RESUMO
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 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, 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
BLUF (blue light sensing using FAD) domains belong to a novel group of blue light sensing receptor proteins found in microorganisms. We have assessed the role of specific aromatic and polar residues in the Synechocystis Slr1694 BLUF protein by investigating site-directed mutants with substitutions Y8W, W91F, and S28A. The W91F and S28A mutants formed the red-shifted signaling state upon blue light illumination, whereas in the Y8W mutant, signaling state formation was abolished. The W91F mutant shows photoactivation dynamics that involve the successive formation of FAD anionic and neutral semiquinone radicals on a picosecond time scale, followed by radical pair recombination to result in the long-lived signaling state in less than 100 ps. The photoactivation dynamics and quantum yield of signaling state formation were essentially identical to those of wild type, which indicates that only one significant light-driven electron transfer pathway is available in Slr1694, involving electron transfer from Y8 to FAD without notable contribution of W91. In the S28A mutant, the photoactivation dynamics and quantum yield of signaling state formation as well as dark recovery were essentially the same as in wild type. Thus, S28 does not play an essential role in the initial hydrogen bond switching reaction in Slr1694 beyond an influence on the absorption spectrum. In the Y8W mutant, two deactivation branches upon excitation were identified: the first involves a neutral semiquinone FADH(*) that was formed in approximately 1 ps and recombines in 10 ps and is tentatively assigned to a FADH(*)-W8(*) radical pair. The second deactivation branch forms FADH(*) in 8 ps and evolves to FAD(*-) in 200 ps, which recombines to the ground state in about 4 ns. In the latter branch, W8 is tentatively assigned as the FAD redox partner as well. Overall, the results are consistent with a photoactivation mechanism for BLUF domains where signaling state formation proceeds via light-driven electron and proton transfer from Y8 to FAD, followed by a hydrogen bond rearrangement and radical pair recombination.
Assuntos
Aminoácidos/metabolismo , Proteínas de Bactérias/efeitos da radiação , Flavina-Adenina Dinucleotídeo , Luz , Fotorreceptores Microbianos/efeitos da radiação , Synechocystis/química , Synechocystis/efeitos da radiação , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sequência de Bases , Benzoquinonas/química , Benzoquinonas/metabolismo , Transporte de Elétrons , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Ligação de Hidrogênio , Mutação , Oxirredução , Processos Fotoquímicos/efeitos da radiação , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/metabolismo , Prótons , Synechocystis/metabolismoRESUMO
BLUF domains constitute a recently discovered class of photoreceptor proteins found in bacteria and eukaryotic algae. BLUF domains are blue-light sensitive through a FAD cofactor that is involved in an extensive hydrogen-bond network with nearby amino acid side chains, including a highly conserved tyrosine and glutamine. The participation of particular amino acid side chains in the ultrafast hydrogen-bond switching reaction with FAD that underlies photoactivation of BLUF domains is assessed by means of ultrafast infrared spectroscopy. Blue-light absorption by FAD results in formation of FAD(*-) and a bleach of the tyrosine ring vibrational mode on a picosecond timescale, showing that electron transfer from tyrosine to FAD constitutes the primary photochemistry. This interpretation is supported by the absence of a kinetic isotope effect on the fluorescence decay on H/D exchange. Subsequent protonation of FAD(*-) to result in FADH(*) on a picosecond timescale is evidenced by the appearance of a N-H bending mode at the FAD N5 protonation site and of a FADH(*) C=N stretch marker mode, with tyrosine as the likely proton donor. FADH(*) is reoxidized in 67 ps (180 ps in D(2)O) to result in a long-lived hydrogen-bond switched network around FAD. This hydrogen-bond switch shows infrared signatures from the C-OH stretch of tyrosine and the FAD C4=O and C=N stretches, which indicate increased hydrogen-bond strength at all these sites. The results support a previously hypothesized rotation of glutamine by approximately 180 degrees through a light-driven radical-pair mechanism as the determinant of the hydrogen-bond switch.
Assuntos
Aminoácidos/química , Flavinas/química , Transdução de Sinal Luminoso , Modelos Químicos , Fotorreceptores Microbianos/química , Aminoácidos/efeitos da radiação , Sítios de Ligação , Simulação por Computador , Flavinas/efeitos da radiação , Ligação de Hidrogênio/efeitos da radiação , Luz , Fotorreceptores Microbianos/efeitos da radiação , Ligação Proteica , Estrutura Terciária de Proteína/efeitos da radiação , Espectrofotometria Infravermelho/métodosRESUMO
OBJECTIVES: The aim of this study was to compare bright blood high spatial resolution (HR) coronary magnetic resonance angiography (MRA) with low spatial resolution (LR) bright blood coronary MRA at 7 T. MATERIALS AND METHODS: Twenty-four healthy volunteers underwent navigator-gated 3-dimensional imaging of the right coronary artery at 7 T using 2 sequences: HR bright blood and LR bright blood. Image postprocessing involved newly developed multiplanar reformatting to straighten the right coronary artery. Image quality was determined by vessel edge sharpness, signal-to-noise ratio, contrast-to-noise ratio, visible vessel length, and vessel diameter. RESULTS: Vessel edge sharpness was statistically significantly higher in HR as compared with LR (0.57 ± 0.1 vs 0.46 ± 0.06; P < 0.001), at the cost of lower signal-to-noise ratio (HR, 32.9 ± 11.0 vs LR, 112.5 ± 48.9; P < 0.001) and contrast-to-noise ratio (HR, 17.9 ± 7.4 vs LR, 50.5 ± 26.1; P < 0.001). Visible vessel length and vessel diameter were similar for both sequences (P > 0.05). CONCLUSIONS: High spatial resolution bright blood coronary MRA at 7 T is feasible and improves vessel edge sharpness as compared with LR bright blood imaging.
