Effect of glycerol and PVA on the conformation of photosystem I

Single-molecule spectroscopy at cryogenic temperatures was used to examine the impact of buffer solution, glycerol/buffer mixtures (25% and 66%), and poly(vinyl alcohol) (PVA) films on the conformation of photosystem I (PSI) from Thermosynechoccocus elongatus. PSI holds a number of chromophores embedded at different places within the protein complex that show distinguishable fluorescence at low temperatures. The fluorescence emission from individual complexes shows inter- and intracomplex heterogeneity depending on the solution wherein PSI was dissolved. Statistical evaluation of spectra of a large number of complexes shows that the fluorescence emission of some of these chromophores can be used as sensors for their local nanoenvironment and some as probe for the conformation of the whole protein complex. Preparation in glycerol/buffer mixtures yields a high homogeneity for all chromophores, indicating a more compact protein conformation with less structural variability. In buffer solution a distinct heterogeneity of the chromophores is observed. PSI complexes in PVA show highly heterogeneous spectra as well as a remarkable blue shift of the fluorescence emission, indicating a destabilization of the protein complex. Photosystem I prepared in PVA cannot be considered fully functional, and conclusions drawn from experiments with PSI in PVA films are of questionable value.     

Purification and characterization of photosystem I complex from Synechocystis sp. PCC 6803 by expressing histidine-tagged subunits

We generated Synechocystis sp. PCC 6803 strains, designated F-His and J-His, which express histidine-tagged PsaF and PsaJ subunits, respectively, for simple purification of the photosystem I (PSI) complex. Six histidine residues were genetically added to the C-terminus of the PsaF subunit in F-His cells and the N-terminus of the PsaJ subunit in J-His cells. The histidine residues introduced had no apparent effect on photoautotrophic growth of the cells or the activity of PSI and PSII in thylakoid membranes. PSI complexes could be simply purified from the F-His and J-His cells by Ni²⁺-affinity column chromatography. When thylakoid membranes corresponding to 20 mg chlorophyll were used, PSI complexes corresponding to about 7 mg chlorophyll could be purified in both strains. The purified PSI complexes could be separated into monomers and trimers by ultracentrifugation in glycerol density gradient and high activity was recorded for trimers isolated from the F-His and J-His strains. Blue-Native PAGE and SDS-PAGE analysis of monomers and trimers indicated the existence of two distinct monomers with different subunit compositions and no contamination of PSI with other complexes, such as PSII and Cyt b₆f. Further analysis of proteins and lipids in the purified PSI indicated the presence of novel proteins in the monomers and about six lipid molecules per monomer unit in the trimers. These results demonstrate that active PSI complexes can be simply purified from the constructed strains and the strains are very useful tools for analysis of PSI.     

Solvent-induced changes in photochemical activity and conformation of photosystem I particles by glycerol

It has been shown that a large number of water molecules coordinate with the pigments and subunits of photosystem I (PSI); however, the function of these water molecules remains to be clarified. In this study, the photosynthetic properties of PSI from spinach were investigated using different spectroscopic and activity measurements under conditions of decreasing water content caused by increasing concentrations of glycerol. The results show that glycerol addition caused pronounced changes in the photochemical activity of PSI particles. At low concentrations (<60%, v/v), glycerol stimulated the rate of oxygen uptake in PSI particles, while higher concentrations of glycerol cause inhibition of PSI activity. The capacity of P700 photooxidation also increased with glycerol concentrations lower than 60%. In contrast, this capacity decreased at higher glycerol concentrations. On the other hand, glycerol addition considerably affected the distribution of the bulk and red antenna chlorophyll (Chl) forms or states, with the population of red-shifted Chl forms augmented with increasing glycerol. In addition, glycerol-treated PSI particles showed a blue shift of the tryptophan fluorescence emission maximum and an increase in their capacity to bind the hydrophobic probe 1-anilino-8-naphthalene sulfonate, indicating a more non-polar environment for tryptophan residues and increased exposure of hydrophobic surfaces.     

Electron transfer between galactose oxidase and an electrode via a redox polymer network

Galactose oxidase from Dactyllium dendroides was purified and immobilised on a carbon electrode in a redox polymer network of a polyvinylpyridine, partially N-complexed with osmiumbis(bipyridine)chloride (POsEA). The current density of the electrodes depended on the concentration of phosphate elution buffer. By additional crosslinking with a 1% glutaraldehyde solution in 50 mM phosphate buffer, pH 7.0, an electrode with an initial current density of 0.8 mA/cm² was obtained. Operational half life times were in the order of 1.2 h. The affinity of the immobilized enzyme for galactose,lactose, raffinose, glycerol and dihydroxyaceton was higher than described in literature for the enzyme in solution. Optimal temperature for the enzyme electrode was 30°C. The pH optimum for the immobilized enzyme was higher than for the enzyme in solution.     

