Characterization of isolated photosystem I from Halomicronema hongdechloris,a chlorophyll f-producing cyanobacterium

Photosynthetica - Halomicronema hongdechloris is a chlorophyll (Chl) f-producing cyanobacterium. Chl f biosynthesis is induced under far-red light, extending its photosynthetically active radiation...     

Synthesis of a photosystem I polypeptide of 15 kilodaltons in isolated etiochloroplasts of wheat

Etiochloroplasts isolated from greening wheat (Triticum aestivum L. cv Norin 61) seedlings synthesized a membrane polypeptide of 15 kilodaltons. One-dimensional peptide mapping with Staphylococcus aureus V8 protease revealed that the 15 kilodaltons polypeptide is the subunit 5 of photosystem I reaction center complex.     

Effect of surfactants on apparent oxygen consumption of photosystem I isolated from Arthrospira platensis

Surfactants play a significant role in solubilization of photosystem I (PSI) in vitro. Triton X-100 (TX), n-Dodecyl-β-d-maltoside (DDM), and sodium dodecyl sulfate (SDS) were employed to solubilize PSI particles in MES buffer to compare the effect of surfactant and its dosage on the apparent oxygen consumption rate of PSI. Through a combined assessment of sucrose density gradient centrifugation, Native PAGE and 77 K fluorescence with the apparent oxygen consumption, the nature of the enhancement of the apparent oxygen consumption activity of PSI by surfactants has been analyzed. Aggregated PSI particles can be dispersed by surfactant molecules into micelles, and the apparent oxygen consumption rate is higher for surfactant-solubilized PSI than for integral PSI particles. For DDM, PSI particles are solubilized mostly as the integral trimeric form. For TX, PSI particles are solubilized as incomplete trimeric and some monomeric forms. For the much harsher surfactant, SDS, PSI particles are completely solubilized as monomeric and its subunit forms. The enhancement of the oxygen consumption rate cannot be explained only by the effects of surfactant on the equilibrium between monomeric and trimeric forms of solubililized PSI. Care must be taken when the electron transfer activity of PSI is evaluated by methods based on oxygen consumption because the apparent oxygen consumption rate is influenced by uncoupled chlorophyll (Chl) from PSI, i.e., the larger the amount of uncoupled Chl, the higher the rate of apparent oxygen consumption. 77 K fluorescence spectra can be used to ensure that there is no uncoupled Chl present in the system. In order to eliminate the effect of trace uncoupled Chl, an efficient physical quencher of ¹O₂, such as 1 mM NaN₃, may be added into the mixture.     

Membrane targeting of a bacterial virulence factor harbouring an extended signal peptide

Filamentous haemagglutinin (FHA) is the major adhesin of Bordetella pertussis, the whooping cough agent. FHA is synthesized as a 367-kDa precursor harbouring a remarkably long signal peptide with an N-terminal extension that is conserved among related virulence proteins. FHA is secreted via the two-partner secretion pathway that involves transport across the outer membrane by a cognate transporter protein. Here we have analyzed the mechanism by which FHA is targeted to, and translocated across, the inner membrane. Studies were performed both in vitro using Escherichia coli inside-out inner membrane vesicles and in vivo by pulse-chase labelling of Bordetella pertussis cells. The data collectively indicate that like classical periplasmic and outer membrane proteins, FHA requires SecA and SecB for its export through the SecYEG translocon in the inner membrane. Although short nascent chains of FHA were found to cross-link to signal recognition particle (SRP), we did not obtain indication for an SRP-dependent, co-translational membrane targeting provoked by the FHA signal sequence. Our results rule out that the extended signal peptide of FHA determines a specific mode of membrane targeting but rather suggest that it might influence the export rate at the inner membrane.     

Electrochemical and spectro-kinetic evidence for an intermediate electron acceptor in photosystem I

Absorption changes accompanying light-induced P-700 formation and its decay in the dark at 15 K in Photosystem-I particles poised at various redox potentials have been examined. In unpoised samples, the light-induced absorption change is practically irreversible. At increasingly negative potentials, an increasing fraction of the absorption change, proportional to the fraction of bound iron-sulfur protein chemically reduced, becomes reversible, and the titration curve has a midpoint potential of --530 mV (vs. normal hydrogen electrode). At --66 mV, the P-700 absorption change is 97% reversible. The total P-700-signal amplitude decreases over the same potential span and levels off at about 43% (to slightly over 50% at a substantially higher excitation intensity). These results provide additional support to previous suggestions of an existence of an intermediate electron acceptor located between the primary donor, P-700, and the more stable primary electron acceptor (P-430 or bound iron-sulfur protein).     

