Joanna Grzyb - Academia.edu (original) (raw)
Papers by Joanna Grzyb
Phytochemistry, 2011
The ferredoxin:NADP+ oxidoreductase (FNR) catalyses the ferredoxin-dependent reduction of NADP+ t... more The ferredoxin:NADP+ oxidoreductase (FNR) catalyses the ferredoxin-dependent reduction of NADP+ to NADPH in linear photosynthetic electron transport. The enzyme also transfers electrons from reduced ferredoxin (Fd) or NADPH to the cytochrome b 6 f complex in cyclic electron transport. In vitro, the enzyme catalyses the NADPH-dependent reduction of various substrates, including ferredoxin, the analogue of its redox centre -ferricyanide, and the analogue of quinones, which is dibromothymoquinone. This paper presents results on the cadmium-induced inhibition of FNR. The K i value calculated for research condition was 1.72 mM.
Journal of Inorganic Biochemistry, 2004
Ferredoxin:NADP þ oxidoreductase (FNR) was treated with cadmium and after that its diaphorase rea... more Ferredoxin:NADP þ oxidoreductase (FNR) was treated with cadmium and after that its diaphorase reaction in the presence of dibromothymoquinone (DBMIB) or ferricyanide (FeCy, K 3 Fe(CN) 6 ) was examined. CdSO 4 (5 mM) caused 50% inhibition after half hour incubation. At least two components were distinguishable in the time-course inhibition, suggesting that more than one amino acid residues were engaged in reaction with the metal ion. The Lineweaver-Burk plots indicate that Cd 2þ is an uncompetitive inhibitor for DBMIB reduction but exerts non-competitive inhibition for the NADPH oxidation. The FeCy reduction did not follow Michaelis-Menten kinetics. Zn 2þ diminished inhibitory effect of Cd 2þ on the DBMIB reduction but enhanced inhibition of the FeCy reduction. Incubation with additional chelator (b-mercaptoethanol, or histidine) abolished inhibitory effect of Cd 2þ on the FeCy reduction but not on the DBMIB reduction. The mode of Cd 2þ action on the diaphorase activity of FNR in the presence of DBMIB or FeCy is briefly discussed with the special reference to the implication of two distinct sites at the FNR molecule, which might be involved in the reduction of various non-physiological substrates.
Chemistry and Physics of Lipids, 2005
The effects of Fe 3+ and Fe 2+ on molecular models of biomembranes were investigated. These consi... more The effects of Fe 3+ and Fe 2+ on molecular models of biomembranes were investigated. These consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and of dimyristoylphosphatidylethanolamine (DMPE), classes of phospholipids located in the outer and inner moieties of cell membranes, respectively. X-ray studies showed that very low concentrations of Fe 3+ affected DMPC organization and 10 −3 M induced a total loss of its multilamellar periodic stacking. Experiments carried out with Fe 2+ on DMPC showed weaker effects than those induced by Fe 3+ ions. Similar experiments were performed on DMPE bilayers. Fe 3+ from 10 −7 M up to 10 −4 M had practically no effect on DMPE structure. However, 10 −3 M Fe 3+ induced a deep perturbation of the multilamellar structure of DMPE. However, 10 −3 M Fe 2+ had no effect on DMPE organization practically. Differential scanning calorimetry measurements also revealed different effects of Fe 3+ and Fe 2+ on the phase transition and other thermal properties of the examined lipids. In conclusion, the results obtained indicate that iron ions interact with phospholipid bilayers perturbing their structures. These findings are consistent with the observation that iron ions change cell membrane fluidity and, therefore, affect its functions.
Acta Physiologiae Plantarum, 2004
2 De part ment of Chem is try, Ped a gog i cal Uni ver sity, ul. Podchorążych 2, 30-084 Kraków, P... more 2 De part ment of Chem is try, Ped a gog i cal Uni ver sity, ul. Podchorążych 2, 30-084 Kraków, Po land Key words: antheraxanthin; lipocalin; mem brane prop er ties; mo lec u lar mech a nism; photopro tection; sig nif i cance; the xanthophyll cy cle; violaxanthin; violaxanthin de-epoxidase; zeaxanthin; zeaxanthin epoxidase
Journal of Plant Physiology, 2013
Prolamellar bodies (PLBs) isolated from etiolated wheat seedlings were studied with the use of at... more Prolamellar bodies (PLBs) isolated from etiolated wheat seedlings were studied with the use of atomic force microscopy (AFM), transmission electron microscopy (TEM) and fluorescence spectroscopy. With AFM, PLBs were seen as spherical structures about 1-2μm in diameter, more elastic than mica and poly-l-lysine substrate. TEM analyses confirmed that PLBs of wheat leaf etioplasts also had an average diameter of appr. 1μm. Illumination induced the photoreduction of photoactive protochlorophyllide (Pchlide), i.e. Pchlide bound to protochlorophyllide oxidoreductase, which was shown in fluorescence spectra. The photoreduction was followed by the disruption of PLB structures, which started with the enlargement of PLB spheres and then their fragmentation into small balls as seen with AFM. Light-induced vesicle formation and the outgrowth of lamellar (pro)thylakoid membranes on the PLB surface were also confirmed by TEM analyses, and resulted in the apparent enlargement of the PLB diameter. The blue-shift of the fluorescence emission maximum of chlorophyllide observed for PLBs at room temperature after Pchlide photoreduction was completed within 25min. However, structural changes in PLBs were still observed after the completion of the blue-shift. The incubation of PLBs in darkness with HgCl2 also resulted in PLB enlargement and a loosening of their structure. AFM provides a unique opportunity to observe PLBs at a physiological temperature without the necessity of fixation.