A Highly Active Oxygen-Evolving Photosystem II Preparation from the Cyanobacterium Anacystis nidulans

A highly active O₂-evolving Photosystem (PS)-II fraction has been isolated from the cyanobacterium, Anacystis nidulans R2, using an isolation buffer containing high concentrations of sucrose and salts and subsequent solubilization of the thylakoid membranes with the detergent Triton X-100. The isolated fraction had very high PSII activity (2500 micromoles O₂ per milligram chlorophyll per hour) and was largely depleted of PSI activity. Fluorescence emission spectra (77 K) and polypeptide analysis indicated that this preparation is highly enriched in PSII, but almost completely devoid of Cyt b₆-f and PSI complexes.     

Glycerol‐induced folding of unstructured disulfide‐deficient lysozyme into a native‐like conformation

2SS[6‐127,64‐80] variant of lysozyme which has two disulfide bridges, Cys6‐Cys127 and Cys64‐Cys80, and lacks the other two disulfide bridges, Cys30‐Cys115 and Cys76‐Cys94, was quite unstructured in water, but a part of the polypeptide chain was gradually frozen into a native‐like conformation with increasing glycerol concentration. It was monitored from the protection factors of amide hydrogens against H/D exchange. In solution containing various concentrations of glycerol, H/D exchange reactions were carried out at pH* 3.0 and 4°C. Then, ¹H‐¹⁵N‐HSQC spectra of partially deuterated protein were measured in a quenching buffer for H/D exchange (95% DMSO/5% D₂O mixture at pH* 5.5 adjusted with dichloroacetate). In a solution of 10% glycerol, the protection factors were nearly equal to 10 at most of residues. With increasing glycerol concentration, some selected regions were further protected, and their protection factors reached about a 1000 in 30% glycerol solution. The highly protected residues were included in A‐, B‐, and C‐helices and β3‐strand, and especially centered on Ile 55 and Leu 56. In 2SS[6‐127,64‐80], long‐range interactions were recovered due to the preferential hydration by glycerol in the hydrophobic box of the α‐domain. Glycerol‐induced recovering of the native‐like structure is discussed from the viewpoint of molten globules growing with the protein folding. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 665–675, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Differential detergent-solubilization of integral thylakoid membrane complexes in spinach chloroplasts. Localization of photosystem II, cytochrome b6-f complex and photosystem I

Progressive solubilization of spinach chloroplast thylakoids by Triton X-100 was employed to investigate the domain organization of the electron transport complexes in the thylakoid membrane. Triton/chlorophyll ratios of 1:1 were sufficient to disrupt fully the continuity of the thylakoid membrane network, but not sufficient to solubilize either photosystem I (PSI), photosystem II (PSII) or the cytochrome b6-f(Cyt b6-f) complex. Progressive with the Triton concentration increase (Triton/Chl greater than 1:1), a differential solubilization of the three electron transport complexes was observed. Solubilization of the Cyt b6-f complex from the thylakoid membrane preceded that of PSI and apparently occurred early in the solubilization of stroma-exposed segments of the chloroplast lamellae. The initial removal of chlorophyll (up to 40% of the total) occurred upon solubilization of PSI from the stroma-exposed lamella regions in which PSI is localized. The tightly appressed membrane of the grana partition regions was markedly resistant to solubilization by Triton X-100. Thus, solubilization of PSII from this membrane region was initiated only after all Cyt b6-f and PSI complexes were removed from the chloroplast lamellae. The results support the notion of extreme lateral heterogeneity in the organization of the electron transport complexes in higher plant chloroplasts and suggest a Cyt b6-f localization in the membrane of the narrow fret regions which serve as a continuum between the grana and stroma lamellae.     

