RESUMEN
The hydrogen-oxidizing "Knallgas" bacterium Ralstonia eutropha can thrive in aerobic and anaerobic environments and readily switches between heterotrophic and autotrophic metabolism, making it an attractive host for biotechnological applications including the sustainable H2-driven production of hydrocarbons. The soluble hydrogenase (SH), one out of four different [NiFe]-hydrogenases in R. eutropha, mediates H2 oxidation even in the presence of O2, thus providing an ideal model system for biological hydrogen production and utilization. The SH reversibly couples H2 oxidation with the reduction of NAD+ to NADH, thereby enabling the sustainable regeneration of this biotechnologically important nicotinamide cofactor. Thus, understanding the interaction of the SH with the cellular NADH/NAD+ pool is of high interest. Here, we applied the fluorescent biosensor Frex to measure changes in cytoplasmic [NADH] in R. eutropha cells under different gas supply conditions. The results show that Frex is well-suited to distinguish SH-mediated changes in the cytoplasmic redox status from effects of general anaerobiosis of the respiratory chain. Upon H2 supply, the Frex reporter reveals a robust fluorescence response and allows for monitoring rapid changes in cellular [NADH]. Compared to the Peredox fluorescence reporter, Frex displays a diminished NADH affinity, which prevents the saturation of the sensor under typical bacterial [NADH] levels. Thus, Frex is a valuable reporter for on-line monitoring of the [NADH]/[NAD+] redox state in living cells of R. eutropha and other proteobacteria. Based on these results, strategies for a rational optimization of fluorescent NADH sensors are discussed.
Asunto(s)
Técnicas Biosensibles/métodos , Cupriavidus necator/metabolismo , Hidrógeno/metabolismo , NAD/análisis , Anaerobiosis , Técnicas Biosensibles/normas , Cupriavidus necator/citología , Hidrogenasas , NAD/metabolismo , Oxidación-ReducciónRESUMEN
The structure of phycobiliproteins of the cyanobacterium Acaryochloris marina was investigated in buffer solution at physiological temperatures, i.e. under the same conditions applied in spectroscopic experiments, using small angle neutron scattering. The scattering data of intact phycobiliproteins in buffer solution containing phosphate can be well described using a cylindrical shape with a length of about 225Å and a diameter of approximately 100Å. This finding is qualitatively consistent with earlier electron microscopy studies reporting a rod-like shape of the phycobiliproteins with a length of about 250 (M. Chen et al., FEBS Letters 583, 2009, 2535) or 300Å (J. Marquart et al., FEBS Letters 410, 1997, 428). In contrast, phycobiliproteins dissolved in buffer lacking phosphate revealed a splitting of the rods into cylindrical subunits with a height of 28Å only, but also a pronounced sample aggregation. Complementary small angle neutron and X-ray scattering experiments on phycocyanin suggest that the cylindrical subunits may represent either trimeric phycocyanin or trimeric allophycocyanin. Our findings are in agreement with the assumption that a phycobiliprotein rod with a total height of about 225Å can accommodate seven trimeric phycocyanin subunits and one trimeric allophycocyanin subunit, each of which having a height of about 28Å. The structural information obtained by small angle neutron and X-ray scattering can be used to interpret variations in the low-energy region of the 4.5K absorption spectra of phycobiliproteins dissolved in buffer solutions containing and lacking phosphate, respectively.
Asunto(s)
Cianobacterias/química , Transferencia de Energía , Dispersión del Ángulo Pequeño , Difracción de Neutrones , Ficobiliproteínas/química , Difracción de Rayos XRESUMEN
Orange carotenoid protein (OCP) is a water-soluble photoactive protein responsible for a photoprotective mechanism of nonphotochemical quenching in cyanobacteria. Under blue-green illumination, OCP converts from the stable orange into the signaling red quenching form; however, the latter form could also be obtained by chemical activation with high concentrations of sodium thiocyanate (NaSCN) or point mutations. In this work, we show that a single replacement of tryptophan-288, normally involved in protein-chromophore interactions, by alanine, results in formation of a new protein form, hereinafter referred to as purple carotenoid protein (PCP). Comparison of resonance Raman spectra of the native photoactivated red form, chemically activated OCP, and PCP reveals that carotenoid conformation is sensitive to the structure of the C-domain, implicating that the chromophore retains some interactions with this part of the protein in the active red form. Combination of differential scanning fluorimetry and picosecond time-resolved fluorescence anisotropy measurements allowed us to compare the stability of different OCP forms and to estimate relative differences in protein rotation rates. These results were corroborated by hydrodynamic analysis of proteins by dynamic light scattering and analytical size-exclusion chromatography, indicating that the light-induced conversion of the protein is accompanied by a significant increase in its size. On the whole, our data support the idea that the red form of OCP is a molten globule-like protein in which, however, interactions between the carotenoid and the C-terminal domain are preserved.