Photosynthesis Research, 2008
Ferredoxin:NADP(+) oxidoreductase is an enzyme associated with the stromal side of the thylakoid ... more Ferredoxin:NADP(+) oxidoreductase is an enzyme associated with the stromal side of the thylakoid membrane in the chloroplast. It is involved in photosynthetic linear electron transport to produce NADPH and is supposed to play a role in cyclic electron transfer, generating a transmembrane pH gradient allowing ATP production, if photosystem II is non-functional or no NADP(+) is available for reduction. Different FNR isoforms have been described in non-photosynthetic tissues, where the enzyme catalyses the NADPH-dependent reduction of ferredoxin (Fd), necessary for some biosynthetic pathways. Here, we report the isolation and purification of two FNR isoproteins from wheat leaves, called FNR-A and FNR-B. These forms of the enzyme were identified as products of two different genes, as confirmed by mass spectrometry. The molecular masses of FNR-A and FNR-B were 34.3 kDa and 35.5 kDa, respectively. The isoelectric point of both FNR-A and FNR-B was about 5, but FNR-B appeared more acidic (of about 0.2 pH unit) than FNR-A. Both isoenzymes were able to catalyse a NADPH-dependent reduction of dibromothymoquinone and the mixture of isoforms catalysed reduction of cytochrome c in the presence of Fd. For the first time, the pH- and ionic strength dependent oligomerization of FNRs is observed. No other protein was necessary for complex formation. The putative role of the two FNR isoforms in photosynthesis is discussed based on current knowledge of electron transport in chloroplasts.
The ferredoxin:NADP + oxidoreductase (FNR) is a plant enzyme, catalyzing the last step of photosy... more The ferredoxin:NADP + oxidoreductase (FNR) is a plant enzyme, catalyzing the last step of photosynthetic linear electron transport, and involved also in cyclic electron transport around photosystem I. In this study we present the first evidence of FNR (isolated from spinach and from wheat) interaction directly with a model membrane without the mediation of any additional protein. The monomolecular layer technique measurements showed a significant increase in surface pressure after the injection of enzyme solution beneath a monolayer consisting of chloroplast lipids: monogalactosyldiacylglycerol or digalactosyldiacylglycerol. An ATR FTIR study revealed also the presence of FNR in a bilayer composed of these lipids. The secondary structure of the protein was significantly impaired by lipids, as with a pH-induced shift. The stabilization of FNR in the presence of lipids leads to an increase in the rate of NADPH-dependent reduction of dibromothymoquinone catalyzed by the enzyme. The biological significance of FNR-membrane interaction is discussed.
Journal of plant physiology, 2006
Lipocalins are a widely distributed group of proteins whose common feature is the presence of six... more Lipocalins are a widely distributed group of proteins whose common feature is the presence of six-or eight-stranded beta-barrel in their tertiary structure and highly conservative motifs short conserved region, (SCR) in their amino acid sequences. The presence of three SCRs is typical for kernel lipocalins, while outlier lipocalins have only one or two such regions. Owing to their ability to bind and transport small, hydrophobic molecules, lipocalins participate in the distribution of such substances. However, the physiological significance of lipocalins is not limited to transfer processes. They play an important role in the regulation of immunological and developmental processes, and are also involved in the reactions of organisms to various stress factors and in the pathways of signal transduction. Of special interest is the enzymatic activity found in a few members of the lipocalin family, as well as the interaction with natural membranes, both directly with lipids and through m...
The ferredoxin:NADP + oxidoreductase (FNR) is a plant enzyme, catalyzing the last step of photosy... more The ferredoxin:NADP + oxidoreductase (FNR) is a plant enzyme, catalyzing the last step of photosynthetic linear electron transport, and involved also in cyclic electron transport around photosystem I. In this study we present the first evidence of FNR (isolated from spinach and from wheat) interaction directly with a model membrane without the mediation of any additional protein. The monomolecular layer technique measurements showed a significant increase in surface pressure after the injection of enzyme solution beneath a monolayer consisting of chloroplast lipids: monogalactosyldiacylglycerol or digalactosyldiacylglycerol. An ATR FTIR study revealed also the presence of FNR in a bilayer composed of these lipids. The secondary structure of the protein was significantly impaired by lipids, as with a pH-induced shift. The stabilization of FNR in the presence of lipids leads to an increase in the rate of NADPH-dependent reduction of dibromothymoquinone catalyzed by the enzyme. The biological significance of FNR-membrane interaction is discussed.