Cyanobacterial Acclimation to Photosystem I or Photosystem II Light

The organization and function of the photochemical apparatus of Synechococcus 6301 was investigated in cells grown under yellow and red light regimes. Broadband yellow illumination is absorbed preferentially by the phycobilisome (PBS) whereas red light is absorbed primarily by the chlorophyll (Chl) pigment beds. Since PBSs are associated exclusively with photosystem II (PSII) and most of the Chl with photosystem I (PSI), it follows that yellow and red light regimes will create an imbalance of light absorption by the two photosystems. The cause and effect relationship between light quality and photosystem stoichiometry in Synechococcus was investigated. Cells grown under red light compensated for the excitation imbalance by synthesis/assembly of more PBS-PSII complexes resulting in high PSII/PSI = 0.71 and high bilin/Chl = 1.30. The adjustment of the photosystem stoichiometry in red light-grown cells was necessary and sufficient to establish an overall balanced absorption of red light by PSII and PSI. Cells grown under yellow light compensated for this excitation imbalance by assembly of more PSI complexes, resulting in low PSII/PSI = 0.27 and low bilin/Chl = 0.42. This adjustment of the photosystem stoichiometry in yellow light-grown cells was necessary but not quite sufficient to balance the absorption of yellow light by the PBS and the Chl pigment beds. A novel excitation quenching process was identified in yellow light-grown cells which dissipated approximately 40% of the PBS excitation, thus preventing over-excitation of PSII under yellow light conditions. It is hypothesized that State transitions in O₂ evolving photosynthetic organisms may serve as the signal for change in the stoichiometry of photochemical complexes in response to light quality conditions.     

Synthesis and Assembly of the Polypeptides of Photosystem I and II in Isolated Etiochloroplasts of Wheat

Synthesis and assembly of photosystems (PS) I and II polypeptides in etiochloroplasts isolated from greening wheat (Triticum aestivum L. cv Norin 61) seedlings were studied. The isolated etiochloroplasts synthesized PSI polypeptides of 66 and 15 kilodaltons, PSII polypeptides of 46 and 42 kilodaltons, and atrazine-binding 34 to 32 kilodalton polypeptide. Their assembly processes in the thylakoid membrane were studied by pulse-chase labeling with [³⁵S]methionine, mild solubilization of the thylakoid membrane with Triton X-100, sucrose density gradient centrifugation, and polyacrylamide gel electrophoresis. The newly synthesized polypeptides of 66, 46, 42, 34, and 32 kilodaltons were first integrated into the complexes of 7.5, 5.9, 7.5, 6.3, and 7.5 Svedberg units, respectively, in 20 minutes. After the chase with excess amount of methionine for 100 min, they were found in complexes of 9.5, 9.1, 9.1, 9.1, and 9.1 Svedberg units, respectively. In this condition, stained polypeptides of PSI and PSII were found in the complexes of 11.1 and 10.3 Svedberg units, respectively. These results indicated that newly synthesized PSI or PSII polypeptides are integrated into intermediate complexes, but not complete complexes in the isolated etiochloroplasts. The relationship between the processing of the atrazine-binding 32 kilodalton polypeptide and its assembly into the PSII complex is also discussed.     

Purification of estradiol-17 beta dehydrogenase from human placenta by affinity chromatography

Estriol 16-hemisuccinate has been synthesized and covalently attached to Sepharose through 1,5-diaminopentane. A crude preparation of estradiol-17β dehydrogenase from human placenta was adsorbed on the gel. After extensive washing, the enzyme was eluted by $\text{M}$ hydroxylamine in 0.1 $\text{M}$ potassium phosphate buffer (20–50% glycerol), pH 7, at room temperature. An apparently homogeneous enzyme with a specific activity of 7.2 U/mg (82% recovery) was obtained. It is stable for weeks in the eluting buffer. The hydroxylamine can be removed by passing the enzyme solution over a Sephadex G-100 column or by dialyzing it against 0.1 $\text{M}$ potassium phosphate buffer containing 20% glycerol. This one-step process makes purification of the enzyme simple and easy.     

Purification and efficient refolding process for recombinant tissue-type plasminogen activator derivative (reteplase) using glycerol and Tranexamic acid

In this study, a novel and economic method for refolding and purifying recombinant tissue plasminogen activator derivative (r-PA; reteplase) was developed. Reteplase with nine disulfide bonds in its complex structure is expressed in the form of inclusion bodies in Escherichia coli and requires tedious dissolving and refolding processes to achieve its biological activity. Among the different refolding additives that were evaluated, glycerol and tranexamic acid (Txa) were found to be more effective in increasing the refolding yield of reteplase. Using response surface methodology, a solution containing 3.5 M urea, 33% (v/v) glycerol, and 400 mM Txa was found to give the highest refolding yield. The synergic effect of urea, glycerol, and Txa under optimum conditions for a reteplase concentration of 25 μg ml⁻¹ resulted in a high refolding yield of 76.41%. Increased reteplase concentration in the refolding buffer was achieved using the pulse-fed method. In the pulse-fed method, a refolding yield of 49.53% was achieved for a final reteplase concentration of 300 μg ml⁻¹. Using Txa as a novel refolding aid for reteplase instead of ionic amino acids like l-Arginine allowed to purify the refolded reteplase directly by cation-exchange chromatography with high purity.     

