Pseudomonas aeruginosa alkaline protease exhibits a high renaturation capability.

Thermally induced unfolding and renaturation capability of alkaline proteases (AprA) of three Pseudomonas aeruginosa strains, i.e. ATCC 27853 and two clinical isolates, was examined. Sequence analyses demonstrated a high level of aprA genes identity (99.24-99.8%) in these bacterial strains. The proteases retained 45-60% and 15% of their activity after pre-treatment at 60oC and 80oC, respectively, whereas pre-incubation at 90-95oC resulted in a higher level of activity than at 80oC. Zymography analyses and immunoblotting with AprA antiserum suggested a high thermostability and renaturation capability of the studied enzymes in comparison to another P. aeruginosa protease, elastase B. An intrinsic capability of renaturation of P. aeruginosa AprA was confirmed by fluorescence spectra of the native, thermally denatured, and renatured enzyme. The value of the fluorescence intensity of the denatured and subsequently cooled enzyme recovered to about 80% of the value of the native protein fluorescence intensity. Moreover, pre-incubation of the enzyme at 60oC and 90oC exerted only a slight effect on the intensity of absorbance and the shape of the amide I band, as demonstrated by Fourier transform infrared (FTIR) spectroscopy performed after subsequent cooling of the pre-treated enzyme. The results indicated a high renaturation capability of the P. aeruginosa AprA proteins.

Light-induced formation of dimeric LHCII.

It emerges from numerous experiments that LHCII, the major photosynthetic antenna complex of plants, can appear not only in the trimeric or monomeric states but also as a dimer. We address the problem whether the dimeric form of the complex is just a simple intermediate element of the trimer-monomer transformation or if it can also be a physiologically relevant molecular organization form? Dimers of LHCII were analyzed with application of native electrophoresis, time-resolved fluorescence spectroscopy, and fluorescence correlation spectroscopy. The results reveal the appearance of two types of LHCII dimers: one formed by the dissociation of one monomer from the trimeric structure and the other formed by association of monomers into a distinctively different molecular organizational form, characterized by a high rate of chlorophyll excitation quenching. The hypothetical structure of such an energy quencher is proposed. The high light-induced LHCII dimerization is discussed as a potential element of the photoprotective response in plants.

A chloroplast "wake up" mechanism: Illumination with weak light activates the photosynthetic antenna function in dark-adapted plants.

The efficient and fluent operation of photosynthesis in plants relies on activity of pigment-protein complexes called antenna, absorbing light and transferring excitations toward the reaction centers. Here we show, based on the results of the fluorescence lifetime imaging analyses of single chloroplasts, that pigment-protein complexes, in dark-adapted plants, are not able to act effectively as photosynthetic antennas, due to pronounced, adverse excitation quenching. It appeared that the antenna function could be activated by a short (on a minute timescale) illumination with light of relatively low intensity, substantially below the photosynthesis saturation threshold. The low-light-induced activation of the antenna function was attributed to phosphorylation of the major accessory light-harvesting complex LHCII, based on the fact that such a mechanism was not observed in the stn7 Arabidopsis thaliana mutant, with impaired LHCII phosphorylation. It is proposed that the protein phosphorylation-controlled change in the LHCII clustering ability provides mechanistic background for this regulatory process.

Light-Driven Reconfiguration of a Xanthophyll Violaxanthin in the Photosynthetic Pigment-Protein Complex LHCII: A Resonance Raman Study.

Resonance Raman analysis of the photosynthetic complex LHCII, immobilized in a polyacrylamide gel, reveals that one of the protein-bound xanthophylls, assigned as violaxanthin, undergoes light-induced molecular reconfiguration. The phototransformation is selectively observed in a trimeric structure of the complex and is associated with a pronounced twisting and a trans-cis molecular configuration change of the polyene chain of the carotenoid. Among several spectral effects accompanying the reconfiguration there are ones indicating a carotenoid triplet state. Possible physiological importance of the light-induced violaxanthin reconfiguration as a mechanism associated with making the pigment available for enzymatic deepoxidation in the xanthophyll cycle is discussed.