In the present study, the solubility and enzymatic de-epoxidation of diadinoxanthin (Ddx) was inv... more In the present study, the solubility and enzymatic de-epoxidation of diadinoxanthin (Ddx) was investigated in three different artificial membrane systems: (1) Unilamellar liposomes composed of different concentrations of the bilayer forming lipid phosphatidylcholine (PC) and the inverted hexagonal phase (H II phase) forming lipid monogalactosyldiacylglycerol (MGDG), (2) liposomes composed of PC and the H II phase forming lipid phosphatidylethanolamine (PE), and (3) an artificial membrane system composed of digalactosyldiacylglycerol (DGDG) and MGDG, which resembles the lipid composition of the natural thylakoid membrane. Our results show that Ddx de-epoxidation strongly depends on the concentration of the inverted hexagonal phase forming lipids MGDG or PE in the liposomes composed of PC or DGDG, thus indicating that the presence of inverted hexagonal structures is essential for Ddx de-epoxidation. The difference observed for the solubilization of Ddx in H II phase forming lipids compared with bilayer forming lipids indicates that Ddx is not equally distributed in the liposomes composed of different concentrations of bilayer versus non-bilayer lipids. In artificial membranes with a high percentage of bilayer lipids, a large part of Ddx is located in the membrane bilayer. In membranes composed of equal proportions of bilayer and H II phase forming lipids, the majority of the Ddx molecules is located in the inverted hexagonal structures. The significance of the pigment distribution and the three-dimensional structure of the H II phase for the de-epoxidation reaction is discussed, and a possible scenario for the lipid dependence of Ddx (and violaxanthin) de-epoxidation in the native thylakoid membrane is proposed.
Iron sulfur cluster Protein de novo design Redox enzyme Coiled-coil Four-helix bundle EPR spectro... more Iron sulfur cluster Protein de novo design Redox enzyme Coiled-coil Four-helix bundle EPR spectroscopy Using a 'metal-first' approach, we computationally designed, prepared, and characterized a four-iron foursulfur (Fe 4 S 4 ) cluster protein with a non-natural α-helical coiled-coil fold. The novelty of this fold lies in the placement of a Fe 4 S 4 cluster within the hydrophobic core of a four-helix bundle, making it unique among previous iron-sulfur (FeS) protein designs, and different from known natural FeS proteins. The apoprotein, recombinantly expressed and purified from E. coli, readily self-assembles with Fe 4 S 4 clusters in vitro. UV-Vis absorption and CD spectroscopy, elemental analysis, gel filtration, and analytical ultracentrifugation confirm that the protein is folded and assembled as designed, namely, α-helical coiled-coil binding a single Fe 4 S 4 cluster. Dithionite-reduced holoprotein samples have characteristic rhombic EPR spectra, typical of lowpotential, [Fe 4 S 4 ] + (S = 1/2), with g values of g z,y = (1.970, 1.975), and g x = 2.053. The temperature, and power dependence of the signal intensity were also characteristic of [Fe 4 S 4 ] + clusters with very efficient spin relaxation, but almost without any interaction between adjacent clusters. The new design is very promising although optimization is required, particularly for preventing aggregation, and adding second shell interactions to stabilize the reduced state. Its main advantage is its extendibility into a multi-FeS cluster protein by simply duplicating and translating the binding site along the coiled-coil axis. This opens new possibilities for designing protein-embedded redox chains that may be used as "wires" for coupling any given set of redox enzymes.
Biochimica Et Biophysica Acta-Bioenergetics, 2012
Biochimica Et Biophysica Acta-Bioenergetics, 2012
Journal of Plant Physiology, 2013
Prolamellar bodies (PLBs) isolated from etiolated wheat seedlings were studied with the use of at... more Prolamellar bodies (PLBs) isolated from etiolated wheat seedlings were studied with the use of atomic force microscopy (AFM), transmission electron microscopy (TEM) and fluorescence spectroscopy. With AFM, PLBs were seen as spherical structures about 1-2μm in diameter, more elastic than mica and poly-l-lysine substrate. TEM analyses confirmed that PLBs of wheat leaf etioplasts also had an average diameter of appr. 1μm. Illumination induced the photoreduction of photoactive protochlorophyllide (Pchlide), i.e. Pchlide bound to protochlorophyllide oxidoreductase, which was shown in fluorescence spectra. The photoreduction was followed by the disruption of PLB structures, which started with the enlargement of PLB spheres and then their fragmentation into small balls as seen with AFM. Light-induced vesicle formation and the outgrowth of lamellar (pro)thylakoid membranes on the PLB surface were also confirmed by TEM analyses, and resulted in the apparent enlargement of the PLB diameter. The blue-shift of the fluorescence emission maximum of chlorophyllide observed for PLBs at room temperature after Pchlide photoreduction was completed within 25min. However, structural changes in PLBs were still observed after the completion of the blue-shift. The incubation of PLBs in darkness with HgCl2 also resulted in PLB enlargement and a loosening of their structure. AFM provides a unique opportunity to observe PLBs at a physiological temperature without the necessity of fixation.