Glycerol inhibition of purified and chromatin-associated mouse liver hepatoma RNA polymerase II activity

The stability of mouse liver hepatoma RNA polymerase II is dependent on the type of buffer, pH and, most importantly, the glycerol concentration of the incubation or storage buffer. Glycerol above 2% or 15% shows a linearly increasing inhibition of enzyme activity with increasing glycerol concentration for purified RNA polymerase II and chromatin-associated RNA polymerase II, respectively. At 25% glycerol the activity of purified enzyme on DNA template was inhibited approximately 50% whereas the chromatin-associated activity was inhibited only approximately 30%. RNA polymerase I activity was not inhibited by glycerol at the concentrations examined.     

Characterization of a redox active cross-linked complex between cyanobacterial photosystem I and soluble ferredoxin.

A covalent stoichiometric complex between photosystem I (PSI) and ferredoxin from the cyanobacterium Synechocystis sp. PCC 6803 was generated by chemical cross-linking. The photoreduction of ferredoxin, studied by laser flash absorption spectroscopy between 460 and 600 nm, is a fast process in 60% of the covalent complexes, which exhibit spectral and kinetic properties very similar to those observed with the free partners. Two major phases with t(1/2) <1 micros and approximately 10-14 micros are observed at two different pH values (5.8 and 8.0). The remaining complexes do not undergo fast ferredoxin reduction and 20-25% of the complexes are still able to reduce free ferredoxin or flavodoxin efficiently, thus indicating that ferredoxin is not bound properly in this proportion of covalent complexes. The docking site of ferredoxin on PSI was determined by electron microscopy in combination with image analysis. Ferredoxin binds to the cytoplasmic side of PSI, with its mass center 77 angstroms distant from the center of the trimer and in close contact with a ridge formed by the subunits PsaC, PsaD and PsaE. This docking site corresponds to a close proximity between the [2Fe- 2S] center of ferredoxin and the terminal [4Fe-4S] acceptor FII of PSI and is very similar in position to the docking site of flavodoxin, an alternative electron acceptor of PSI.     

Stability of native and cross-linked crystalline glucose isomerase.

Stabilities of native and cross-linked crystalline forms of Streptomyces rubiginosus glucose isomerase were compared in buffer and in 45% glucose/fructose solutions. The cross-linked crystalline form of the enzyme was more stable in the presence of substrate while in a buffer solution the native enzyme was more stable. Inactivation of native enzyme in buffer did not obey first-order kinetics but proceeded with a rapid first phase followed by a stable phase. This stabilization is interpreted to be a result of a conformational change in the protein structure. Inactivation of the native enzyme in buffer was directly related to protein precipitation. In the presence of high substrate concentration, the inactivation was related to browning reactions between the enzyme and the reactive sugar, resulting in soluble sugar-protein complexes.     

Isoelectric focusing of proteins using a pH gradient created by a concentration gradient of nonelectrolytes in solution.

A new method of producing a stable pH gradient in buffer solutions is suggested, obtained by the concentration gradient of a nonelectrolyte in buffer solutions as a result of the gradual change in the dielectric properties of the solution. The maximal concentrations of nonelectrolyte which do not influence the protein configuration allow a pH gradient with a range of two pH units to be produced. It is suggested that the properties of some polyols (i.e. glycerol or mannitol) be used to change the pH of the borate buffer for the production of greater pH gradient with a range of up to 4-5 pH units. Creating the gradient of concentration of polyols, one can obtain a pH gradient in borate buffer solutions. Though the polylydroxyl compound-borate complexes posses mobility in an electric field, a stable pH gradient can be achieved during 12 days of electrophoresis. The isoelectric focusing of haemoglobin, human serum albumin and immunoglobulins was carried out in both systems suggested. These findings were compared with isoelectric focusing in Ampholines. There was a good agreement between the methods compared. The possible differences are discussed.     