The xanthophyll cycle pigments, violaxanthin and zeaxanthin, modulate molecular organization of the photosynthetic antenna complex LHCII.

The effect of violaxanthin and zeaxanthin, two main carotenoids of the xanthophyll cycle, on molecular organization of LHCII, the principal photosynthetic antenna complex of plants, was studied in a model system based on lipid-protein membranes, by means of analysis of 77 K chlorophyll a fluorescence and "native" electrophoresis. Violaxanthin was found to promote trimeric organization of LHCII, contrary to zeaxanthin which was found to destabilize trimeric structures. Moreover, violaxanthin was found to induce decomposition of oligomeric LHCII structures formed in the lipid phase and characterized by the fluorescence emission band at 715 nm. Both pigments promoted formation of two-component supramolecular structures of LHCII and xanthophylls. The violaxanthin-stabilized structures were composed mostly of LHCII trimers while, the zeaxanthin-stabilized supramolecular structures of LHCII showed more complex organization which depended periodically on the xanthophyll content. The effect of the xanthophyll cycle pigments on molecular organization of LHCII was analyzed based on the results of molecular modeling and discussed in terms of a physiological meaning of this mechanism. Supramolecular structures of LHCII stabilized by violaxanthin, prevent uncontrolled oligomerization of LHCII, potentially leading to excitation quenching, therefore can be considered as structures protecting the photosynthetic apparatus against energy loses at low light intensities.

Is It Beneficial for the Major Photosynthetic Antenna Complex of Plants To Form Trimers?

The process of primary electric charge separation in photosynthesis takes place in the reaction centers, but photosynthesis can operate efficiently and fluently due to the activity of several pigment-protein complexes called antenna, which absorb light quanta and transfer electronic excitations toward the reaction centers. LHCII is the major photosynthetic pigment-protein antenna complex of plants and appears in the trimeric form. Several recent reports point to trimeric organization of LHCII as a key factor responsible for the chloroplast architecture via stabilization of granal organization of the thylakoid membranes. In the present work, we address the question of whether such an organization could also directly influence the antenna properties of this pigment-protein complex. Chlorophyll fluorescence analysis reveals that excitation energy transfer in LHCII is substantially more efficient in trimers and dissipative energy losses are higher in monomers. It could be concluded that trimers are exceptionally well suited to perform the antenna function. Possibility of fine regulation of the photosynthetic antenna function via the LHCII trimer-monomer transition is also discussed, based on the fluorescence lifetime analysis in a single chloroplast.

Molecular architecture of plant thylakoids under physiological and light stress conditions: a study of lipid-light-harvesting complex II model membranes.

In this study, we analyzed multibilayer lipid-protein membranes composed of the photosynthetic light-harvesting complex II (LHCII; isolated from spinach [Spinacia oleracea]) and the plant lipids monogalcatosyldiacylglycerol and digalactosyldiacylglycerol. Two types of pigment-protein complexes were analyzed: those isolated from dark-adapted leaves (LHCII) and those from leaves preilluminated with high-intensity light (LHCII-HL). The LHCII-HL complexes were found to be partially phosphorylated and contained zeaxanthin. The results of the x-ray diffraction, infrared imaging microscopy, confocal laser scanning microscopy, and transmission electron microscopy revealed that lipid-LHCII membranes assemble into planar multibilayers, in contrast with the lipid-LHCII-HL membranes, which form less ordered structures. In both systems, the protein formed supramolecular structures. In the case of LHCII-HL, these structures spanned the multibilayer membranes and were perpendicular to the membrane plane, whereas in LHCII, the structures were lamellar and within the plane of the membranes. Lamellar aggregates of LHCII-HL have been shown, by fluorescence lifetime imaging microscopy, to be particularly active in excitation energy quenching. Both types of structures were stabilized by intermolecular hydrogen bonds. We conclude that the formation of trans-layer, rivet-like structures of LHCII is an important determinant underlying the spontaneous formation and stabilization of the thylakoid grana structures, since the lamellar aggregates are well suited to dissipate excess energy upon overexcitation.