Asunto(s)
Proteínas Bacterianas/fisiología , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Cromatografía en Gel , Clonación Molecular , Cianobacterias/fisiología , Fluorescencia , Polarización de Fluorescencia , Fluorometría , Espectrometría Raman , Synechocystis/fisiologíaRESUMEN
The time courses of the photosystem II (PSII) redox states were analyzed with a model scheme supposing a fraction of 11-25 % semiquinone (with reduced [Formula: see text]) RCs in the dark. Patterns of single flash-induced transient fluorescence yield (SFITFY) measured for leaves (spinach and Arabidopsis (A.) thaliana) and the thermophilic alga Chlorella (C.) pyrenoidosa Chick (Steffen et al. Biochemistry 44:3123-3132, 2005; Belyaeva et al. Photosynth Res 98:105-119, 2008, Plant Physiol Biochem 77:49-59, 2014) were fitted with the PSII model. The simulations show that at high-light conditions the flash generated triplet carotenoid (3)Car(t) population is the main NPQ regulator decaying in the time interval of 6-8 µs. So the SFITFY increase up to the maximum level [Formula: see text]/F 0 (at ~50 µs) depends mainly on the flash energy. Transient electron redistributions on the RC redox cofactors were displayed to explain the SFITFY measured by weak light pulses during the PSII relaxation by electron transfer (ET) steps and coupled proton transfer on both the donor and the acceptor side of the PSII. The contribution of non-radiative charge recombination was taken into account. Analytical expressions for the laser flash, the (3)Car(t) decay and the work of the water-oxidizing complex (WOC) were used to improve the modeled P680(+) reduction by YZ in the state S 1 of the WOC. All parameter values were compared between spinach, A. thaliana leaves and C. pyrenoidosa alga cells and at different laser flash energies. ET from [Formula: see text] slower in alga as compared to leaf samples was elucidated by the dynamics of [Formula: see text] fractions to fit SFITFY data. Low membrane energization after the 10 ns single turnover flash was modeled: the ∆Ψ(t) amplitude (20 mV) is found to be about 5-fold smaller than under the continuous light induction; the time-independent lumen pHL, stroma pHS are fitted close to dark estimates. Depending on the flash energy used at 1.4, 4, 100 % the pHS in stroma is fitted to 7.3, 7.4, and 7.7, respectively. The biggest ∆pH difference between stroma and lumen was found to be 1.2, thus pH- dependent NPQ was not considered.
Asunto(s)
Arabidopsis/metabolismo , Chlorella/metabolismo , Transporte de Electrón/efectos de la radiación , Complejo de Proteína del Fotosistema II/metabolismo , Spinacia oleracea/metabolismo , Animales , Arabidopsis/efectos de la radiación , Chlorella/efectos de la radiación , Electrones , Fluorescencia , Rayos Láser , Luz , Simulación de Dinámica Molecular , Oxidación-Reducción , Complejo de Proteína del Fotosistema II/efectos de la radiación , Hojas de la Planta/metabolismo , Hojas de la Planta/efectos de la radiación , Protones , Espectrometría de Fluorescencia , Spinacia oleracea/efectos de la radiaciónRESUMEN
Measurements of Single Flash Induced Transient Fluorescence Yield (SFITFY) on spinach leaves and whole cells of green thermophilic alga Chlorella pyrenoidosa Chick were analyzed for electron transfer (ET) steps and coupled proton transfer (PT) on both the donor and the acceptor side of the reaction center (RC) of photosystem II (PS II). A specially developed PS II model (Belyaeva et al., 2008, 2011a) allowed the determination of ET steps that occur in a hierarchically ordered time scale from nanoseconds to several seconds. Our study demonstrates that our SFITFY data is consistent with the concept of the reduction of P680(+) by YZ in both leaves and algae (studied on spinach leaves and cells of Chlorella pyrenoidosa Chick). The multiphasic P680(+) reduction kinetics by YZ in PS II core complexes with high oxygen evolution capacity was seen in both algae and leaves. Model simulation to fit SFITFY curves for dark adapted species used here gives the rate constants to verify nanosecond kinetic stages of P680(+) reduction by YZ in the redox state S1 of the water oxidizing complex (WOC) shown in Kühn et al. (2004). Then a sequence of relaxation steps in the redox state S1, outlined by Renger (2012), occurs in both algae and leaves as a similar non-adiabatic ET reactions. Coupled PT is discussed briefly to understand a rearrangement of hydrogen bond protons in the protein matrix of the WOC (Umena et al., 2011). On the other hand, present studies showed a slower reoxidation of reduced QA by QB in algal cells as compared with that in a leaf that might be regarded as a consequence of differences of spatial domains at the QB-site in leaves compared to algae. Our comparative study helped to correlate theory with experimental data for molecular photosynthetic mechanisms in thylakoid membranes.
