A comparative study of the thermal stability of plastocyanin, cytochrome c6 and Photosystem I in thermophilic and mesophilic cyanobacteria

Cytochrome c₆ (Cyt) from the thermophilic cyanobacterium Phormidium laminosum has been purified and characterized. It is a mildly acidic protein, with physicochemical properties very similar to those of plastocyanin (Pc). This is in agreement with the functional interchangeability of the two metalloproteins as electron donors to Photosystem I (PS I). The kinetic analyses of the interaction of Pc and Cyt with Photosystem I show that both metalloproteins reduce PS I with similar efficiencies, according to an oriented collisional kinetic model involving repulsive electrostatic interactions. The thermostability study of the Phormidium Pc/PS I system compared with those from mesophilic cyanobacteria (Synechocystis, Anabaena and Pseudanabaena) reveals that Pc is the partner limiting the thermostability of the Phormidium couple. The cross-reactions between Pc and PS I from different organisms demonstrate not only that Phormidium Pc enhances the stability of the Pc/PS I system using PS I from mesophilic cyanobacteria, but also that Phormidium PS I possesses a higher thermostability than the other photosystems. This revised version was published online in June 2006 with corrections to the Cover Date.     

Gene Control in Somatic Embryos of Digitalis lanata: Expression of the β-Glucuronidase Gene Fused to a Plastocyanin Promoter

Digitalis lanata was transformed by agrobacteria-mediated gene transfer with a chimeric reporter gene encoding for β-glucuronidase (CUS) from Escherichia coll under the control of the plastocyanin 3 (Pc3) promoter from Spinada oleracea (Pc3::uidA fusion gene). Transformed cell lines were regenerated to plants via somatic embryos. CUS activity was determined fluorometrically and histochemically. The Pc3::uidA fusion gene was expressed in the late globular and bipolar stages of somatic embryos. Expression started in globular embryos (stage-1-globules) in that part of the parenchymatic tissue which later on formed the cotyledons. No GUS activity was detectable in the parenchymatic tissue forming the root pole, in cells of the developing procambium or in epidermal cells. These tissues were free of GUS activity also in bipolar embryos. The parenchymatic cells of the cotyledons and the primary cortex of the hypocotyl of germinating embryos showed GUS activity, in contrast to the epidermal cells and the cells of the central cylinder.     

Developmentally regulated plant genes: the nucleotide sequence of a wheat gliadin genomic clone

Gliadins, the major wheat seed storage proteins, are encoded by a multigene family. Northern blot analysis shows that gliadin genes are transcribed in endosperm tissue into two classes of poly(A)+ mRNA, 1400 bases (class I) and 1600 bases (class II) in length. Using poly(A)+ RNA from developing wheat endosperm we constructed a cDNA library from which a number of clones coding for alpha/beta and gamma gliadins were identified by hybrid-selected mRNA translation and DNA sequencing. These cDNA clones were used as probes for the isolation of genomic gliadin clones from a wheat genomic library. One such genomic clone was characterized in detail and its DNA sequence determined. It contains a gene for a 33-kd alpha/beta gliadin protein (a 20 amino acid signal peptide and a 266 amino acid mature protein) which is very rich in glutamine (33.8%) and proline (15.4%). The gene sequence does not contain introns. A typical eukaryotic promoter sequence is present at -104 (relative to the translation initiation codon) and there are two normal polyadenylation signals 77 and 134 bases downstream from the translation termination codon. The coding sequence contains some internal sequence repetition, and is highly homologous to several alpha/beta gliadin cDNA clones. Homology to a gamma-gliadin cDNA clone is low, and there is no homology with known glutenin or zein cDNA sequences.     

Three-dimensional model of stellacyanin and its implications for electron transfer reactivity

Experimental data were combined with computational methods in constructing a hypothetical three-dimensional model for the blue single copper protein Rhus stellacyanin (St). The known sequence of stellacyanin and its homology with plastocyanin (Pc) were used together with the results of spectroscopic studies of the protein that yielded the current assignment of two histidines, one cysteine and a disulfide sulfur as copper ligands in stellacyanin. By computer graphics and energy minimization the folding of the protein was predicted. The model structure is somewhat less regular than Pc as judged by surface area and energy comparisons, but it is a stable structure. Besides rotation of one imidazole ring the copper site undergoes no change even in the absence of the copper ion and the model shows that the site can be constructed with the four assumed copper ligands without forming a strained system. The structure also indicates that a carbonyl oxygen atom is near the copper, thus the site may have analogy to the Alcaligenes denitrificans azurin (Az) site, although the amino acid sequence is more homologous to that of Pc. The model indicates that aspartate 49, reductively labeled by Cr(III), is near the copper center and homologous to the site labeled by Cr(III) on Pc. Also homologous to Pc is a tyrosine residue adjacent to the aspartate. This tyrosine has been implicated in Pc electron transfer and thus is probably involved in electron transfer reactivity of St as well. The higher reactivity of St with small-molecule redox reagents compared to Az and Pc, may be due to the proximity of the above-mentioned aspartate 49 to the Cu, or the greater exposure of one of the Cu cysteine ligands, in the predicted structure as compared to that in the known Pc and Az structures.     