Studies on photosystem I. Characteristics of "310 material" isolated from spinach chloroplasts

Exposure of osmotically shocked chloroplasts to dilute pyridine and sonic oscillation results in the extraction of a small molecular-weight factor. Purification of the factor was accomplished using gel filtration chromatography. Due to the spectral nature of the purified species (λ_max at 310 nm) the factor was named “310 material.”Physiologically, the 310 material was found to inhibit a variety of ferredoxin-dependent photoreductions catalyzed by isolated spinach chloroplasts but stimulate both pseudocyclic photophosporylation and the ferredoxin-independent photoreduction of mammalian cytochrome c. The latter reaction was found to involve, at least partially, the formation of a Superoxide radical. Dark-reduction studies have further established that the 310 material is an autooxidizable electron carrier.Chemically, the 310 material is a water-soluble, low molecular-weight phenolic-type compound; possibly a derivative of coumaric acid. No proteinaceous material is observed in physiologically active preparations of 310 material.Based on these findings, it is concluded that the isolated 310 material acts on the reducing side of Photosystem I at or near the site of reduction of ferredoxin and competes with ferredoxin for the reducing power generated by the Photosystem I reaction center. The exact physiological role of the 310 material in the intact photosynthetic system, however, remains unknown.The similarities between the 310 material and a variety of other factors previously isolated from chloroplasts are discussed.     

A urokinase-type plasminogen activator-inhibiting cyclic peptide with an unusual P2 residue and an extended protease binding surface demonstrates new modalities for enzyme inhibition

To find new principles for inhibiting serine proteases, we screened phage-displayed random peptide repertoires with urokinase-type plasminogen activator (uPA) as the target. The most frequent of the isolated phage clones contained the disulfide bridge-constrained sequence CSWRGLENHRMC, which we designated upain-1. When expressed recombinantly with a protein fusion partner, upain-1 inhibited the enzymatic activity of uPA competitively with a temperature and pH-dependent K(i), which at 25 degrees C and pH 7.4 was approximately 500 nm. At the same conditions, the equilibrium dissociation constant K(D), monitored by displacement of p-aminobenzamidine from the specificity pocket of uPA, was approximately 400 nm. By an inhibitory screen against other serine proteases, including trypsin, upain-1 was found to be highly selective for uPA. The cyclical structure of upain-1 was indispensable for uPA binding. Alanine-scanning mutagenesis identified Arg(4) of upain-1 as the P(1) residue and indicated an extended binding interaction including the specificity pocket and the 37-, 60-, and 97-loops of uPA and the P(1), P(2), P(3)', P(4)', and the P(5)' residues of upain-1. Substitution with alanine of the P(2) residue, Trp(3), converted upain-1 into a distinct, although poor, uPA substrate. Upain-1 represents a new type of uPA inhibitor that achieves selectivity by targeting uPA-specific surface loops. Most likely, the inhibitory activity depends on its cyclical structure and the unusual P(2) residue preventing the scissile bond from assuming a tetrahedral geometry and thus from undergoing hydrolysis. Peptide-derived inhibitors such as upain-1 may provide novel mechanistic information about enzyme-inhibitor interactions and alternative methodologies for designing effective protease inhibitors.     

Conjugates between photosystem I and a carbon nanotube for a photoresponse device

Photosystem I (PS I) is a large pigment–protein complex embedded in the thylakoid membranes that performs light-driven electron transfer across the thylakoid membrane. Carbon nanotubes exhibit excellent electrical conductivities and excellent strength and stiffness. In this study, we generated PSI–carbon nanotube conjugates dispersed in a solution aimed at application in artificial photosynthesis. PS I complexes in which a carbon nanotube binding peptide was introduced into the middle of the PsaE subunit were conjugated on a single-walled carbon nanotube, orienting the electron acceptor side to the nanotube. Spectral and photoluminescence analysis showed that the PS I is bound to a single-walled carbon nanotube, which was confirmed by transmission electron microscopy. Photocurrent observation proved that the photoexcited electron originated from PSI and transferred to the carbon nanotube with light irradiation, which also confirmed its orientated conjugation. The PS I–carbon nanotube conjugate will be a useful nano-optoelectronic device for the development of artificial systems.     

Wiring an [FeFe]-hydrogenase with photosystem I for light-induced hydrogen production

A molecular wire is used to connect two proteins through their physiologically relevant redox cofactors to facilitate direct electron transfer. Photosystem I (PS I) and an [FeFe]-hydrogenase (H(2)ase) serve as the test bed for this new technology. By tethering a photosensitizer with a hydrogen-evolving catalyst, attached by Fe-S coordination bonds between the F(B) iron-sulfur cluster of PS I and the distal iron-sulfur cluster of H(2)ase, we assayed electron transfer between the two components via light-induced hydrogen generation. These hydrogen-producing nanoconstructs self-assemble when the PS I variant, the H(2)ase variant, and the molecular wire are combined.     