Asunto(s)
Chlorella/metabolismo , Transporte de Electrón , Fluorescencia , Luz , Fotosíntesis , Complejo de Proteína del Fotosistema II/metabolismo , Spinacia oleracea/metabolismo , Chlorella/química , Clorofila/metabolismo , Rayos Láser , Modelos Biológicos , Oxidación-Reducción , Hojas de la Planta/química , Hojas de la Planta/metabolismo , Protones , Espectrometría de Fluorescencia , Spinacia oleracea/química , Agua/químicaRESUMEN
Photoprotective mechanisms were studied on the tripartite lichen Peltigera aphthosa that exhibits external cephalodia. Using the methods of steady-state and time-resolved fluorescence microscopy, we studied the dynamics of the rehydration process in different parts of the lichen thalli. It was found that apical, medial and basal parts of the thallus are not only morphologically different, but also show completely different chlorophyll induction curves and other spectral characteristics. In dry state, significant contribution to the fluorescence spectrum of lichen gives a green fluorescence of hyphae forming the upper crust, which is rapidly and almost completely quenched during the rehydration process. Probably this is one of the protective mechanisms that reduce the amount of light reaching the PS II reaction centers in the dry state. In the process of rehydration, we observed an increase in the intensity of the chlorophyll fluorescence of the photobiont at 680 nm, with significant changes of the fluorescence lifetimes and the amplitude ratios of fast and slow components of fluorescence decay kinetics. While in dry state, chlorophyll fluorescence is strongly quenched (opposite to the fluorescence of the hyphae), and the fluorescence time constants recover to the typical decay times of active photosynthetic organisms during rehydration. The quantitative behavior of these changes differs largely between the apical, medial and basal parts of the thallus, probably due to the complex interactions of the fungus, algae and cyanobacteria.
Asunto(s)
Líquenes/citología , Microscopía Fluorescente/métodos , Clorofila/metabolismo , Desecación , Fluorescencia , Cinética , Líquenes/fisiología , Complejo de Proteína del Fotosistema II/fisiologíaRESUMEN
As high-intensity solar radiation can lead to extensive damage of the photosynthetic apparatus, cyanobacteria have developed various protection mechanisms to reduce the effective excitation energy transfer (EET) from the antenna complexes to the reaction center. One of them is non-photochemical quenching (NPQ) of the phycobilisome (PB) fluorescence. In Synechocystis sp. PCC6803 this role is carried by the orange carotenoid protein (OCP), which reacts to high-intensity light by a series of conformational changes, enabling the binding of OCP to the PBs reducing the flow of energy into the photosystems. In this paper the mechanisms of energy migration in two mutant PB complexes of Synechocystis sp. were investigated and compared. The mutant CK is lacking phycocyanin in the PBs while the mutant ΔPSI/PSII does not contain both photosystems. Fluorescence decay spectra with picosecond time resolution were registered using a single photon counting technique. The studies were performed in a wide range of temperatures - from 4 to 300 K. The time course of NPQ and fluorescence recovery in darkness was studied at room temperature using both steady-state and time-resolved fluorescence measurements. The OCP induced NPQ has been shown to be due to EET from PB cores to the red form of OCP under photon flux densities up to 1000 µmolphotonsm⻲s⻹. The gradual changes of the energy transfer rate from allophycocyanin to OCP were observed during the irradiation of the sample with blue light and consequent adaptation to darkness. This fact was interpreted as the revelation of intermolecular interaction between OCP and PB binding site. At low temperatures a significantly enhanced EET from allophycocyanin to terminal emitters has been shown, due to the decreased back transfer from terminal emitter to APC. The activation of OCP not only leads to fluorescence quenching, but also affects the rate constants of energy transfer as shown by model based analysis of the decay associated spectra. The results indicate that the ability of OCP to quench the fluorescence is strongly temperature dependent. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy.