Import into chloroplasts of a yeast mitochondrial protein directed by ferredoxin and plastocyanin transit peptides

Many chloroplast proteins are synthesized in the cytoplasm as precursors which contain an amino terminal transit peptide. These precursors are subsequently imported into chloroplast and targeted to one of several organellar locations. This import is mediated by the transit peptide, which is cleaved off during import. We have used the transit peptides of ferredoxin (chloroplast stroma) and plastocyanin (thylakoid lumen) to study chloroplast protein import and intra-organellar routing toward different compartments. Chimeric genes were constructed that encode precursor proteins in which the transit peptides are linked to yeast mitochondrial manganese superoxide dismutase. Chloroplast protein import and localization experiments show that both chimeric proteins are imported into the chloroplast stroma and processed. The plastocyanin transit sequence did not direct superoxide dismutase to the thylakoids; this protein was found in the stroma as an intermediate that still contains part of the plastocyanin transit peptide. The organelle specificity of these chimeric precursors reflected the transit peptide parts of the molecules, because neither the ferredoxin and plastocyanin precursors nor the chimeric proteins were imported into isolated yeast mitochondria.     

Expression of spinach plastocyanin in E. coli

An expression vector designed for overexpression of plastocyanin in the periplasmic space of E. coli has been developed. The vector contains the signal peptide sequence of Pseudomonas aeruginosa azurin and the mature sequence of spinach plastocyanin. The precursor is efficiently translocated to the periplasmic space and correctly processed to mature plastocyanin. No detectable amount of plastocyanin was present in the cytoplasmic or in the membrane fraction. A large scale preparation of the recombinant plastocyanin in a 20 litre fermentor yielded approximately 30 mg of pure plastocyanin. The recombinant protein obtained from E. coli shows CD, EPR and optical properties identical to plastocyanin isolated from spinach.     

Folding of peptide fragments comprising the complete sequence of proteins. Models for initiation of protein folding. II. Plastocyanin.

In an attempt to understand the earliest events in the protein folding pathway, the complete sequence of French bean plastocyanin has been synthesized as a series of short peptide fragments, and the conformational preferences of each peptide examined in aqueous solution using proton n.m.r. methods. Plastocyanin consists largely of beta-sheet, with reverse turns and loops between the strands of the sheet, and one short helix. The n.m.r. experiments indicate that most of the peptides derived from the plastocyanin sequence have remarkably little propensity to adopt folded conformations in aqueous solution, in marked contrast to the peptides derived from the helical protein, myohemerythrin (accompanying paper). For most plastocyanin peptides, the backbone dihedral angles are predominantly in the beta-region of conformational space. Some of the peptides show weak NOE connectivities between adjacent amide protons, indicative of small local populations of backbone conformations in the a region of (phi,psi) space. A conformational preference for a reverse turn is seen in the sequence Ala65-Pro-Gly-Glu68, where a turn structure is found in the folded protein. Significantly, the peptide sequences that populate the alpha-region of (phi,psi) space are mostly derived from turn and loop regions in the protein. The addition of trifluoroethanol does not drive the peptides into helical conformations. In one region of the sequence, the n.m.r. spectra provide evidence of the formation of a hydrophobic cluster involving aromatic and aliphatic side-chains. These results have significance for understanding the initiation of protein folding. From these studies of the fragments of plastocyanin (this paper) and myohemerythrin (accompanying paper), it appears that there is a pre-partitioning of the conformational space sampled by the polypeptide backbone that is related to the secondary structure in the final folded state.     

Cloning and comparative bioinformatic analysis of feline glucose-6-phosphatase catalytic subunit cDNA.

Glucose-6-phosphatase is a multicomponent enzyme composed of a transporter subunit and a catalytic subunit that is involved in hepatic glucose production. The objective of the present study was to determine the complete nucleotide sequence of feline hepatic glucose-6-phosphatase catalytic subunit (G6Pc) cDNA and to perform comparative analysis of the molecular features of the feline G6Pc cDNA and protein. Feline G6Pc cDNA contains 2261 bases and encodes a 357 aa protein. The feline cDNA and protein are highly conserved with overall identity ranging from 73-86% to 86-95%, respectively, among mammalian species. Membrane topology, phosphatase consensus sequence, ER retention sequence, N-glycosylation sites and active site residues are conserved in the feline protein. Analysis of the putative feline G6Pc protein did not reveal any species-specific features to explain the unusual in vivo regulation of G6Pase activity reported in feline liver.     

