Fd Complex Research Articles

LEAFY, a Pioneer Transcription Factor in Plants: A Mini-Review.

A subset of eukaryotic transcription factors (TFs) possess the ability to reprogram one cell type into another. Genes important for cellular reprograming are typically located in closed chromatin, which is covered by nucleosomes. Pioneer factors are a special class of TFs that can initially engage their target sites in closed chromatin prior to the engagement with, opening of, or modification of the sites by other factors. Although many pioneer factors are known in animals, a few have been characterized in plants. The TF LEAFY (LFY) acts as a pioneer factor specifying floral fate in Arabidopsis. In response to endogenous and environmental cues, plants produce appropriate floral inducers (florigens). During the vegetative phase, LFY is repressed by the TERMINAL FLOWER 1 (TFL1)–FD complex, which functions as a floral inhibitor, or anti-florigen. The florigen FLOWERING LOCUS T (FT) competes with TFL1 to prevent the binding of the FD TF to the LFY locus. The resulting FT–FD complex functions as a transient stimulus to activate its targets. Once LFY has been transcribed in the appropriate spatiotemporal manner, LFY binds to nucleosomes in closed chromatin regions. Subsequently, LFY opens the chromatin by displacing H1 linker histones and recruiting the SWI/SNF chromatin-remodeling complex. Such local changes permit the binding of other TFs, leading to the expression of the floral meristem identity gene APETALA1. This mini-review describes the latest advances in our understanding of the pioneer TF LFY, providing insight into the establishment of gene expression competence through the shaping of the plant epigenetic landscape.

Thermodynamics of ferredoxin binding to cyanobacterial nitrate reductase.

The role of the seven negatively charged amino acids of Synechocystis sp. PCC 6803 ferredoxin (Fd), i.e., Glu29, Glu30, Asp60, Asp65, Asp66, Glu92, and Glu93, predicted to form complex with nitrate reductase (NR), was investigated using site-directed mutagenesis and isothermal titration calorimetry (ITC). These experiments identified four Fd amino acids, i.e., Glu29, Asp60, Glu92, and Glu93, that are essential for the Fd binding and efficient electron transfer to the NR. ITC measurements showed that the most likely stoichiometry for the wild-type NR/wild-type Fd complex is 1:1, a Kd value 4.7μM for the complex at low ionic strength residues and both the enthalpic and entropic components are associated with complex formation. ITC titrations of wild-type NR with four Fd variants, E29N, D60N, E92Q, and E93N demonstrated that the complex formation, although favorable, was less energetically favorable when compared to complex formation between the two wild-type proteins, suggesting that these negatively charged Fd residues at these positions are important for the effective and productive interaction with wild-type enzyme.

Transient activity of the florigen complex during the floral transition in Arabidopsis thaliana.

FLOWERING LOCUS T (FT) is an essential component of florigen in Arabidopsis thaliana Transcription of FT is induced in leaves, and the resulting FT protein is transported to the shoot apex, in which it initiates floral development. Previous analyses suggest that, together with the b-ZIP transcription factor FD, FT regulates the transcription of downstream targets such as APETALA1 (AP1) in floral anlagen. However, conclusive in vivo evidence that FT is transported to the shoot apex to form an FT-FD complex is lacking. Here, using an innovative in vivo imaging technique, we show that the FT-FD complex and AP1 colocalise in floral anlagen. In addition, the FT-FD complex disappears soon after the floral transition owing to a reduction in FD transcripts in the shoot apex. We further show that misinduction of FD activity after the transition leads to defective reproductive development. Taken together, our results indicate that the FT-FD complex functions as a transient stimulus and imply that a regulatory mechanism exists during the floral transition that reduces FT-FD complex levels via modulation of FD expression.

Investigation of inclusion behaviour of gefitinib with epichlorohydrin-β-cyclodextrin polymer: preparation of binary complex, stoichiometric determination and characterization

Gefitinib is anticancer drug which is sparingly soluble in water. This limits its dissolution and bioavailability. Binary inclusion complex of gefitinib with Epi-β-CD was prepared by freeze-drying method. Stoichiometric ratio of 1:1 M was established by continuous variation (Job’s) plot. The stability constant of complex as determined by phase solubility study was found to be 15,871.3 M−1. Complex was characterized by FTIR, DSC, DTA and dissolution study. Results revealed that in complex the drug no longer exist in crystalline state and is converted into amorphous form; which shows higher dissolution efficiency as compared to crystalline drug. The solubilizing efficiency for freeze dried complex was found to be 175.57 and the relative drug crystallinity degree was 87.91% as estimated by thermal analysis. Complexation led to decrease in surface tension; from 54.8 dynes/cm (pure gefitinib) to 40.3 dynes/cm (FD complex) due to adsorption phenomenon. The results obtained in this study confirmed that complexation of gefitinib with Epi-β-CD is a prominent approach and suitable tool for tailoring the issue related to its delivery and can be explored for development of an effective delivery system.