Asunto(s)
Fluorometría/métodos , Ficobilisomas/química , Synechocystis/metabolismo , Transferencia de Energía , Fluorescencia , Conformación ProteicaRESUMEN
The present work describes results obtained on hybrid systems formed in aqueous buffer solution by self-assembly of different CdSe quantum dots (QDs) surrounded by a ZnS shell and functionalized by covering the surface with anionic and cationic groups and various isolated pigment-protein complexes from the light-harvesting antennae of photosynthetic organisms (light-harvesting complexes 1 and 2 (LH1 and LH2, respectively) from purple bacteria, phycobiliproteins (PBPs) from cyanobacteria and the rod-shaped PBP from the cyanobacterium Acaryochloris marina). Excitation energy transfer (EET) from QDs to PBP rods was found to take place with varying and highly temperature-dependent efficiencies of up to 90%. Experiments performed at room temperature on hybrid systems with different QDs show that no straightforward correlation exists between the efficiency of EET and the parameter J/(R(12)(6)) given by the theory of Förster resonance energy transfer (FRET), where J is the overlap integral of the normalized QD emission and PBP absorption and R(12) the distance between the transition dipole moments of donor and acceptor. The results show that the hybrid systems cannot be described as randomly orientated aggregates consisting of QDs and photosynthetic pigment-protein complexes. Specific structural parameters are inferred to play an essential role. The mode of binding and coupling seems to change with the size of QDs and with temperature. Efficient EET and fluorescence enhancement of the acceptor was observed at particular stoichiometric ratios between QDs and trimeric phycoerythrin (PE). At higher concentrations of PE, a quenching of its fluorescence is observed in the presence of QDs. This effect is explained by the existence of additional quenching channels in aggregates formed within hybrid systems. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Complejos de Proteína Captadores de Luz/química , Nanopartículas/química , Puntos Cuánticos , Transferencia de EnergíaRESUMEN
Persistent spectral hole burning at 4.5 K has been used to investigate the excitonic energy level structure and the excited state dynamics of the recombinant class-IIa water-soluble chlorophyll-binding protein (WSCP) from cauliflower. The hole-burned spectra are composed of four main features: (i) a narrow zero-phonon hole (ZPH) at the burn wavelength, (ii) a number of vibrational ZPHs, (iii) a broad low-energy hole at ~665 and ~683 nm for chlorophyll b- and chlorophyll a-WSCP, respectively, and (iv) a second satellite hole at ~658 and ~673 nm for chlorophyll b- and chlorophyll a-WSCP, respectively. The doublet of broad satellite holes is assigned to an excitonically coupled chlorophyll dimer. The lower-energy holes at ~665 and ~683 nm for chlorophyll b- and chlorophyll a-WSCP, respectively, represent the lower exciton states. Taking into account the parameters of electron-phonon coupling, the lower exciton state can be assigned as the fluorescence origin. The lower exciton state is populated by two processes: (i) exciton relaxation from the higher exciton state and (ii) vibrational relaxation within the lower exciton state. Assuming identical site energies for the two excitonically coupled chlorophyll molecules, the dipole-dipole interaction energy J is directly determined to be 85 and 100 cm(-1) for chlorophyll b- and chlorophyll a-WSCP, respectively, based on the positions of the satellite holes. The Gaussian low-energy absorption band identified by constant fluence hole burning at 4.5 K has a width of ~150 cm(-1) and peaks at 664.9 and 682.7 nm for chlorophyll b- and chlorophyll a-WSCP, respectively. The action spectrum is broader and blue-shifted compared to the fluorescent lower exciton state. This finding can be explained by a slow protein relaxation between energetically inequivalent conformational substates within the lowest exciton state in agreement with the results of Schmitt et al. (J. Phys. Chem. B2008, 112, 13951).