Properties of photosystem I antenna protein complexes of the diatom Cyclotella meneghiniana

Analysis of photosystem I (PSI) complexes from Cyclotella meneghiniana cultured under different growth conditions led to the identification of three groups of antenna proteins, having molecular weights of around 19, 18, and 17 kDa. The 19-kDa proteins have earlier been demonstrated to be more peripherally bound to PSI, and their amount in the PSI complexes was significantly reduced when the iron supply in the growth medium was lowered. This polypeptide was almost missing, and thus the total amount of fucoxanthin-chlorophyll proteins (Fcps) bound to PSI was reduced as well. When treating cells with high light in addition, no further changes in antenna polypeptide composition were detected. Xanthophyll cycle pigments were found to be bound to all Fcps of PSI. However, PSI of high light cultures had a significantly higher diatoxanthin to diadinoxanthin ratio, which is assumed to protect against a surplus of excitation energy. PSI complexes from the double-stressed cultures (high light plus reduced iron supply) were slightly more sensitive against destruction by the detergent treatment. This could be seen as a higher 674-nm emission at 77 K in comparison to the PSI complexes isolated from other growth conditions. Two major emission bands of the Fcps bound to PSI at 77 K could be identified, whereby chlorophyll a fluorescing at 697 nm was more strongly coupled to the PSI core than those fluorescing at 685 nm. Thus, the build up of the PSI antenna of several Fcp components enables variable reactions to several stress factors commonly experienced by the diatoms in vivo, in particular diatoxanthin enrichment under high light and reduction of antenna size under reduced iron conditions.     

Regulation of energy balance in photosystems in response to changes in CO2 concentrations and light intensities during growth in extremely-high-CO2-tolerant green microalgae

Regulation of energy balance in photosystems in response to extremely-high-CO2 (40%) and low-CO2 (0.04%) stress was studied in extremely-high-CO2-tolerant green microalgae, Chlorococcum littorale and Chlorella sp. UK001. To investigate the energy input process, we assessed an F714/F685-ratio in a 77K fluorescence emission spectrum induced by 440-nm excitation in intact cells, which represents a ratio of fluorescence intensities derived from light-harvesting chlorophyll complexes in PSI and PSII. The F714/F685-ratio increased in several days after transferring C. littorale cells from air to 40% CO2, from 3% to 40% CO2 and from 3% to air. In all cases, the increase in the F714/F685-ratio was observed in high cell density culture, but no or a little increase was apparent in sparse cell density culture, when these cultures were illuminated at 250 micromol photon m-2 s-1. Even in the sparse culture, however, a similar increase in the F714/F685-ratio was observed when C. littorale cells were transferred from 3% to 40% CO2 at 20 micromol photon m-2 s-1. The cell density did not affect the F714/F685-ratio when CO2 concentration was kept at 3%. The activity of PSI electron (e-) transport was much higher in 40% CO2-grown cells than in 3% CO2-grown cells irrespective of the cell density during the culture, whereas the difference in PSII activity between them was small. The PSI activity at high cell density was higher also in air-grown cells than that in 3% CO2-grown cells. In both dense and sparse culture, 40% CO2-grown cells and air-grown cells showed higher relative quantum yield of PSI in the presence of DCMU than 3% CO2-grown cells, suggesting an increase in cyclic electron flow around PSI. Likewise, the increase in the F714/F685-ratio in response to the transfer to 40% CO2 was observed also in another extremely-high-CO2-tolerant alga, Chlorella sp. UK001. The possible role of the increases in the F714/F685-ratio, PSI/PSII activity ratio and cyclic e- transport activity in extremely-high-CO2 acclimation is discussed in comparison with low-CO2 acclimation.     

Effects of low-molecular-weight fractions (LMWF) from milk, egg yolk, and seminal plasma on freezability of bovine spermatozoa

The effects of the dialyzable fractions from bovine seminal plasma, egg yolk, and milk and of two buffer systems (TEST and sodium citrate) on post-thaw sperm motility were studied. Each basic salt solution was used in the experimental design. These solutions were used as extender systems in combination with egg yolk and glycerol. After collection, semen samples were extended (1:20), cooled to 5 degrees C in 1.5 hr, and frozen in 0.5-cc French straws after 3 hr of equilibration. Post-thaw samples were assayed for percentage of motile cells immediately after thawing and after 4 hr of incubation at room temperature (22 degrees C). Egg yolk (25%) provided the same protection as did the combination of colloidal material present in the skim milk-yolk extenders. The use of TEST as a buffer provided significantly higher (P less than 0.01) sperm post-thaw motility than milk salts or Na citrate. Sperm survival in extenders containing high concentrations of seminal plasma and/or egg yolk salts was significantly lower (P less than 0.01). Spermatozoa frozen in the presence of 6% glycerol resulted in sperm motility significantly (P less than 0.05) higher than that of spermatozoa frozen with 3% glycerol. However, no difference was observed between these two concentrations when TEST solution was used.