature.
Assuntos
Flavoproteínas/química , Proteínas de Plantas/química , Avena , Criptocromos , Temperatura Alta , Ligação de Hidrogênio , Luz , Modelos Químicos , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura , Água/químicaRESUMO
Phototropins, major blue-light receptors in plants, are sensitive to blue light through a pair of flavin mononucleotide (FMN)-binding light oxygen and voltage (LOV) domains, LOV1 and LOV2. LOV2 undergoes a photocycle involving light-driven covalent adduct formation between a conserved cysteine and the FMN C(4a) atom. Here, the primary reactions of Avena sativa phototropin 1 LOV2 (AsLOV2) were studied using ultrafast mid-infrared spectroscopy and quantum chemistry. The singlet excited state (S1) evolves into the triplet state (T1) with a lifetime of 1.5 ns at a yield of approximately 50%. The infrared signature of S1 is characterized by absorption bands at 1657 cm(-1), 1495-1415 cm(-1), and 1375 cm(-1). The T1 state shows infrared bands at 1657 cm(-1), 1645 cm(-1), 1491-1438 cm(-1), and 1390 cm(-1). For both electronic states, these bands are assigned principally to C=O, C=N, C-C, and C-N stretch modes. The overall downshifting of C=O and C=N bond stretch modes is consistent with an overall bond-order decrease of the conjugated isoalloxazine system upon a pi-pi* transition. The configuration interaction singles (CIS) method was used to calculate the vibrational spectra of the S1 and T1 excited pipi* states, as well as respective electronic energies, structural parameters, electronic dipole moments, and intrinsic force constants. The harmonic frequencies of S1 and T1, as calculated by the CIS method, are in satisfactory agreement with the evident band positions and intensities. On the other hand, CIS calculations of a T1 cation that was protonated at the N(5) site did not reproduce the experimental FMN T1 spectrum. We conclude that the FMN T1 state remains nonprotonated on a nanosecond timescale, which rules out an ionic mechanism for covalent adduct formation involving cysteine-N(5) proton transfer on this timescale. Finally, we observed a heterogeneous population of singly and doubly H-bonded FMN C(4)=O conformers in the dark state, with stretch frequencies at 1714 cm(-1) and 1694 cm(-1), respectively.
Assuntos
Flavoproteínas/química , Proteínas de Plantas/química , Avena , Criptocromos , Flavinas/química , Ligação de Hidrogênio , Raios Infravermelhos , Cinética , Modelos Moleculares , Teoria Quântica , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral , VibraçãoRESUMO
The orange carotenoid protein (OCP), which is essential in cyanobacterial photoprotection, is the first photoactive protein containing a carotenoid as an active chromophore. Static and time-resolved Fourier transform infrared (FTIR) difference spectroscopy under continuous illumination at different temperatures was applied to investigate its photoactivation mechanism. Here, we demonstrate that in the OCP, the photo-induced conformational change involves at least two different steps, both in the second timescale at 277 K. Each step involves partial reorganization of α-helix domains. At early illumination times, the disappearance of a nonsolvent-exposed α-helix (negative 1651 cm-1 band) is observed. At longer times, a 1644 cm-1 negative band starts to bleach, showing the disappearance of a solvent-exposed α-helix, either the N-terminal extension and/or the C-terminal tail. A kinetic analysis clearly shows that these two events are asynchronous. Minor modifications in the overall FTIR difference spectra confirm that the global protein conformational change consists of-at least-two asynchronous contributions. Comparison of spectra recorded in H2O and D2O suggests that internal water molecules may contribute to the photoactivation mechanism.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Fatores de TempoRESUMO
Carotenoid-containing oil droplets in the avian retina act as cut-off filters to enhance colour discrimination. We report a confocal resonance Raman investigation of the oil droplets of the domestic chicken, Gallus gallus domesticus. We show that all carotenoids present are in a constrained conformation, implying a locus in specific lipid binding sites. In addition, we provide proof of a recent conclusion that all carotenoid-containing droplets contain a mixture of all carotenoids present, rather than only a subset of them-a conclusion that diverges from the previously-held view. Our results have implications for the mechanism(s) giving rise to these carotenoid mixtures in the differently-coloured droplets.
Assuntos
Carotenoides/química , Galinhas/fisiologia , Visão de Cores/fisiologia , Gotículas Lipídicas/química , Retina/citologia , Animais , Carotenoides/análise , Gotículas Lipídicas/fisiologia , Microscopia Confocal , Conformação Molecular , Retina/fisiologia , Análise Espectral RamanRESUMO
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.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Flavoproteínas/química , Flavoproteínas/ultraestrutura , Modelos Químicos , Modelos Moleculares , Fotoquímica/métodos , Proteínas de Bactérias/efeitos da radiação , Simulação por Computador , Flavoproteínas/efeitos da radiação , Luz , Conformação Proteica/efeitos da radiação , Estrutura Terciária de Proteína/efeitos da radiação , Doses de RadiaçãoRESUMO
Angiopoietin-like 2 (ANGPTL2) is an inflammatory adipokine linking obesity to insulin resistance. Intermittent fasting, on the other hand, is a lifestyle intervention able to prevent obesity and diabetes but difficult to implement and maintain. Our objectives were to characterize a link between ANGPTL2 and intermittent fasting and to investigate whether the knockdown of ANGPTL2 reproduces the benefits of intermittent fasting on weight gain and insulin responsiveness in knockdown and wild-type littermates mice. Intermittent fasting, access to food ad libitum once every other day, was initiated at the age of three months and maintained for four months. Intermittent fasting decreased by 63% (p < 0.05) gene expression of angptl2 in adipose tissue of wild-type mice. As expected, intermittent fasting improved insulin sensitivity (p < 0.05) and limited weight gain (p < 0.05) in wild-type mice. Knockdown mice fed ad libitum, however, were comparable to wild-type mice following the intermittent fasting regimen: insulin sensitivity and weight gain were identical, while intermittent fasting had no additional impact on these parameters in knockdown mice. Energy intake was similar between both wild-type fed intermittent fasting and ANGPTL2 knockdown mice fed ad libitum, suggesting that intermittent fasting and knockdown of ANGPTL2 equally lower feeding efficiency. These results suggest that the reduction of ANGPTL2 could be a useful and promising strategy to prevent obesity and insulin resistance, although further investigation of the mechanisms linking ANGPTL2 and intermittent fasting is warranted. Impact statement Intermittent fasting is an efficient diet pattern to prevent weight gain and improve insulin sensitivity. It is, however, a difficult regimen to follow and compliance is expected to be very low. In this work, we demonstrate that knockdown of ANGPTL2 in mice fed ad libitum mimics the beneficial effects of intermittent fasting on weight gain and insulin sensitivity in wild-type mice. ANGPTL2 is a cytokine positively associated with fat mass in humans, which inactivation in mice improves resistance to a high-fat metabolic challenge. This study provides a novel pathway by which IF acts to limit obesity despite equivalent energy intake. The development of a pharmacological ANGPTL2 antagonist could provide an efficient tool to reduce the burden of obesity.