Gallium ferredoxin as a tool to study the effects of ferredoxin binding to photosystem I without ferredoxin reduction.

Reduction of ferredoxin by photosystem I (PSI) involves the [4Fe-4S] clusters FA and FB harbored by PsaC, with FB being the direct electron transfer partner of ferredoxin (Fd). Binding of the redox-inactive gallium ferredoxin to PSI was investigated by flash-absorption spectroscopy, studying both the P700+ decay and the reduction of the native iron Fd in the presence of FdGa. FdGa binding resulted in a faster recombination between P700+ and (FA, FB)-, a slower electron escape from (FA, FB)- to exogenous acceptors, and a decreased amount of intracomplex FdFe reduction, in accordance with competitive binding between FdFe and FdGa. [FdGa] titrations of these effects revealed that the dissociation constant for the PSI:FdGa complex is different whether (FA, FB) is oxidized or singly reduced. This difference in binding, together with the increase in the recombination rate, could both be attributed to a c. -30mV shift of the midpoint potential of (FA, FB), considered as a single electron acceptor, due to FdGa binding. This effect of FdGa binding, which can be extrapolated to FdFe because of the highly similar structure and the identical charge of the two Fds, should help irreversibility of electron transfer within the PSI:Fd complex. The effect of Fd binding on the individual midpoint potentials of FA and FB is also discussed with respect to the possible consequences on intra-PSI electron transfer and on the escape process.

Structural basis for the isotype-specific interactions of ferredoxin and ferredoxin: NADP+ oxidoreductase: an evolutionary switch between photosynthetic and heterotrophic assimilation

In higher plants, ferredoxin (Fd) and ferredoxin-NADP+ reductase (FNR) are each present as distinct isoproteins of photosynthetic type (leaf type) and non-photosynthetic type (root type). Root-type Fd and FNR are considered to facilitate the electron transfer from NADPH to Fd in the direction opposite to that occurring in the photosynthetic processes. We previously reported the crystal structure of the electron transfer complex between maize leaf FNR and Fd (leaf FNR:Fd complex), providing insights into the molecular interactions of the two proteins. Here we show the 2.49Å crystal structure of the maize root FNR:Fd complex, which reveals that the orientation of FNR and Fd remarkably varies from that of the leaf FNR:Fd complex, giving a structural basis for reversing the redox path. Root FNR was previously shown to interact preferentially with root Fd over leaf Fd, while leaf FNR retains similar affinity for these two types of Fds. The structural basis for such differential interaction was investigated using site-directed mutagenesis of the isotype-specific amino acid residues on the interface of Fd and FNR, based on the crystal structures of the FNR:Fd complexes from maize leaves and roots. Kinetic and physical binding analyses of the resulting mutants lead to the conclusion that the rearrangement of the charged amino acid residues on the Fd-binding surface of FNR confers isotype-specific interaction with Fd, which brings about the evolutional switch between photosynthetic and heterotrophic redox cascades.

Mechanostability of the Single-Electron-Transfer Complexes of Anabaena Ferredoxin-NADP(+) Reductase.

The complexes formed between the flavoenzyme ferredoxin-NADP(+) reductase (FNR; NADP(+) =nicotinamide adenine dinucleotide phosphate) and its redox protein partners, ferredoxin (Fd) and flavodoxin (Fld), have been analysed by using dynamic force spectroscopy through AFM. A strategy is developed to immobilise proteins on a substrate and AFM tip to optimise the recognition ability. The differences in the recognition efficiency regarding a random attachment procedure, together with nanomechanical results, show two binding models for these systems. The interaction of the reductase with the natural electron donor, Fd, is threefold stronger and its lifetime is longer and more specific than that with the substitute under iron-deficient conditions, Fld. The higher bond probability and two possible dissociation pathways in Fld binding to FNR are probably due to the nature of this complex, which is closer to a dynamic ensemble model. This is in contrast with the one-step dissociation kinetics that has been observed and a specific interaction described for the FNR:Fd complex.