Organization and functionality of chlorophyll-protein complexes in thylakoid membranes isolated from Pb-treated Secale cereale.

In this study, Secale cereale seedlings cultivated under 0 (control), 2 or 5mM Pb(NO3)2 concentrations were used to examine alterations in the organization and functionality of chlorophyll-protein complexes in thylakoid membranes under Pb ion stress. The studies were conducted on whole leaves of rye seedlings or thylakoid membranes isolated from Pb-treated and control plants. Using non-denaturing electrophoresis, it was assessed that increasing Pb concentrations resulted in an increase in the value of the ratio of the content of LHCII oligomers (mainly trimers) to the content of LHCII monomers. The parameters of chlorophyll fluorescence induction (q(p) and q(n)) indicated that the change in the LHCII supramolecular organization in the presence of Pb ions was connected with an increase in non-photochemical fluorescence quenching. Quantification of photosynthetic pigments showed that both Pb concentrations decreased the content of chlorophyll a, chlorophyll b, and carotenoids. The changes in the pigment content led to a significant reduction in light absorption by antenna complexes. However, the absorption spectra showed that red light was preferentially absorbed by antenna complexes in thylakoid membranes isolated from the Pb-treated plants. Examination of fluorescence emission spectra revealed that Pb ions decreased the fluorescence quantum yield of PSII. The emission spectra of thylakoids indicated a relative increase in the intensity of fluorescence emission from the trimeric and aggregated forms of the LHCII complexes in comparison to the intensity of fluorescence emission from PSI antenna complexes under excitation at 440 nm. Simultaneously, under excitation at 470 nm, we observed a rise in fluorescence intensity from the LHCII trimer after addition of 5mM Pb as well as a decrease in fluorescence intensity from the LHCII aggregates and PSI core and LHCI antenna complexes under both Pb concentrations. Pb treatments also reduced excitation energy absorbed by chlorophyll b and carotenoids within antenna complexes and transferred to chlorophyll a species emitting at 680 nm.

The negative feedback molecular mechanism which regulates excitation level in the plant photosynthetic complex LHCII: towards identification of the energy dissipative state.

Overexcitation of the photosynthetic apparatus is potentially dangerous because it can cause oxidative damage. Photoprotection realized via the feedback de-excitation in the pigment-protein light-harvesting complex LHCII, embedded in the chloroplast lipid environment, was studied with use of the steady-state and time-resolved fluorescence spectroscopy techniques. Illumination of LHCII results in the pronounced singlet excitation quenching, demonstrated by decreased quantum yield of the chlorophyll a fluorescence and shortening of the fluorescence lifetimes. Analysis of the 77K chlorophyll a fluorescence emission spectra reveals that the light-driven excitation quenching in LHCII is associated with the intensity increase of the spectral band in the region of 700nm, relative to the principal band at 680nm. The average chlorophyll a fluorescence lifetime at 700nm changes drastically upon temperature decrease: from 1.04ns at 300K to 3.63ns at 77K. The results of the experiments lead us to conclude that: (i) the 700nm band is associated with the inter-trimer interactions which result in the formation of the chlorophyll low-energy states acting as energy traps and non-radiative dissipation centers; (ii) the Arrhenius analysis, supported by the results of the FTIR measurements, suggests that the photo-reaction can be associated with breaking of hydrogen bonds. Possible involvement of photo-isomerization of neoxanthin, reported previously (Biochim. Biophys. Acta 1807 (2011) 1237-1243) in generation of the low-energy traps in LHCII is discussed.

Strong light-induced reorganization of pigment-protein complexes of thylakoid membranes in rye (spectroscopic study).