Journal of Physics: Condensed Matter, 2013
This paper reports the observation of Tb 3+ 4f-4f emission gain in ZrO 2 nanocrystals stabilized ... more This paper reports the observation of Tb 3+ 4f-4f emission gain in ZrO 2 nanocrystals stabilized by Y 2 O 3 as the amount of stabilizer increases from 0% to 10% mol. The nanocrystals were obtained via microwave solvothermal technology. The photoluminescence properties of as-grown samples are investigated. The possibility of biological applications of the material is tested on living organisms (mice). The result indicates the potential use of the studied material as a luminescent nanomarker.
Journal of Physics: Condensed Matter, 2013
Ferredoxin:NADP + oxidoreductase (FNR) is a plant and cyanobacterial photosynthetic enzyme, also ... more Ferredoxin:NADP + oxidoreductase (FNR) is a plant and cyanobacterial photosynthetic enzyme, also found in non-photosynthetic tissues, where it is involved in redox reactions of biosynthetic pathways. In vivo it transfers electrons to nicotinamide adenine dinucleotide phosphate (NADP + ), forming its reduced version, NADPH, while in vitro it can also use NADPH to reduce several substrates, such as ferricyanide, various quinones and nitriles. As an oxidoreductase catalyzing reaction of a broad range of substrates, FNR may be used in biotechnological processes. Quantum dots are semiconductor nanocrystals of a few to several nanometers diameter, having very useful luminescent properties. We present the spectroscopic and functional characteristics of a covalent conjugation of FNR and CdSe/ZnS quantum dots. Two types of quantum dots, of different diameter and emission maximum (550 and 650 nm), were used for comparison. Steady-state fluorescence and gel electrophoresis confirmed efficient conjugation, while fluorescence correlation spectroscopy (FCS) allowed for determination of the conjugates' radii. The nanohybrids sustained enzymatic activity; however, changes in maximal reaction rates and Michaelis constant were found. Detailed analysis of the kinetic parameters showed that the changes in the enzyme activity depend on the substrate used for activity measurement but also on the size of the quantum dots. The presented nanohybrids, as the first example using plant and photosynthetic enzyme as a protein partner, may became a tool to study photosynthesis as well as other biosynthetic and biotechnological processes, involving enzymatically catalyzed electron transfer.
This study deals with the influence of cadmium on the structure and function of ferredoxin:NADP +... more This study deals with the influence of cadmium on the structure and function of ferredoxin:NADP + oxidoreductase (FNR), one of the key photosynthetic enzymes. We describe changes in the secondary and tertiary structure of the enzyme upon the Electronic supplementary material The online version of this article (416 J. Grzyb et al.
In the present studies, we have found a fragment of amino acid sequence, called TFT motif, both i... more In the present studies, we have found a fragment of amino acid sequence, called TFT motif, both in light-dependent protochlorophyllide oxidoreductase (LPOR) and in the L subunit of dark-operative (light-independent) protochlorophyllide oxidoreductases (DPOR). Amino acid residues of this motif shared similar physicochemical properties in both types of the enzymes. In the present paper, physicochemical properties of amino acid residues of this common motif, its spatial arrangement and a possible physiological role are being discussed. This is the first report when similarity between LPOR and DPOR, phylogenetically unrelated, but functionally redundant enzymes, is described.
Here, we compare two approaches of protein design. A computational approach was used in the desig... more Here, we compare two approaches of protein design. A computational approach was used in the design of the coiled-coil iron-sulfur protein, CCIS, as a four helix bundle binding an iron-sulfur cluster within its hydrophobic core. An empirical approach was used for designing the redox-chain maquette, RCM as a four-helix bundle assembling iron-sulfur clusters within loops and one heme in the middle of its hydrophobic core. We demonstrate that both ways of design yielded the desired proteins in terms of secondary structure and cofactors assembly. Both approaches, however, still have much to improve in predicting conformational changes in the presence of bound cofactors, controlling oligomerization tendency and stabilizing the bound iron-sulfur clusters in the reduced state. Lessons from both ways of design and future directions of development are discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.
Effects of selective reagents of amino groups (fluorescamine, Fc) and thiol [5,5'-dithio-bis(2-ni... more Effects of selective reagents of amino groups (fluorescamine, Fc) and thiol [5,5'-dithio-bis(2-nitrobenzoic) acid, DTNB] groups on the diaphorase activity of spinach ferredoxin:NADP + oxidoreductase (FNR, E.C 1.18.1.2) in the presence of dibromothymoquinone (DBMIB) as an electron acceptor were studied. The incubation of FNR with 250 µM Fc in the time range from 0 to 120 min led to the gradual decrease of FNR activity according to biphasic kinetics. At the initial phase the activity (defined as the rate of NADPH oxidation) decreased about 4-time faster than at the subsequent second slower phase. Incubation of FNR simultaneously with Fc and DBMIB for more than 20 min caused restoration of the activity to about 80 % of the control. The inhibitory effect of Fc on the FNR-catalysed DBMIB reduction had non-competitive character. Incubation of FNR with DTNB led also to a gradual decrease of the enzyme activity, which reached about 45 % of the control after 2 h of incubation. Thus neither amino nor thiol groups in the FNR molecule are involved directly in the DBMIB reduction. However, the presence of DBMIB in the incubation medium influenced the inhibitory pattern of Fc and DTNB, and this suggests that DBMIB modified the conformational state of the FNR molecule.