Assuntos
Proteínas Semelhantes a Angiopoietina/metabolismo , Jejum , Resistência à Insulina , Obesidade/prevenção & controle , Proteína 2 Semelhante a Angiopoietina , Animais , Técnicas de Silenciamento de Genes , Humanos , Hipoglicemiantes/metabolismo , Insulina/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Obesidade/complicações , Redução de PesoRESUMO
In Cu-containing nitrite reductase from Alcaligenes faecalis S-6 the axial methionine ligand of the type-1 site was replaced (M150G) to make the copper ion accessible to external ligands that might affect the enzyme's catalytic activity. The type-1 site optical spectrum of M150G (A(460)/A(600)=0.71) differs significantly from that of the native nitrite reductase (A(460)/A(600)=1.3). The midpoint potential of the type-1 site of nitrite reductase M150G (E(M)=312(+/-5)mV versus hydrogen) is higher than that of the native enzyme (E(M)=213(+/-5)mV). M150G has a lower catalytic activity (k(cat)=133(+/-6)s(-1)) than the wild-type nitrite reductase (k(cat)=416(+/-10)s(-1)). The binding of external ligands to M150G restores spectral properties, midpoint potential (E(M)<225mV), and catalytic activity (k(cat)=374(+/-28)s(-1)). Also the M150H (A(460)/A(600)=7.7, E(M)=104(+/-5)mV, k(cat)=0.099(+/-0.006)s(-1)) and M150T (A(460)/A(600)=0.085, E(M)=340(+/-5)mV, k(cat)=126(+/-2)s(-1)) variants were characterized. Crystal structures show that the ligands act as allosteric effectors by displacing Met62, which moves to bind to the Cu in the position emptied by the M150G mutation. The reconstituted type-1 site has an otherwise unaltered geometry. The observation that removal of an endogenous ligand can introduce allosteric control in a redox enzyme suggests potential for structural and functional flexibility of copper-containing redox sites.
Assuntos
Nitrito Redutases/química , Nitrito Redutases/metabolismo , Alcaligenes faecalis/enzimologia , Alcaligenes faecalis/genética , Regulação Alostérica , Sítio Alostérico/genética , Substituição de Aminoácidos , Sequência de Bases , Cristalografia por Raios X , DNA Bacteriano/genética , História Antiga , Cinética , Ligantes , Modelos Moleculares , Mutagênese Sítio-Dirigida , Nitrito Redutases/genética , Oxirredução , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , EspectrofotometriaRESUMO
The two light, oxygen, and voltage domains of phototropin are blue-light photoreceptor domains that control various functions in plants and green algae. The key step of the light-driven reaction is the formation of a photoadduct between its FMN chromophore and a conserved cysteine, where the canonical reaction proceeds through the FMN triplet state. Here, complete photoreaction mapping of CrLOV2 from Chlamydomonas reinhardtii phototropin and AsLOV2 from Avena sativa phototropin-1 was realized by ultrafast broadband spectroscopy from femtoseconds to microseconds. We demonstrate that in CrLOV2, a direct photoadduct formation channel originates from the initially excited singlet state, in addition to the canonical reaction through the triplet state. This direct photoadduct reaction is coupled by a proton or hydrogen transfer process, as indicated by a significant kinetic isotope effect of 1.4 on the fluorescence lifetime. Kinetic model analyses showed that 38% of the photoadducts are generated from the singlet excited state.
Assuntos
Chlamydomonas reinhardtii/química , Mononucleotídeo de Flavina/química , Fotoquímica/métodos , Fototropinas/químicaRESUMO
Bacteriophytochromes (BphPs) constitute a class of photosensory proteins that toggle between Pr and Pfr functional states through absorption of red and far-red light. The photosensory core of BphPs is composed of PAS, GAF, and PHY domains. Here, we apply FTIR spectroscopy to investigate changes in the secondary structure of Rhodopseudomonas palustris BphP2 (RpBphP2) upon Pr to Pfr photoconversion. Our results indicate conversion from a ß-sheet to an α-helical element in the so-called tongue region of the PHY domain, consistent with recent X-ray structures of Deinococcus radiodurans DrBphP in dark and light states (Takala H.; et al. Nature2014, 5, 245-248). A conserved Asp in the GAF domain that noncovalently connects with the PHY domain and a conserved Pro in the tongue region of the PHY domain are essential for the ß-sheet-to-α-helix conversion.