The supramolecular reorganization of LHCII complexes within the thylakoid membrane in Secale cereale leaves under low and high light condition was examined. Rye seedlings were germinated hydroponically in a climate chamber with a 16 h daylight photoperiod, photosynthetic photon flux density (PPFD) of 150 µmo lm(-2)s(-1) and 24/16°C day/night temperature. The influence of pre-illumination of the plants with high light intensity on the PSII antenna complexes was studied by comparison of the structure and function of the LHCII complexes and organization of thylakoid membranes isolated from 10-day-old plants illuminated with low (150 µmo lm(-2)s(-1)) or high (1200 µmo lm(-2)s(-1)) light intensity. Aggregated and trimeric with monomeric forms of LHCII complexes were separated from the whole thylakoid membranes using non-denaturing electrophoresis. Analyses of fluorescence emission spectra of these different LHCII forms showed that the monomer was the most effective aggregating antenna form. Moreover, photoprotection connected with LHCII aggregation was more effective upon LHCII monomers in comparison to trimer aggregation. Light stress induced specific organization of neighboring LHCII complexes, causing an increase in fluorescence yield of the long-wavelength bands (centered at 701 and 734 nm). The changes in the organization of the thylakoid membrane under light stress, observed by analysis of absorbance spectra obtained by Fourier transform infrared spectroscopy, also indicated light-induced LHCII aggregation.

Structural and functional modifications of the major light-harvesting complex II in cadmium- or copper-treated Secale cereale.

The effects of 50 microM cadmium (Cd) or copper (Cu) ions on the supramolecular conformation of the light-harvesting pigment-protein complex of PSII (LHCII) isolated from rye seedlings were studied. It was found that the action of these two metal ions on the LHCII structure and organization is dissimilar. The Fourier transform infrared (FTIR) measurements indicated inhibition or stimulation of formation of parallel beta-structures and aggregates in the presence of Cd or Cu ions, respectively. The Chl a fluorescence excitation spectra of LHCII extracted from Cd-treated plants showed that the decreased aggregation of complexes was correlated with a decline in efficiency of quenching of excitation energy. From the results of mass spectrometry, changes in LHCII aggregation in the presence of Cd ions might be based on decreases in the molecular mass of Lhcb1 and Lhcb2 proteins. An increase in the content of LHCII aggregates under Cu ion excess was associated with changes in the LHCII xanthophyll pigment pool. In the complexes isolated from Cu-treated plants, all-trans violaxanthin and 9'-cis neoxanthin content declined and the simultaneous appearance of the fraction of 9-cis violaxanthin was observed. 9-cis violaxanthin formation under Cu ion excess might facilitate LHCII inter-trimer interaction and, therefore, aggregation of complexes. RLS (resonance light scattering) spectra indicated that the excitonic interaction between Chl molecules and between Chls and xanthophylls was responsible for the effective dissipation of excitation energy in LHCII isolated from Cu-treated plants. Also, changes in singlet excitation energy transfer between carotenoids and Chls under the action of heavy metals were observed.

The photoprotective mechanisms in Secale cereale leaves under Cu and high light stress condition.

The influence of excess Cu ions and high light treatment on the function of photosystem II was investigated in order to examine how this heavy metal modifies the photoprotective mechanisms operating at the molecular level in Secale cereale plants. Thus, non-treated plants and those treated with 5 or 50 microM Cu, simultaneously illuminated with 150 micromol m(-2) s(-1) or 1200 micromol m(-2) s(-1) light intensity, were studied. To analyze the PSII reaction to the stress conditions, Chl a fluorescence induction was applied. An increase in the value of Phi(PSII) and R(fd) parameters indicated that the photosynthetic apparatus adapted to the high light condition by effective utilization of excitation energy in the light and dark phases of photosynthesis. This phenomenon was accompanied by dissipation of excitation energy within the antenna complexes. The xanthophyll cycle pigments in Secale cereale leaves were separated and quantified by the HPLC technique. The results showed that, under high light irradiance, both 5 and 50 microM Cu induced the process of violaxanthin de-epoxidation and zeaxanthin accumulation. The significant zeaxanthin accumulation was found to be involved in photoprotective energy dissipation as heat, which was supported by correlation between the rate of violaxanthin de-epoxidation and the value of SV parameters. Interestingly, Cu treatment caused violaxanthin isomerization from its trans to 15-, 13- and 9-cis forms in proportional correlation to the metal concentration. This phenomenon was confirmed by a study of Cu-induced violaxanthin isomerization in vitro, which suggests a direct metal-pigment molecule interaction. We also observed that the violaxanthin trans-cis isomerization increased simultaneously with anteraxanthin content. On the basis of these findings, it can be speculated that violaxanthin isomerization is the basic process responsible for the xanthophyll cycle operation.