Asunto(s)
Clorofila/química , Complejos de Proteína Captadores de Luz/química , Brassica/metabolismo , Clorofila A , Electrones , Complejos de Proteína Captadores de Luz/genética , Complejos de Proteína Captadores de Luz/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Termodinámica , Vibración , Agua/químicaRESUMEN
This short review paper describes spectroscopic studies on pigment-pigment and pigment-protein interactions of chlorophyll (Chl) a and b bound to the recombinant protein of class IIa water soluble chlorophyll protein (WSCP) from cauliflower. Two Chls form a strongly excitonically coupled open sandwich dimer within the tetrameric protein matrix. In marked contrast to the mode of excitonic coupling of Chl and bacterio-Chl molecules in light harvesting complexes and reaction centers of all photosynthetic organisms, the unique structural pigment array in the Chl dimer of WSCP gives rise to an upper excitonic state with a large oscillator strength. This property opens the way for thorough investigations on exciton relaxation processes in Chl-protein complexes. Lifetime measurements of excited singlet states show that the unusual stability towards photodamage of Chls bound to WSCP, which lack any protective carotenoid molecule, originates from a high diffusion barrier to interaction of molecular dioxygen with Chl triplets. Site selective spectroscopic methods provide a wealth of information on the interactions of the Chls with the protein matrix and on the vibronic structure of the pigments. The presented data and discussions illustrate the great potential of WSCP as a model system for systematic experimental and theoretical studies on the functionalizing of Chls by the protein matrix. It opens the way for further detailed analyses and a deeper understanding of the properties of pigment protein complexes.
Asunto(s)
Clorofila/metabolismo , Complejos de Proteína Captadores de Luz/metabolismo , Modelos Biológicos , Plantas/metabolismo , Agua/metabolismo , TemperaturaRESUMEN
Our recently presented PS II model (Belyaeva et al., 2008) was improved in order to permit a consistent simulation of Single Flash Induced Transient Fluorescence Yield (SFITFY) traces that were earlier measured by Steffen et al. (2005) on whole leaves of Arabidopsis (A.) thaliana at four different energies of the actinic flash. As the essential modification, the shape of the actinic flash was explicitly taken into account assuming that an exponentially decaying rate simulates the time dependent excitation of PS II by the 10 ns actinic flash. The maximum amplitude of this excitation exceeds that of the measuring light by 9 orders of magnitude. A very good fit of the SFITFY data was achieved in the time domain from 100 ns to 10s for all actinic flash energies (the maximum energy of 7.5 × 10¹6 photons/(cm²flash) is set to 100%, the relative energies of weaker actinic flashes were of â¼8%, 4%, â¼1%). Our model allows the calculation and visualization of the transient PS II redox state populations ranging from the dark adapted state, via excitation energy and electron transfer steps induced by pulse excitation, followed by final relaxation into the stationary state eventually attained under the measuring light. It turned out that the rate constants of electron transfer steps are invariant to intensity of the actinic laser flash. In marked contrast, an increase of the actinic flash energy by more than two orders of magnitude from 5.4×10¹4 photons/(cm²flash) to 7.5×10¹6 photons/(cm²flash), leads to an increase of the extent of fluorescence quenching due to carotenoid triplet (³Car) formation by a factor of 14 and of the recombination reaction between reduced primary pheophytin (Phe(-)) and P680(+) by a factor of 3 while the heat dissipation in the antenna complex remains virtually constant. The modified PS II model offers new opportunities to compare electron transfer and dissipative parameters for different species (e.g. for the green algae and the higher plant) under varying illumination conditions.
Asunto(s)
Arabidopsis/metabolismo , Transporte de Electrón/fisiología , Fluorescencia , Modelos Biológicos , Complejo de Proteína del Fotosistema II/metabolismo , Hojas de la Planta/metabolismo , Simulación por Computador , CinéticaRESUMEN
The crystal structure of the class IIb water-soluble chlorophyll binding protein (WSCP) from Lepidium virginicum is used to model linear absorption and circular dichroism spectra as well as excited state decay times of class IIa WSCP from cauliflower reconstituted with chlorophyll (Chl) a and Chl b. The close agreement between theory and experiment suggests that both types of WSCP share a common Chl binding motif, where the opening angle between pigment planes in class IIa WSCP should not differ by more than 10 degrees from that in class IIb. The experimentally observed (Schmitt et al. J. Phys. Chem. B 2008, 112, 13951) decrease in excited state lifetime of Chl a homodimers with increasing temperature is fully explained by thermally activated superradiance via the upper exciton state of the dimer. Whereas a temperature-independent intersystem crossing (ISC) rate is inferred for WSCP containing Chl a homodimers, that of WSCP with Chl b homodimers is found to increase above 100 K. Our quantum chemical/electrostatic calculations suggest that a thermally activated ISC via an excited triplet state T4 is responsible for the latter temperature dependence.