Identification and Characterization of Putative Receptors for Sperm-Activating Peptide I (SAP-I) in Spermatozoa of the Sea Urchin Hemicentrotus pulcherrimus1

We characterized putative receptors specific for sperm-activating peptide I (SAP-I: GFDLNGGGVG) in spermatozoa of the sea urchin Hemicentrotus pulcherrimus, using both binding and crosslinking techniques. Analysis of the data obtained from the equilibrium binding of a radioiodinated SAP-I analogue [GGGY(¹²⁵I)-SAP-I] to H. pulcherrimus spermatozoa showed the presence of two classes of receptors specific for SAP-I in the spermatozoa. The incubation of intact spermatozoa as well as sperm tails or sperm membranes prepared from H. pulcherrimus spermatozoa with GGGY(¹²⁵I)-SAP-I and a chemical crosslinking reagent, disuccinimidyl suberate, resulted in the radiolabelling of a 71 kDa protein. The protein appears to be associated with a 220 kDa wheat germ agglutinin (WGA)-binding protein. A cDNA encoding the 71 kDa protein was isolated from a H. pulcherrimus testis cDNA library. The cDNA was 2443 bp long and an open reading frame predicted a protein of 532 amino acids containing a 30-residue amino-terminal signal peptide, followed by the same sequence as the N-terminal sequence of the 71 kDa protein. The amino acid sequence of the matured 71 kDa protein is strikingly similar to the 77 kDa protein of Strongylocentrotus purpuratus (95.5% identical) and also similar to cysteine rich domain of a human macrophage scavenger receptor. Northern blot analysis demonstrated that mRNA of 2.6 kb encoding the 71 kDa protein was expressed only in the testis.     

Identification of the plastocyanin binding subunit of photosystem I

Plastocyanin is specifically cross-linked by incubation with N-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) to a subunit of photosystem I in stroma lamellae and in isolated photosystem I complex. SDS-PAGE shows the disappearance of a 18.5 kDa subunit and the appearance of a new 31.5 kDa protein which was recognized by anti-plastocyanin antibodies. The isolated subunit was identified by its N-terminal amino acid sequence as the mature peptide coded by the nuclear gene psaF [Steppuhn et al. (1988) FEBS Lett. 237, 218–224]. P700⁺ was reduced by cross-linked plastocyanin with the same halftime of 13 μs as found in the native complex. This is evidence that cross-linking conserved the orientation of the complex and that the 18.5 kDa subunit provides the conformation of photosystem I necessary for the extremely rapid electron transfer from plastocyanin to P700⁺.     

A gene coding for an RPS2 protein is present in the mitochondrial genome of several cereals, but not in dicotyledons

A gene coding for a protein that shows homologies to prokaryotic ribosomal protein S2 is present in the mitochondrial (mt) genome of wheat (Triticum aestivum). The wheat gene is transcribed as a single mRNA which is edited by C-to-U conversions at seven positions, all resulting in alteration of the encoded amino acid. Homologous gene sequences are also present in the mt genomes of rice and maize, but we failed to identify the corresponding sequences in the mtDNA of all dicotyledonous species tested; in these species the mitochondrial RPS2 is probably encoded in the nucleus. The protein sequence deduced from the wheat rps2 gene sequence has a long C-terminal extension when compared to other prokaryotic RPS2 sequences. This extension presents no similarity with any known sequence and is not conserved in the maize or rice mitochondrial rps2 gene. Most probably, after translation, this peptide extension is processed by a specific peptidase to give rise to the mature wheat mitochondrial RPS2. Received: 20 November 1997 / Accepted: 29 January 1998     

Structural and antifungal properties of a pathogenesis-related protein from wheat kernel

We have purified and characterized a protein from the water-soluble fraction of wheat kernel (Triticum aestivum cv. S. Pastore) consisting of a single polypeptide chain blocked at its N-terminus by a pyroglutamate residue; the complete amino acid sequence has been determined by automated sequence analysis performed on peptide fragments obtained by enzymatic hydrolyses of the protein. Homology studies have shown that this protein is very similar (97% sequence identity) to the previously characterized wheatwin1 as well as to other members of the pathogenesis-related (PR) proteins of class 4; in analogy with wheatwin1, we have termed this protein wheatwin2. Both wheatwin1 and wheatwin2 have specific antifungal activity toward the wide-host-range pathogenBotrytis cinerea and the wheat-specific pathogenic fungi of wheatFusarium culmorum andFusarium graminearum of groups 1 and 2. On the basis of their structural and functional properties, wheatwin1 and wheatwin2 can be classified as members of the PR4 protein family; this represents the first report concerning the presence of this kind of protein in wheat.     