Blue-light-controlled photoprotection in plants at the level of the photosynthetic antenna complex LHCII.

Plants have developed several adaptive regulatory mechanisms, operating at all the organization levels, to optimize utilization of light energy and to protect themselves against over-excitation-related damage. We report activity of a previously unknown possible regulatory mechanism that operates at the molecular level of the major photosynthetic pigment-protein complexes of plants, LHCII. This mechanism is driven exclusively by blue light, operates in the trimeric but not in the monomeric complex, and results in singlet excitation quenching leading to thermal energy dissipation. The conclusions are based on single molecule fluorescence lifetime analysis, direct measurements of thermal energy dissipation by photo-thermal spectroscopy, and on fluorescence spectroscopy. Possible molecular mechanisms involved in the blue-light-induced photoprotective effect are discussed, including xanthophyll photo-isomerization and the thermo-optic effect.

Supramolecular organization of the main photosynthetic antenna complex LHCII: a monomolecular layer study.

The light-harvesting pigment-protein complex LHCII is a main antenna complex of the photosynthetic apparatus of plants, responsible for collecting light energy and also for photoprotection against overexcitation-induced damage. Realization of both functions depends on molecular organization of the complex. Monolayer technique has been applied to address the problem of supramolecular organization of LHCII. Analysis of the isotherms of compression of monomolecular films formed at the argon-water interface shows that LHCII appears in two phases: one characterized by the specific molecular area characteristic of trimeric and one of monomeric organization of LHCII. Monolayers of LHCII were deposited by means of the Langmuir-Blodgett technique to solid supports and examined by means of AFM, FTIR, fluorescence spectroscopy, and fluorescence lifetime imaging microscopy (FLIM). FTIR analysis shows that organization of the trimers of LHCII within a monolayer is associated with formation of intermolecular hydrogen bonds between neighboring polypeptides. The linear-dichroism FTIR analysis reveals that polypeptide fragments involved in intermolecular interactions are oriented at an angle of 67 degrees with respect to the normal axis to the plane of the layer. Fluorescence and fluorescence lifetime analysis reveal that the organization of LHCII within monolayers is associated with formation of the low-lying excitonic energy levels that can be potentially responsible for excess excitation quenching. FLIM and AFM reveal heterogeneous organization of LHCII monolayers, in particular, formation of ring-like structures. The potential of LHCII to form molecular structures characterized by pigment excitonic interactions is discussed in terms of regulation of the photosynthetic accessory function and photoprotection against overexcitation-induced damage.

The xanthophyll cycle pigments in Secale cereale leaves under combined Cd and high light stress conditions.

Leaves of Secale cereale seedlings were exposed to high light illumination (1200micromolm(-2)s(-1)) and Cd ions at 5 or 50microM concentrations. Influence of these stress factors on violaxanthin cycle pigments content was analysed chromatographically. Chlorophyll a fluorescence induction was used to analyse response of PSII to stress conditions and contribution of light-harvesting complex (LHCII) in non-photochemical quenching of excitation energy. The Cd-induced all-trans violaxanthin isomerization was analysed by HPLC technique in acetonitrile:methanol:water (72:8:3, v/v) solvent mixture. Interestingly, in the control and Cd-treated leaves subjected to high light, photochemical utilization of absorbed energy increased. This indicates plant adaptation to high light stress. In control plants high light caused zeaxanthin formation, however, the presence of Cd in the nutrient solution resulted in reduction of the second step of violaxanthin de-epoxidation process and anteraxanthin accumulation. In this study we have also shown, that non-photochemical quenching can be independent of anteraxanthin and zeaxanthin content. The particular increase in the cis isomers fraction in Cd-treated leaves has been explained in terms of a direct metal-pigment interaction as confirmed by Cd-induced all-trans violaxanthin isomerization in organic solvent, leading to formation of 13-cis, 9-cis and 15-cis isomers.