Specification of reproductive meristems requires the combined function of SHOOT MERISTEMLESS and floral integrators FLOWERING LOCUS T and FD during Arabidopsis inflorescence development

In Arabidopsisfloral meristems are specified on the periphery of the inflorescence meristem by the combined activities of the FLOWERING LOCUS T (FT)–FD complex and the flower meristem identity gene LEAFY. The floral specification activity of FT is dependent upon two related BELL1-like homeobox (BLH) genes PENNYWISE (PNY) and POUND-FOOLISH (PNF) which are required for floral evocation. PNY and PNF interact with a subset of KNOTTED1-LIKE homeobox proteins including SHOOT MERISTEMLESS (STM). Genetic analyses show that these BLH proteins function with STM to specify flowers and internodes during inflorescence development. In this study, experimental evidence demonstrates that the specification of flower and coflorescence meristems requires the combined activities of FT–FD and STM. FT and FD also regulate meristem maintenance during inflorescence development. In plants with reduced STM function, ectopic FT and FD promote the formation of axillary meristems during inflorescence development. Lastly, gene expression studies indicate that STM functions with FT–FD and AGAMOUS-LIKE 24 (AGL24)–SUPPRESSOR OF OVEREXPRESSION OF CONTANS1 (SOC1) complexes to up-regulate flower meristem identity genes during inflorescence development

Regulatory networks that function to specify flower meristems require the function of homeobox genes PENNYWISE and POUND‐FOOLISH in Arabidopsis

Flowering is a major developmental phase change that transforms the fate of the shoot apical meristem (SAM) from a leaf-bearing vegetative meristem to that of a flower-producing inflorescence meristem. In Arabidopsis, floral meristems are specified on the periphery of the inflorescence meristem by the combined activities of the FLOWERING LOCUS T (FT)-FD complex and the flower meristem identity gene, LEAFY (LFY). Two redundant functioning homeobox genes, PENNYWISE (PNY) and POUND-FOOLISH (PNF), which are expressed in the vegetative and inflorescence SAM, regulate patterning events during reproductive development, including floral specification. To determine the role of PNY and PNF in the floral specification network, we characterized the genetic relationship of these homeobox genes with LFY and FT. Results from this study demonstrate that LFY functions downstream of PNY and PNF. Ectopic expression of LFY promotes flower formation in pny pnf plants, while the flower specification activity of ectopic FT is severely attenuated. Genetic analysis shows that when mutations in pny and pnf genes are combined with lfy, a synergistic phenotype is displayed that significantly reduces floral specification and alters inflorescence patterning events. In conclusion, results from this study support a model in which PNY and PNF promote LFY expression during reproductive development. At the same time, the flower formation activity of FT is dependent upon the function of PNY and PNF.

A single in vivo-selected point mutation in the active center of Toxoplasma gondii ferredoxin-NADP + reductase leads to an inactive enzyme with greatly enhanced affinity for ferredoxin

Electron transfer between plant-type [2Fe–2S] ferredoxin (Fd) and ferredoxin-NADP + reductase (FNR) depends on the physical interaction between both proteins. We have applied a random mutagenesis approach with subsequent in vivo selection using the yeast two-hybrid system to obtain mutants of Toxoplasma gondii FNR with higher affinity for Fd. One mutant showed a 10-fold enhanced binding using affinity chromatography on immobilized Fd. A single serine-to-arginine exchange in the active site was responsible for its increased affinity. The mutant reductase was also enzymatically inactive. Homology modeling of the mutant FNR–Fd complex predicts substantial alterations of protein–FAD interactions in the active site of the enzyme with subsequent structural changes. Collectively, for the first time a point mutation in this important class of enzymes is described which leads to greatly enhanced affinity for its protein ligand.

Electrostatic and Hydrophobic Interactions during Complex Formation and Electron Transfer in the Ferredoxin/Ferredoxin:NADP+Reductase System fromAnabaena

Transient kinetics and protein-protein binding measurements over a wide range of ionic strength (I) have been used to characterize the interactions occurring during complex formation and electron transfer (et) between recombinant ferredoxin (Fd) and both native and recombinant ferredoxin: NADP{sup +} reductase (FNR) from the cyanobacterium Anabaena. Between I = 12 mM and I = 100 mM, the dissociation constant (K{sub d}) for the complex formed between oxidized Fd and oxidized native FNR increases from 4.5 to 8.1 {mu}M, whereas K{sub d} for the Fd complex with recombinant FNR increases from 0.3 to 3.3 {mu}M. For both pairs of proteins, the ionic strength dependence of k{sub obs} for the et reaction is biphasic. Electrostatic analysis of the kinetic data above I = 100 mM allows a prediction of the ionic strength dependence of the K{sub d} values, if electrostatic interactions are the only determinant of complex stability. The predicted dependence is dramatically larger than the observed one, indicating that hydrophobic interactions make an important contribution to complex stability. The differences in binding between native and recombinant FNR are ascribed to proteolytic cleavage at the N-terminus, which occurs during preparation of the native enzyme and which removes two positively charged residues,more » thereby decreasing the electrostatic interactions with Fd. 39 refs., 4 figs., 1 tab.« less

Effect of Ferredoxin on the Diaphorase Activity of Cyanobacterial Ferredoxin-NADP Reductase

The interaction of ferredoxin-NADP reductase (FNR) and ferredoxin (Fd) results in an enhanced rate of reaction and a shift of the pH optimum for the FNR-mediated diaphorase reaction. Low concentrations of NaCl (<100 millimolar), favorable for formation of the FNR:Fd complex, further magnify the alteration of the diaphorase reaction; the activity is enhanced 3-fold and pH optimum is shifted from 9.5 to 7.8. The Fd-stimulated diaphorase activity of FNR may result either from a conformational change of the enzyme and/or from a transition from a two electron to a one electron reaction.