Asunto(s)
Complejos de Proteína Captadores de Luz/química , Temperatura , Agua/química , Dicroismo Circular , Cristalografía por Rayos X , Lepidium/química , Modelos Moleculares , Teoría Cuántica , Solubilidad , Electricidad EstáticaRESUMEN
The present study describes the fluorescence emission properties of recombinant water-soluble chlorophyll (Chl) protein (WSCP) complexes reconstituted with either Chl a or Chl b alone (Chl a only or Chl b only WSCP, respectively) or mixtures of both pigments at different stoichiometrical ratios. Detailed investigations were performed with time and space correlated ps fluorescence spectroscopy within the temperature range from 10 to 295 K. The following points were found: (a) The emission spectra at room temperature (295 K) are well characterized by bands with a dominating Lorentzian profile broadened due to phonon scattering and peak positions located at 677, 684 and 693 nm in the case of Chl a only WSCP and at 665, 675 and 689 nm for Chl b only WSCP. In addition, all spectra contain minor bands in the longer wavelength region. (b) The emission spectra at 10 K of samples suspended in buffer containing 50% glycerol are dominated by bands peaking at 668 nm for Chl b only WSCP and at 685 nm for Chl a only WSCP and samples reconstituted with mixtures of Chl a and Chl b. (c) At 10 K and in buffer with 50% glycerol the decay kinetics of WSCP samples with Chl a only are dominated by a component with a time constant of 6.2 (+/-0.2) ns at 685 nm while those of WSCP containing mixtures of Chl a and Chl b are characterized by a slightly shorter value of 6.0 (+/-0.2) ns. WSCP containing Chl b only exhibits a distinctly longer value of 7.0 (+/-0.3) ns at an emission wavelength of 668 nm. (d) The decay associated emission spectra at 10 K of all samples exhibit at least 3 decay components with time constants of 80-120 ps, 2-4 ns and 6-7 ns in 50% glycerol. These results are consistently described within the framework of our previously presented model (J. Phys. Chem. B 2007, 111, No. 46, 13325; J. Phys. Chem. B 2007, 111, No. 35, 10487) , for the structural motifs of chlorophyll binding to the tetrameric protein matrix of WSCP. It is shown that formation of strongly coupled open sandwich dimers does not lead to quenching of 1Chl a* or 1Chl b*.
Asunto(s)
Brassica/enzimología , Complejos de Proteína Captadores de Luz/química , Agua/química , Complejos de Proteína Captadores de Luz/metabolismo , Proteínas de Plantas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidad , Espectrometría de Fluorescencia , Temperatura , Factores de TiempoRESUMEN
The set up described in Steffen et al. (Biochemistry 40:173-180, 2001) was used to monitor in the time domain from 100 ns to 10 s single turnover flash-induced transients of the normalized fluorescence yield (SFITFY) on dark-adapted cells of the thermophilic algae Chlorella pyrenoidosa Chick. Perfect data fit was achieved within the framework of a previously proposed model for the PS II reaction pattern (Lebedeva et al., Biophysics 47:968-980, 2002; Belyaeva et al., Biophysics 51:860-872, 2006) after its modification by taking into account nonradiative decay processes including nonphotochemical quenching due to time-dependent populations of P680(+*) and (3)Car. On the basis of data reported in the literature, a consistent set of rate constants was obtained for electron transfer at the donor and acceptor sides of PS II, pH in lumen and stroma, the initial redox state of plastoquinone pool and the rate of plastoquinone oxidation. The evaluation of the rate constant values of dissipative processes due to quenching by carotenoid triplets in antennae and P680(+*)Q(A)(-*) recombination as well as the initial state populations after excitation with a single laser flash are close to that outlined in (Steffen et al., Biochemistry 44:3123-3133, 2005a). The simulations based on the model of the PS II reaction pattern provide information on the time courses of population probabilities of different PS II states. We analyzed the maximum (F(m)(STF)) and minimum (F(0)) of the normalized FL yield dependence on the rate of the recombination processes (radiative and dissipative nonradiative) and of P680(+*) reduction. The developed PS II model provides a basis for theoretical comparative analyses of time-dependent fluorescence signals, observed at different photosynthetic samples under various conditions (e.g. presence of herbicides, other stress conditions, excitation with actinic pulses of different intensity, and duration).