Phytochemistry, 2011
The ferredoxin:NADP+ oxidoreductase (FNR) catalyses the ferredoxin-dependent reduction of NADP+ t... more The ferredoxin:NADP+ oxidoreductase (FNR) catalyses the ferredoxin-dependent reduction of NADP+ to NADPH in linear photosynthetic electron transport. The enzyme also transfers electrons from reduced ferredoxin (Fd) or NADPH to the cytochrome b 6 f complex in cyclic electron transport. In vitro, the enzyme catalyses the NADPH-dependent reduction of various substrates, including ferredoxin, the analogue of its redox centre -ferricyanide, and the analogue of quinones, which is dibromothymoquinone. This paper presents results on the cadmium-induced inhibition of FNR. The K i value calculated for research condition was 1.72 mM.
Journal of Inorganic Biochemistry, 2004
Ferredoxin:NADP þ oxidoreductase (FNR) was treated with cadmium and after that its diaphorase rea... more Ferredoxin:NADP þ oxidoreductase (FNR) was treated with cadmium and after that its diaphorase reaction in the presence of dibromothymoquinone (DBMIB) or ferricyanide (FeCy, K 3 Fe(CN) 6 ) was examined. CdSO 4 (5 mM) caused 50% inhibition after half hour incubation. At least two components were distinguishable in the time-course inhibition, suggesting that more than one amino acid residues were engaged in reaction with the metal ion. The Lineweaver-Burk plots indicate that Cd 2þ is an uncompetitive inhibitor for DBMIB reduction but exerts non-competitive inhibition for the NADPH oxidation. The FeCy reduction did not follow Michaelis-Menten kinetics. Zn 2þ diminished inhibitory effect of Cd 2þ on the DBMIB reduction but enhanced inhibition of the FeCy reduction. Incubation with additional chelator (b-mercaptoethanol, or histidine) abolished inhibitory effect of Cd 2þ on the FeCy reduction but not on the DBMIB reduction. The mode of Cd 2þ action on the diaphorase activity of FNR in the presence of DBMIB or FeCy is briefly discussed with the special reference to the implication of two distinct sites at the FNR molecule, which might be involved in the reduction of various non-physiological substrates.
Chemistry and Physics of Lipids, 2005
The effects of Fe 3+ and Fe 2+ on molecular models of biomembranes were investigated. These consi... more The effects of Fe 3+ and Fe 2+ on molecular models of biomembranes were investigated. These consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and of dimyristoylphosphatidylethanolamine (DMPE), classes of phospholipids located in the outer and inner moieties of cell membranes, respectively. X-ray studies showed that very low concentrations of Fe 3+ affected DMPC organization and 10 −3 M induced a total loss of its multilamellar periodic stacking. Experiments carried out with Fe 2+ on DMPC showed weaker effects than those induced by Fe 3+ ions. Similar experiments were performed on DMPE bilayers. Fe 3+ from 10 −7 M up to 10 −4 M had practically no effect on DMPE structure. However, 10 −3 M Fe 3+ induced a deep perturbation of the multilamellar structure of DMPE. However, 10 −3 M Fe 2+ had no effect on DMPE organization practically. Differential scanning calorimetry measurements also revealed different effects of Fe 3+ and Fe 2+ on the phase transition and other thermal properties of the examined lipids. In conclusion, the results obtained indicate that iron ions interact with phospholipid bilayers perturbing their structures. These findings are consistent with the observation that iron ions change cell membrane fluidity and, therefore, affect its functions.
Acta Physiologiae Plantarum, 2004
2 De part ment of Chem is try, Ped a gog i cal Uni ver sity, ul. Podchorążych 2, 30-084 Kraków, P... more 2 De part ment of Chem is try, Ped a gog i cal Uni ver sity, ul. Podchorążych 2, 30-084 Kraków, Po land Key words: antheraxanthin; lipocalin; mem brane prop er ties; mo lec u lar mech a nism; photopro tection; sig nif i cance; the xanthophyll cy cle; violaxanthin; violaxanthin de-epoxidase; zeaxanthin; zeaxanthin epoxidase
Journal of Plant Physiology, 2013
Prolamellar bodies (PLBs) isolated from etiolated wheat seedlings were studied with the use of at... more Prolamellar bodies (PLBs) isolated from etiolated wheat seedlings were studied with the use of atomic force microscopy (AFM), transmission electron microscopy (TEM) and fluorescence spectroscopy. With AFM, PLBs were seen as spherical structures about 1-2μm in diameter, more elastic than mica and poly-l-lysine substrate. TEM analyses confirmed that PLBs of wheat leaf etioplasts also had an average diameter of appr. 1μm. Illumination induced the photoreduction of photoactive protochlorophyllide (Pchlide), i.e. Pchlide bound to protochlorophyllide oxidoreductase, which was shown in fluorescence spectra. The photoreduction was followed by the disruption of PLB structures, which started with the enlargement of PLB spheres and then their fragmentation into small balls as seen with AFM. Light-induced vesicle formation and the outgrowth of lamellar (pro)thylakoid membranes on the PLB surface were also confirmed by TEM analyses, and resulted in the apparent enlargement of the PLB diameter. The blue-shift of the fluorescence emission maximum of chlorophyllide observed for PLBs at room temperature after Pchlide photoreduction was completed within 25min. However, structural changes in PLBs were still observed after the completion of the blue-shift. The incubation of PLBs in darkness with HgCl2 also resulted in PLB enlargement and a loosening of their structure. AFM provides a unique opportunity to observe PLBs at a physiological temperature without the necessity of fixation.