A nucleoprotein complex in bacteria infected with Pf1 filamentous virus: Identification and electron microscopic analysis

We report the discovery, partial purification, and high-resolution electron microscopic characterization of an intracellular complex from Pseudomonas aeruginosa bacteria infected by Pf1 filamentous virus. The Pf1 complex resembles the virion precursor complex of DNA and viral gene 5 protein formed by fd virus of Escherichia coli, but the two complexes differ in structure. Image reconstruction indicates that both complexes are single-start morphological helices; specimen tilting shows the Pf1 helix to be right-handed. Although the Pf1 and fd complexes contain a similar number of nucleotides per axial unit length, the mean distance between helical turns is 61 Å for Pf1 but 91 Å for fd under the conditions used for our measurements; two turns of the fd nucleoprotein helix contain about as many nucleotides as do three turns of the Pf1 helix. The Pf1 complex is much shorter than are Pf1 virions, in contrast to the similar lengths of the fd virion and complex. The fd complex is extremely flexible, but the Pf1 complex is more highly regular in structure. Most significant, calculations based on our data indicate that the DNA in the Pf1 complex is probably located at a smaller radius than in the bulk of the protein. If the DNA and morphological helices coincide, the DNA in the Pf1 complex must be well inside of (axial to) the outeer protein surfaces of the complex, rather than being wrapped around the protein subunits as proposed by others for fd complex.

A nucleoprotein complex in bacteria infected with Pf1 filamentous virus: Isolation and biochemical characterization

A nucleoprotein complex has been isolated from Pseudomonas aeruginosa infected with filamentous virus Pf1, and purified to homogeneity. The complex has a sedimentation coefficient of 48 S in low ionic strength and neutral pH. It consists of single-stranded circular viral DNA combined with about 1500 copie of a virus-specific protein weighing about 16,500 daltons. The complex is similar to one containing viral DNA and gene 5 protein of virus fd. However, the Pf1 DNA-binding protein has a mass about 1.7-fold greater than that of the analogous fd protein, and the Pf1 complex is considerably more resistant to dissociation by high ionic strength than the fd complex. Although the amino acid composition of the Pf1 protein is quite different from that of the fd protein, the two proteins have about the same number of aromatic amino acids, which are thought to be involved in binding single-stranded DNA.

Carbamylation with cyanate of basic phospholipase A 2 from the venom of Naja nigricollis (Spitting cobra)

The most basic and toxic phospholipase A 2 purified from Naja nigricollis venom was subjected to lysine modification with cyanate at pH 8.0. The carbamylated derivatives were separated by chromatography on a column of SP-Sephadex C-25 and five fractions were obtained. Amino acid analysis showed that the numbers of Lys-residues modified for fractions Fa to Fe were 10, 9.2, 9.0, 8.2 and 7.4 respectively. The pI values decrease with increasing carbamylation, converting the basic enzyme into an acidic protein. It is noteworthy that even after 9 out of 10 Lys-residues had been modified (Fc) and the pI of the enzyme decreased from 10.6 to 4.4, the enzyme still possessed 75% of its enzymatic activity and 51% of its antigenicity. Although both native and Lys-modified N. nigricollis phospholipase A 2 were perturbed by the presence of Ca 2+, the difference spectra of Lys-modified Fd differ greatly from that of the native enzyme and become similar to that of the acidic and less toxic phospholipase A 2 from N. naja atra venom. At pH 8.0, the effect of Ca 2+ on the fluorescence emission intensity of 8-anilinonaphthalenesulfonate-Lys-modified Fd complex was different from that of 8-anilinonaphthalenesulfonate-native N. nigricollis phospholipase A 2 complex and was similar to that of 8-anilinonaphthalenesulfonate- N. naja atra phospholipase A 2 complex. It is clear that the different conformational changes induced by Ca 2+ might be attributable to the charge properties of the enzyme, which depend on the pH of the solution and the pI of the enzyme. These data are discussed in relationship to the decreased lethal potency and pharmacological effects induced by carbamylation ( Condrea et al., 1981b).