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Chlorella/fisiología , Fluorescencia , Modelos Biológicos , Complejo de Proteína del Fotosistema II/metabolismo , Adaptación Fisiológica , Simulación por Computador , Oscuridad , Nanotecnología , Fotosíntesis , Factores de TiempoRESUMEN
Plants contain water-soluble chlorophyll-binding proteins (WSCPs) that function neither as antennas nor as components of light-induced electron transfer of photosynthesis but are likely constituents of regulatory protective pathways in particular under stress conditions. This study presents results on the spectroscopic properties of recombinant WSCP from cauliflower reconstituted with chlorophyll b (Chl b) alone or with mixtures of Chl a and Chl b. Two types of experiments were performed: (a) measurements of stationary absorption spectra at 77 and 298 K and CD spectra at 298 K and (b) monitoring of laser flash-induced transient absorption changes with a resolution of 200 fs in the time domain of up to 100 ps. On the basis of a theoretical analysis outlined by Renger et al. (J. Phys. Chem. B 2007, 111, 10487) the data obtained in part (a) are interpreted within a model where tetrameric WSCP binds predominantly two Chl molecules in the form of an excitonically coupled "open sandwich" dimer with a tilt angle of about 30 degrees between the chlorin planes. The time-resolved measurements on Chl a/Chl b heterodimers are described by two exponential kinetics with time constants of 400 fs and 7 ps. These kinetics are assumed to reflect a heterogeneous population of WSCPs with Chl dimers either in excitonic coupled "open sandwich" or weakly coupled geometric arrays. The 400 fs component is assigned to excited-state relaxations from the upper to the lower excitonic level of the strongly coupled "open sandwich" dimer, while the 7-8 ps component probably indicates excitation energy transfer from 1Chl b* to Chl a in a dimer array with weak coupling due to significantly longer mutual distances between the chlorin rings.
Asunto(s)
Brassica/química , Complejos de Proteína Captadores de Luz/química , Pigmentos Biológicos/química , Dicroismo Circular , Cinética , Rayos Láser , Transferencia Lineal de Energía , Proteínas Recombinantes/química , Espectrofotometría UltravioletaRESUMEN
Appropriate values for the orientation of the amide transition dipoles are essential to the growing use of isotopically edited vibrational spectroscopy generally in structural biology and to infrared dichroism measurements on membrane-associated alpha-helices, in particular. The orientations of the transition moments for the amide vibrations of an alpha-helix have been determined from the ratio of intensities of the A- and E(1)-symmetry modes in the infrared spectra of poly(gamma-methyl-L-glutamate)(x)-co-(gamma-n-octadecyl-L-glutamate)( y) oriented on silicon substrates. Samples possessing a high degree of alignment were used to facilitate band fitting. Consistent results were obtained from both attenuated total reflection and transmission experiments with polarized radiation, yielding values of Theta(I) = 38 degrees, Theta(II) = 73 degrees, and Theta(A) = 29 degrees, relative to the helix axis, for the amide I, amide II, and amide A bands, respectively. The measurements are discussed both in the context of the somewhat divergent older determinations, and in relation to the helix geometry and results on model amide compounds, to resolve current uncertainties in the literature.
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Péptidos/química , Fenómenos Biofísicos , Biofisica , Técnicas In Vitro , Modelos Químicos , Ácido Poliglutámico/análogos & derivados , Ácido Poliglutámico/química , Estructura Secundaria de Proteína , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
The strength of receptor-mediated cell adhesion is directly controlled by the mechanism of cohesive failure between the cell surface and underlying substrate. Unbinding can occur either at the locus of the specific bond or within the bilayer, which results in tearing the hydrophobic anchors from the membrane interior. In this work, the surface force apparatus has been used to investigate the relationship between the receptor-ligand bond affinities and the dominant mechanism of receptor-coupled membrane detachment. The receptors and ligands used in this study were membrane-bound streptavidin and biotin analogs, respectively, with solution affinities ranging over 10 orders of magnitude. With the optical technique of the surface force apparatus, the occurrence of membrane rupture was directly visualized in situ. The latter observations together with measurements of the corresponding intermembrane adhesive strengths were used to identify the dominant failure pathway for each streptavidin-analog pair. Even in cases where the membrane pull-out energy exceeded the equilibrium bond energy, cohesive failure occurred within the membrane interior at nearly all bond affinities considered. These results are consistent with previous findings and provide direct support for the commonly held view that, under nonequilibrium conditions of applied external stress, the gradient of the bond energy, not the equilibrium bond energy alone, determines the adhesive strength. Furthermore, our findings directly demonstrate that, in the presence of competing failure mechanisms, the preferred detachment mechanism- hence, the adhesive strength-will be determined by the bond that exhibits the weakest tensile strength. Because the tensile strength is determined by the gradient of the unbinding energy, the critical detachment force will be determined by both the bond energy and the effective bond length.