Photosynthesis Research, 2008
Ferredoxin:NADP(+) oxidoreductase is an enzyme associated with the stromal side of the thylakoid ... more Ferredoxin:NADP(+) oxidoreductase is an enzyme associated with the stromal side of the thylakoid membrane in the chloroplast. It is involved in photosynthetic linear electron transport to produce NADPH and is supposed to play a role in cyclic electron transfer, generating a transmembrane pH gradient allowing ATP production, if photosystem II is non-functional or no NADP(+) is available for reduction. Different FNR isoforms have been described in non-photosynthetic tissues, where the enzyme catalyses the NADPH-dependent reduction of ferredoxin (Fd), necessary for some biosynthetic pathways. Here, we report the isolation and purification of two FNR isoproteins from wheat leaves, called FNR-A and FNR-B. These forms of the enzyme were identified as products of two different genes, as confirmed by mass spectrometry. The molecular masses of FNR-A and FNR-B were 34.3 kDa and 35.5 kDa, respectively. The isoelectric point of both FNR-A and FNR-B was about 5, but FNR-B appeared more acidic (of about 0.2 pH unit) than FNR-A. Both isoenzymes were able to catalyse a NADPH-dependent reduction of dibromothymoquinone and the mixture of isoforms catalysed reduction of cytochrome c in the presence of Fd. For the first time, the pH- and ionic strength dependent oligomerization of FNRs is observed. No other protein was necessary for complex formation. The putative role of the two FNR isoforms in photosynthesis is discussed based on current knowledge of electron transport in chloroplasts.
The ferredoxin:NADP + oxidoreductase (FNR) is a plant enzyme, catalyzing the last step of photosy... more The ferredoxin:NADP + oxidoreductase (FNR) is a plant enzyme, catalyzing the last step of photosynthetic linear electron transport, and involved also in cyclic electron transport around photosystem I. In this study we present the first evidence of FNR (isolated from spinach and from wheat) interaction directly with a model membrane without the mediation of any additional protein. The monomolecular layer technique measurements showed a significant increase in surface pressure after the injection of enzyme solution beneath a monolayer consisting of chloroplast lipids: monogalactosyldiacylglycerol or digalactosyldiacylglycerol. An ATR FTIR study revealed also the presence of FNR in a bilayer composed of these lipids. The secondary structure of the protein was significantly impaired by lipids, as with a pH-induced shift. The stabilization of FNR in the presence of lipids leads to an increase in the rate of NADPH-dependent reduction of dibromothymoquinone catalyzed by the enzyme. The biological significance of FNR-membrane interaction is discussed.
Journal of plant physiology, 2006
Lipocalins are a widely distributed group of proteins whose common feature is the presence of six... more Lipocalins are a widely distributed group of proteins whose common feature is the presence of six-or eight-stranded beta-barrel in their tertiary structure and highly conservative motifs short conserved region, (SCR) in their amino acid sequences. The presence of three SCRs is typical for kernel lipocalins, while outlier lipocalins have only one or two such regions. Owing to their ability to bind and transport small, hydrophobic molecules, lipocalins participate in the distribution of such substances. However, the physiological significance of lipocalins is not limited to transfer processes. They play an important role in the regulation of immunological and developmental processes, and are also involved in the reactions of organisms to various stress factors and in the pathways of signal transduction. Of special interest is the enzymatic activity found in a few members of the lipocalin family, as well as the interaction with natural membranes, both directly with lipids and through m...
The ferredoxin:NADP + oxidoreductase (FNR) is a plant enzyme, catalyzing the last step of photosy... more The ferredoxin:NADP + oxidoreductase (FNR) is a plant enzyme, catalyzing the last step of photosynthetic linear electron transport, and involved also in cyclic electron transport around photosystem I. In this study we present the first evidence of FNR (isolated from spinach and from wheat) interaction directly with a model membrane without the mediation of any additional protein. The monomolecular layer technique measurements showed a significant increase in surface pressure after the injection of enzyme solution beneath a monolayer consisting of chloroplast lipids: monogalactosyldiacylglycerol or digalactosyldiacylglycerol. An ATR FTIR study revealed also the presence of FNR in a bilayer composed of these lipids. The secondary structure of the protein was significantly impaired by lipids, as with a pH-induced shift. The stabilization of FNR in the presence of lipids leads to an increase in the rate of NADPH-dependent reduction of dibromothymoquinone catalyzed by the enzyme. The biological significance of FNR-membrane interaction is discussed.