Competitive inhibition of electron donation to photosystem 1 by metal-substituted plastocyanin

The electron transfer from wild-type spinach plastocyanin (Pc) to photosystem 1 has been studied by flash-induced absorption changes at 830 nm. The decay kinetics of photo-oxidized P700 are drastically slower in the presence of Ag(I)-substituted Pc, while addition of Zn(II)-substituted Pc has a weaker effect. The metal-substituted forms of Pc act as competitive inhibitors of the reaction between normal, Cu-containing, Pc and P700. The inhibition constants obtained from an analysis of the kinetic data were 30 and 410 μM for Ag(I)- and Zn(II)-substituted Pc, respectively. When the Gly8Asp mutant form of Pc was used instead of the wild-type form, the corresponding values were found to be 77 and 442 μM. If the Ag- and Zn-derivatives can be considered as structural mimics of reduced and oxidized CuPc, respectively, our results imply that there is a redox-induced decrease in the affinity between Pc and photosystem 1 that follows the electron donation to P700. Our data also imply that the Gly8Asp mutation can diminish the magnitude of this change. The findings reported here are consistent with a reaction mechanism where the electron transfer in the complex between Pc and photosystem 1 is assumed to be reversible.     

Ionic strength and pH dependence of the reaction between plastocyanin and Photosystem 1. Evidence of a rate-limiting conformational change

The electron-transfer reaction between spinach wild-type plastocyanin (Pc(WT)) two site-directed mutants, Pc(Thr79His) and Pc(Lys81His), and spinach Photosystem 1 particles, has been studied as a function of protein concentration, ionic strength and pH by using laser-flash absorption spectroscopy. The kinetic data are interpreted using the simplest possible three-step model, involving a rate-limiting conformational change preceding intracomplex electron transfer. The three proteins show similar concentration, pH and ionic strength dependencies. The effects of ionic strength and pH on the reaction indicate a strong influence of complementary charges on complex formation and stabilization. Studies with apoprotein support the opinion that the hydrophobic patch is critical for an productive interaction with the reaction center of Photosystem 1. Together with earlier site-directed mutagenesis studies, the absence of a detectable Photosystem 1 reaction in the presence of reduced azurin, stellacyanin, cytochrome c and cytochrome c₅₅₁, demonstrates the existence of a high level of specificity in the protein-protein interface in the formation of an efficient electron-transfer complex.     

Redox- and pH-dependent association of plastocyanin with lipid bilayers: effect on protein conformation and thermal stability

The effect of electrostatic interactions on the conformation and thermal stability of plastocyanin (Pc) was studied by infrared spectroscopy. Association of any of the two redox states of the protein with positively charged membranes at neutral pH does not significantly change the secondary structure of Pc. However, upon membrane binding, the denaturation temperature decreases, regardless of the protein redox state. The extent of destabilization depends on the proportion of positively charged lipid headgroups in the membrane, becoming greater as the surface density of basic phospholipids increases. In contrast, at pH 4.8 the membrane binding-dependent conformational change becomes redox-sensitive. While the secondary structures and thermal stabilities of free and membrane-bound oxidized Pc are similar under acidic conditions, the conformation of the reduced form of the protein drastically rearranges upon membrane association. This rearrangement does not depend on electrostatic interactions to occur, since it is also observed in the presence of uncharged lipid bilayers. The conformational transition, only observed for reduced Pc, involves the exposure of hydrophobic regions that leads to intermolecular interactions at the membrane surface. Membrane-mediated partial unfolding of reduced Pc can be reversed by readjusting the pH to neutrality, in the absence of electrostatic interactions. This redox-dependent behavior might reflect specific structural requirements for the interaction of Pc with its redox partners.     

Antibacterial Activity of Synthetic Peptides Against Plant Pathogenic Pectobacterium Species

The aim of the work was to check the antibacterial activity of three synthetic peptides: CAMEL, Iseganan and Pexiganan as well as their possible application against plant pathogenic bacteria from the species Pectobacterium carotovorum (Pc) and Pectobacterium chrysanthemi (Pch). The antibacterial activity of the three chosen synthetic peptides was evaluated with the use of two tests: minimal inhibitory concentration and minimal bactericidal concentration. The CAMEL proved to be the most effective peptide, inhibiting the growth of different species of Pectobacterium in concentrations ranging from 2 to 8 μg/ml. Iseganan and Pexiganan also demonstrated activity against Pectobacterium sp., but it was lower than CAMEL. The CAMEL was able to inhibit Pc and Pch bacterial growth and tissue maceration in pathogenicity tests performed on potato tuber slices.