Asunto(s)
Adhesión Celular , Membrana Celular/fisiología , Membrana Dobles de Lípidos , Modelos Biológicos , Receptores de Superficie Celular/fisiología , Animales , Compuestos Azo , Proteínas Bacterianas , Colorantes Fluorescentes , Cinética , Modelos Estructurales , Conformación Molecular , Fosfatidiletanolaminas/química , Estreptavidina , Ácido TiócticoRESUMEN
The association of lipid molecules into spherical vesicles in solution as a result of non-specific intermolecular forces constitutes a primary self-assembly process. Such vesicles can undergo a secondary self-assembly into higher order structures in a controlled and reversible manner by means of site-specific ligand-receptor (biotin-streptavidin) coupling. Cryoelectron microscopy shows these structures to be composed of tethered, rather than adhering, vesicles in their original, unstressed state. In contrast, vesicles aggregated by nonspecific, such as van der Waals, forces are deformed and stressed, producing unstable structures. Vesicle association by site-specific binding provides a practical mechanism for the production of stable, yet controllable, microstructured biomaterials.
Asunto(s)
Liposomas/química , Adhesividad , Proteínas Bacterianas/química , Biotina/análogos & derivados , Biotina/química , Coloides , Técnica de Fractura por Congelación , Membrana Dobles de Lípidos , Matemática , Microscopía Electrónica , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Estreptavidina , Propiedades de SuperficieRESUMEN
Streptavidin-biotin (receptor-ligand) interaction forces were measured directly as a function of their intermolecular separation in various salt solutions and at various temperatures with a surface forces apparatus. Electrostatic and van der Waals forces were found to dominate the long-range streptavidin-biotin interaction at > 20 A. At intermediate separations, down to approximately 10 A, the interaction is governed by repulsive steric and attractive van der Waals and hydrophobic forces. A much stronger short-range attraction giving rise to the strong, specific adhesive binding was measured at molecular separations of less than 5 A. A decrease in the pH from 7.2 to 6.0 resulted in complete charge reversal on the binding surface of streptavidin (pK approximately 6) from net negative to net positive, while leaving the negatively charged biotin surface (pK approximately 3.0) unchanged, and the long-range interaction switched from repulsive to attractive. This observed behavior can be attributed to the titration of two histidines on the biotin binding surface of streptavidin. These results reveal a strong sensitivity of the long-range interaction forces to the detailed amino acid composition of the biotin binding surface. They also demonstrate the powerful regulatory potential conferred by small changes in local surface ionic conditions on protein interaction forces over different distance regimes. The effects of temperature on receptor-ligand dynamics and on the strength of intermembrane adhesion forces were studied by measuring the long-range force profiles and short-range adhesion forces above and below the chain melting temperature (Tc approximately 30 degrees C) of the lipids in the supporting bilayers. Increased bilayer fluidity due to a temperature increase to 33 degrees C (T > Tc) increased short-range adhesion by 7-fold relative to bilayers in the gel state at 25 degrees C (T < Tc). This effect was attributed to the enhanced rates of lateral diffusion and molecular rearrangements on the more fluid bilayer surfaces, which resulted in greater and more rapid intermembrane bond formation. A change in the rates of molecular rearrangements was also found to affect the repulsive part of the interaction potential at intermediate separations (10-20 A) via modulation of the steric repulsion between streptavidin and the highly flexible, polymer-like biotin molecules. This is expected to have a large effect on the association rates of receptor-ligand binding, even if it does not change the equilibrium binding energy.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Proteínas Bacterianas/química , Biotina/química , Proteínas de la Membrana/química , Adsorción , Fenómenos Químicos , Química Física , Electroquímica , Concentración de Iones de Hidrógeno , Punto Isoeléctrico , Membrana Dobles de Lípidos/química , Fluidez de la Membrana , Estreptavidina , Temperatura , TermodinámicaRESUMEN
An atomic force microscope (AFM) was used to investigate a specific recognition reaction: the binding of streptavidin to a biotinylated lipid bilayer. Prior to the recognition reaction, the phase coexistence of the lipid bilayer was clearly observed: fluid domains were lower than the crystalline domains. After introducing to the bilayer a very dilute solution of streptavidin to give a final concentration of approximately 0.5 microM, the recognition reaction was imaged in real time. Several hours later, we observed a contrast reversal, i.e., the previously lower fluid domains grew so much in height that they became higher than the crystalline domains. We found that the streptavidin molecules bound almost exclusively to the biotin in the fluid domain (less than 0.25% coverage of the crystalline domains). The apparent structure of the few streptavidin molecules bound to the crystalline domain of the bilayer is shown to depend on the applied force. Finally, in a 2-dimensional quasi-crystal in which the streptavidin molecules were compressed at the air-water interface molecular resolution was achieved.