Assuntos
Angiografia Coronária/métodos , Vasos Coronários/anatomia & histologia , Processamento de Imagem Assistida por Computador/métodos , Angiografia por Ressonância Magnética/métodos , Adulto , Feminino , Humanos , Imageamento Tridimensional/métodos , Masculino , Valores de Referência , Razão Sinal-RuídoRESUMO
BACKGROUND: Diabetic patients have increased interstitial myocardial fibrosis on histological examination. Magnetic resonance imaging (MRI) T(1) mapping is a previously validated imaging technique that can quantify the burden of global and regional interstitial fibrosis. However, the association between MRI T(1) mapping and subtle left ventricular (LV) dysfunction in diabetic patients is unknown. METHODS AND RESULTS: Fifty diabetic patients with normal LV ejection fraction (EF) and no underlying coronary artery disease or regional macroscopic scar on MRI delayed enhancement were prospectively recruited. Diabetic patients were compared with 19 healthy controls who were frequency matched in age, sex and body mass index. There were no significant differences in mean LV end-diastolic volume index, end-systolic volume index and LVEF between diabetic patients and healthy controls. Diabetic patients had significantly shorter global contrast-enhanced myocardial T(1) time (425±72 ms vs. 504±34 ms, P<0.001). There was no correlation between global contrast-enhanced myocardial T(1) time and LVEF (r=0.14, P=0.32) in the diabetic patients. However, there was good correlation between global contrast-enhanced myocardial T(1) time and global longitudinal strain (r=-0.73, P<0.001). Global contrast-enhanced myocardial T(1) time was the strongest independent determinant of global longitudinal strain on multivariate analysis (standardized ß=-0.626, P<0.001). Similarly, there was good correlation between global contrast-enhanced myocardial T(1) time and septal E' (r=0.54, P<0.001). Global contrast-enhanced myocardial T(1) time was also the strongest independent determinant of septal E' (standardized ß=0.432, P<0.001). CONCLUSIONS: A shorter global contrast-enhanced myocardial T(1) time was associated with more impaired longitudinal myocardial systolic and diastolic function in diabetic patients.
Assuntos
Cardiomiopatias/patologia , Meios de Contraste , Diabetes Mellitus , Aumento da Imagem/métodos , Imageamento por Ressonância Magnética/métodos , Disfunção Ventricular Esquerda/patologia , Cardiomiopatias/complicações , Cardiomiopatias/diagnóstico por imagem , Ecocardiografia , Feminino , Fibrose , Gadolínio DTPA , Humanos , Masculino , Pessoa de Meia-Idade , Variações Dependentes do Observador , Projetos Piloto , Estudos Prospectivos , Disfunção Ventricular Esquerda/complicações , Disfunção Ventricular Esquerda/diagnóstico por imagemRESUMO
The BlrB protein from Rhodobacter sphaeroides is a small 136 amino acid photoreceptor belonging to the BLUF family of blue light receptors. It contains merely the conserved BLUF fold responsible for binding the flavin pigment and a short C-terminal extension of unknown function. We investigated the primary photoreactions of BlrB by picosecond fluorescence and transient absorption spectroscopy. After excitation of the flavin the fluorescence decays in an H/D isotope independent manner with time constants of 21 and 390 ps, indicating a BLUF characteristic heterogeneous excited state quenched by electron transfer. By transient absorption spectroscopy, we observed a rapid relaxation of a vibrationally hot excited state within 6 ps upon excitation at 400 nm. The relaxed excited state evolves biexponentially with 18 ps (27%) and 216 ps (73%) into the signaling state spectrum indicated by a growing absorptive feature at 492 nm. Additionally, a broad triplet feature is observed as residual absorbance at a delay of 5 ns, which we attribute to derive from a significant fraction of free flavin in the sample. The photochemistry of BlrB is similar to other small BLUF proteins in respect to the fast formation of the photoproduct but does not resolve any further intermediates. We compare the photoreaction with other BLUF proteins on the basis of available spectroscopic data and crystal structures. An arginine close to the C2âO carbonyl of the flavin is likely to be a key determinant for the fast electron transfer in BlrB. Additionally, the orientation of the electron-donating tyrosine in respect to the flavin might play a role in the so far unique kinetic separation of the semiquinonic intermediates in Slr1694.