In the present study, the solubility and enzymatic de-epoxidation of diadinoxanthin (Ddx) was inv... more In the present study, the solubility and enzymatic de-epoxidation of diadinoxanthin (Ddx) was investigated in three different artificial membrane systems: (1) Unilamellar liposomes composed of different concentrations of the bilayer forming lipid phosphatidylcholine (PC) and the inverted hexagonal phase (H II phase) forming lipid monogalactosyldiacylglycerol (MGDG), (2) liposomes composed of PC and the H II phase forming lipid phosphatidylethanolamine (PE), and (3) an artificial membrane system composed of digalactosyldiacylglycerol (DGDG) and MGDG, which resembles the lipid composition of the natural thylakoid membrane. Our results show that Ddx de-epoxidation strongly depends on the concentration of the inverted hexagonal phase forming lipids MGDG or PE in the liposomes composed of PC or DGDG, thus indicating that the presence of inverted hexagonal structures is essential for Ddx de-epoxidation. The difference observed for the solubilization of Ddx in H II phase forming lipids compared with bilayer forming lipids indicates that Ddx is not equally distributed in the liposomes composed of different concentrations of bilayer versus non-bilayer lipids. In artificial membranes with a high percentage of bilayer lipids, a large part of Ddx is located in the membrane bilayer. In membranes composed of equal proportions of bilayer and H II phase forming lipids, the majority of the Ddx molecules is located in the inverted hexagonal structures. The significance of the pigment distribution and the three-dimensional structure of the H II phase for the de-epoxidation reaction is discussed, and a possible scenario for the lipid dependence of Ddx (and violaxanthin) de-epoxidation in the native thylakoid membrane is proposed.
Iron sulfur cluster Protein de novo design Redox enzyme Coiled-coil Four-helix bundle EPR spectro... more Iron sulfur cluster Protein de novo design Redox enzyme Coiled-coil Four-helix bundle EPR spectroscopy Using a 'metal-first' approach, we computationally designed, prepared, and characterized a four-iron foursulfur (Fe 4 S 4 ) cluster protein with a non-natural α-helical coiled-coil fold. The novelty of this fold lies in the placement of a Fe 4 S 4 cluster within the hydrophobic core of a four-helix bundle, making it unique among previous iron-sulfur (FeS) protein designs, and different from known natural FeS proteins. The apoprotein, recombinantly expressed and purified from E. coli, readily self-assembles with Fe 4 S 4 clusters in vitro. UV-Vis absorption and CD spectroscopy, elemental analysis, gel filtration, and analytical ultracentrifugation confirm that the protein is folded and assembled as designed, namely, α-helical coiled-coil binding a single Fe 4 S 4 cluster. Dithionite-reduced holoprotein samples have characteristic rhombic EPR spectra, typical of lowpotential, [Fe 4 S 4 ] + (S = 1/2), with g values of g z,y = (1.970, 1.975), and g x = 2.053. The temperature, and power dependence of the signal intensity were also characteristic of [Fe 4 S 4 ] + clusters with very efficient spin relaxation, but almost without any interaction between adjacent clusters. The new design is very promising although optimization is required, particularly for preventing aggregation, and adding second shell interactions to stabilize the reduced state. Its main advantage is its extendibility into a multi-FeS cluster protein by simply duplicating and translating the binding site along the coiled-coil axis. This opens new possibilities for designing protein-embedded redox chains that may be used as "wires" for coupling any given set of redox enzymes.
Biochimica Et Biophysica Acta-Bioenergetics, 2012
Biochimica Et Biophysica Acta-Bioenergetics, 2012
Journal of Plant Physiology, 2013
Prolamellar bodies (PLBs) isolated from etiolated wheat seedlings were studied with the use of at... more Prolamellar bodies (PLBs) isolated from etiolated wheat seedlings were studied with the use of atomic force microscopy (AFM), transmission electron microscopy (TEM) and fluorescence spectroscopy. With AFM, PLBs were seen as spherical structures about 1-2μm in diameter, more elastic than mica and poly-l-lysine substrate. TEM analyses confirmed that PLBs of wheat leaf etioplasts also had an average diameter of appr. 1μm. Illumination induced the photoreduction of photoactive protochlorophyllide (Pchlide), i.e. Pchlide bound to protochlorophyllide oxidoreductase, which was shown in fluorescence spectra. The photoreduction was followed by the disruption of PLB structures, which started with the enlargement of PLB spheres and then their fragmentation into small balls as seen with AFM. Light-induced vesicle formation and the outgrowth of lamellar (pro)thylakoid membranes on the PLB surface were also confirmed by TEM analyses, and resulted in the apparent enlargement of the PLB diameter. The blue-shift of the fluorescence emission maximum of chlorophyllide observed for PLBs at room temperature after Pchlide photoreduction was completed within 25min. However, structural changes in PLBs were still observed after the completion of the blue-shift. The incubation of PLBs in darkness with HgCl2 also resulted in PLB enlargement and a loosening of their structure. AFM provides a unique opportunity to observe PLBs at a physiological temperature without the necessity of fixation.