A novel peptide microarray for protein detection and analysis utilizing a dry peptide array system

A novel dry peptide microarray system has been constructed that affords a practical solution for protein detection and analysis. This system is an array preparation and assay procedure under dry conditions that uses designed peptides as non-immobilized capture agents for the detection of proteins. The system has several advantages that include its portability and ease-of-use, as well as the fact that vaporization of sample solutions need not be considered. In this study, various proteins have been characterized with an alpha-helical peptide mini-library. When proteins were added to the peptide library array, the fluorescent peptides showed different fluorescent intensities depending on their sequences. The patterns of these responses could be regarded as 'protein fingerprints' (PFPs), which are sufficient to establish the identities of the target proteins. Furthermore, statistical analysis of the resulting PFPs was performed using cluster analysis. The PFPs of the proteins were clustered successfully depending on their families and binding properties. Additionally, the target protein was characterized using a nanolitre system and could be detected down to 1.2 fmol. These studies imply that the dry peptide array system is a promising tool for detecting and analyzing target proteins. The dry peptide array will play a role in development of high-throughput protein-detecting nano/micro arrays for proteomics and ligand screening studies.     

Interaction of an ionic complementary peptide with a hydrophobic graphite surface

Protein adsorption on a surface plays an important role in biomaterial science and medicine. It is strongly related to the interaction between the protein residues and the surface. Here we report all‐atom molecular dynamics simulations of the adsorption of an ionic complementary peptide, EAK16‐II, to the hydrophobic highly ordered pyrolytic graphite surface. We find that, the hydrophobic interaction is the main force to govern the adsorption, and the peptide interchain electrostatic interaction affects the adsorption rate. Under neutral pH condition, the interchain electrostatic attraction facilitates the adsorption, whereas under acidic and basic conditions, because of the protonation and deprotonation of glutamic acid and lysine residues, respectively, the resulting electrostatic repulsion slows down the adsorption. We also found that under basic condition, during the adsorption peptide Chain II will be up against a choice to adsorb to the surface through the hydrophobic interaction or to form a temporary hydrophobic core with the deposited peptide Chain I. These results provide a basis for understanding some of the fundamental interactions governing peptide adsorption on the surface, which can shed new light on novel applications, such as the design of implant devices and drug delivery materials.     

Anticooperativity in a Glu-Lys-Glu salt bridge triplet in an isolated alpha-helical peptide

Salt bridges between oppositely charged side chains are well-known to stabilize protein structure, though their contributions vary considerably. Here we study Glu-Lys and Lys-Glu salt bridges, formed when the residues are spaced i, i + 4 surface of an isolated alpha-helix in aqueous solution. Both are stabilizing by -0.60 and -1.02 kcal/mol, respectively, when the interacting residues are fully charged. When the side chains are spaced i, i + 4, i + 8, forming a Glu-Lys-Glu triplet, the second salt bridge provides no additional stabilization to the helix. We attribute this to the inability of the central Lys to form two salt bridges simultaneously. Analysis of these salt bridges in protein structures shows that the Lys-Glu interaction is dominant, with the side chains of the Glu-Lys pair far apart.     

The primary structure of a cDNA for PsaN,encoding an extrinsic lumenal polypeptide of barley photosystem I

A cDNA clone encoding a 15.501 Da photosystem I (PSI) subunit of barley was isolated using an oligonucleotide based on the NH₂-terminal amino acid sequence of the isolated protein. The polypeptide, which migrates with an apparent molecular mass of 9.5 kDa on denaturing SDS-PAGE, has been designated PSI-N, and the corresponding gene is PsaN. Analysis of the deduced protein sequence indicates a mature protein of 85 amino acid residues and a molecular mass of 9818 Da. PSI-N is a hydrophilic, extrinsic protein with no predicted membrane-spanning regions. The transit peptide of 60 residues (5683 Da) contains a predicted hydrophobic -helix, suggesting that the protein is routed into the thylakoid lumen. Thus, PSI-N is the second known lumenal protein component associated with PSI, together with PSI-F.     

In vitro oligomerization of a membrane protein complex. liposome-based reconstitution of trimeric photosystem I from isolated monomers.