Journal of Physics: Condensed Matter, 2013
This paper reports the observation of Tb 3+ 4f-4f emission gain in ZrO 2 nanocrystals stabilized ... more This paper reports the observation of Tb 3+ 4f-4f emission gain in ZrO 2 nanocrystals stabilized by Y 2 O 3 as the amount of stabilizer increases from 0% to 10% mol. The nanocrystals were obtained via microwave solvothermal technology. The photoluminescence properties of as-grown samples are investigated. The possibility of biological applications of the material is tested on living organisms (mice). The result indicates the potential use of the studied material as a luminescent nanomarker.
Journal of Physics: Condensed Matter, 2013
Ferredoxin:NADP + oxidoreductase (FNR) is a plant and cyanobacterial photosynthetic enzyme, also ... more Ferredoxin:NADP + oxidoreductase (FNR) is a plant and cyanobacterial photosynthetic enzyme, also found in non-photosynthetic tissues, where it is involved in redox reactions of biosynthetic pathways. In vivo it transfers electrons to nicotinamide adenine dinucleotide phosphate (NADP + ), forming its reduced version, NADPH, while in vitro it can also use NADPH to reduce several substrates, such as ferricyanide, various quinones and nitriles. As an oxidoreductase catalyzing reaction of a broad range of substrates, FNR may be used in biotechnological processes. Quantum dots are semiconductor nanocrystals of a few to several nanometers diameter, having very useful luminescent properties. We present the spectroscopic and functional characteristics of a covalent conjugation of FNR and CdSe/ZnS quantum dots. Two types of quantum dots, of different diameter and emission maximum (550 and 650 nm), were used for comparison. Steady-state fluorescence and gel electrophoresis confirmed efficient conjugation, while fluorescence correlation spectroscopy (FCS) allowed for determination of the conjugates' radii. The nanohybrids sustained enzymatic activity; however, changes in maximal reaction rates and Michaelis constant were found. Detailed analysis of the kinetic parameters showed that the changes in the enzyme activity depend on the substrate used for activity measurement but also on the size of the quantum dots. The presented nanohybrids, as the first example using plant and photosynthetic enzyme as a protein partner, may became a tool to study photosynthesis as well as other biosynthetic and biotechnological processes, involving enzymatically catalyzed electron transfer.
This study deals with the influence of cadmium on the structure and function of ferredoxin:NADP +... more This study deals with the influence of cadmium on the structure and function of ferredoxin:NADP + oxidoreductase (FNR), one of the key photosynthetic enzymes. We describe changes in the secondary and tertiary structure of the enzyme upon the Electronic supplementary material The online version of this article (416 J. Grzyb et al.
In the present studies, we have found a fragment of amino acid sequence, called TFT motif, both i... more In the present studies, we have found a fragment of amino acid sequence, called TFT motif, both in light-dependent protochlorophyllide oxidoreductase (LPOR) and in the L subunit of dark-operative (light-independent) protochlorophyllide oxidoreductases (DPOR). Amino acid residues of this motif shared similar physicochemical properties in both types of the enzymes. In the present paper, physicochemical properties of amino acid residues of this common motif, its spatial arrangement and a possible physiological role are being discussed. This is the first report when similarity between LPOR and DPOR, phylogenetically unrelated, but functionally redundant enzymes, is described.
Here, we compare two approaches of protein design. A computational approach was used in the desig... more Here, we compare two approaches of protein design. A computational approach was used in the design of the coiled-coil iron-sulfur protein, CCIS, as a four helix bundle binding an iron-sulfur cluster within its hydrophobic core. An empirical approach was used for designing the redox-chain maquette, RCM as a four-helix bundle assembling iron-sulfur clusters within loops and one heme in the middle of its hydrophobic core. We demonstrate that both ways of design yielded the desired proteins in terms of secondary structure and cofactors assembly. Both approaches, however, still have much to improve in predicting conformational changes in the presence of bound cofactors, controlling oligomerization tendency and stabilizing the bound iron-sulfur clusters in the reduced state. Lessons from both ways of design and future directions of development are discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.
Effects of selective reagents of amino groups (fluorescamine, Fc) and thiol [5,5'-dithio-bis(2-ni... more Effects of selective reagents of amino groups (fluorescamine, Fc) and thiol [5,5'-dithio-bis(2-nitrobenzoic) acid, DTNB] groups on the diaphorase activity of spinach ferredoxin:NADP + oxidoreductase (FNR, E.C 1.18.1.2) in the presence of dibromothymoquinone (DBMIB) as an electron acceptor were studied. The incubation of FNR with 250 µM Fc in the time range from 0 to 120 min led to the gradual decrease of FNR activity according to biphasic kinetics. At the initial phase the activity (defined as the rate of NADPH oxidation) decreased about 4-time faster than at the subsequent second slower phase. Incubation of FNR simultaneously with Fc and DBMIB for more than 20 min caused restoration of the activity to about 80 % of the control. The inhibitory effect of Fc on the FNR-catalysed DBMIB reduction had non-competitive character. Incubation of FNR with DTNB led also to a gradual decrease of the enzyme activity, which reached about 45 % of the control after 2 h of incubation. Thus neither amino nor thiol groups in the FNR molecule are involved directly in the DBMIB reduction. However, the presence of DBMIB in the incubation medium influenced the inhibitory pattern of Fc and DTNB, and this suggests that DBMIB modified the conformational state of the FNR molecule.