Many membrane proteins can be isolated in different oligomeric forms. Photosystem I (PSI), for example, exists in cyanobacteria either as a monomeric or as a trimeric complex. Neither the factors responsible for the specific trimerization process nor its biological role are known at present. In the filamentous cyanobacterium Spirulina platensis, trimers in contrast to monomers show chlorophyll fluorescence emission at 760 nm. To investigate the oligomerization process as well as the nature of the long wavelength chlorophylls, we describe here an in vitro reconstitution procedure to assemble trimeric PS I from isolated purified PS I monomers. Monomers (and trimers) were extracted from S. platensis with n-dodecyl beta-D-maltoside and further purified by perfusion chromatography steps. The isolated complexes had the same polypeptide composition as other cyanobacteria (PsaA-PsaF and PsaI-PsaM), as determined from high resolution gels and immunoblotting. They were incorporated into proteoliposomes, which had been prepared by the detergent absorption method, starting from a phosphatidylcholine:phosphatidic acid mixture solubilized by octylglucoside. After the addition of monomeric PS I (lipid:chlorophyll, 25:1), octylglucoside was gradually removed by the stepwise addition of Biobeads. The 77 K fluorescence emission spectrum of these proteoliposomes displays a long wavelength emission at 760 nm that is characteristic of PS I trimers, which indicates for the first time the successful in vitro reconstitution of PS I trimers. In addition, a high performance liquid chromatography analysis of complexes extracted from these proteoliposomes confirms the formation of structural trimers. We also could show with this system 1) that at least one of the stromal subunits PsaC, -D, and -E is necessary for trimer formation and 2) that the extreme long wavelength emitting chlorophyll is formed as a result of trimer formation.     

Biogenesis of a photosystem I light-harvesting complex. Evidence for a membrane intermediate.

CAB-7p is a chlorophyll a/b binding protein of photosystem I (PSI). It is found in light-harvesting complex I 680 (LHCI-680), one of the chlorophyll complexes produced by detergent solubilization of PSI. Two types of evidence are presented to indicate that assembly of CAB-7p into PSI proceeds through a membrane intermediate. First, when CAB-7p is briefly imported into chloroplasts or isolated thylakoids, we initially observe a fast-migrating membrane form of CAB-7p that is subsequently converted into PSI. The conversion of the fast-migrating form into PSI does not require stroma or ATP. Second, trypsin treatment of thylakoids containing radiolabeled CAB-7p indicates that there are at least two membrane forms of the mature 23-kD protein. The predominant form is completely resistant to proteolysis; a second form of the protein is cleaved by trypsin into 12- and 7-kD polypeptides. We interpret this to mean that the intermediate is a cleavable form that becomes protease resistant during assembly. This notion is supported by the observation that CAB-7p in LHCI-680 is largely cleaved by trypsin into 12- and 7-kD polypeptides, whereas CAB-7p in isolated PSI particles is trypsin resistant. In vitro, we generated a mutant form of CAB-7p, CAB-7/BgI2p, that was able to integrate into thylakoid membranes but was unable to assemble into PSI. The membrane form of CAB-7/BgI2p, like LHCI-680, was predominantly cleaved by trypsin into 12- and 7-kD fragments. We suggest that the mutant protein is arrested at an intermediate stage in the assembly pathway of PSI. Based on its mobility in nondenaturing gels and its susceptibility to protease cleavage, we suggest that the intermediate form is LHCI-680. We propose the following distinct stages in the biogenesis of LHCI: (a) apoprotein is integrated into the thylakoid, (b) chlorophyll is rapidly bound to apoprotein forming LHCI-680, and (c) LHCI-680 assembles into the native PSI complex.     

Mutants for photosystem I subunit D of Arabidopsis thaliana: effects on photosynthesis, photosystem I stability and expression of nuclear genes for chloroplast functions

In Arabidopsis thaliana, the D-subunit of photosystem I (PSI-D) is encoded by two functional genes, PsaD1 and PsaD2, which are highly homologous. Knock-out alleles for each of the loci have been identified by a combination of forward and reverse genetics. The double mutant psad1-1 psad2-1 is seedling-lethal, high-chlorophyll-fluorescent and deficient for all tested PSI subunits, indicating that PSI-D is essential for photosynthesis. In addition, psad1-1 psad2-1 plants show a defect in the accumulation of thylakoid multiprotein complexes other than PSI. Of the single-gene mutations, psad2 plants behave like wild-type (WT) plants, whereas psad1-1 markedly affects the accumulation of PsaD mRNA and protein, and photosynthetic electron flow. Additional effects of the psad1-1 mutation include a decrease in growth rate under greenhouse conditions and downregulation of the mRNA expression of most genes involved in the light phase of photosynthesis. In the same mutant, a marked decrease in the levels of PSI and PSII polypeptides is evident, as well as a light-green leaf coloration and increased photosensitivity. Increased dosage of PsaD2 in the psad1-1 background restores the WT phenotype, indicating that PSI-D1 and PSI-D2 have redundant functions.     

Probing the kinetics of photosystem I and photosystem II fluorescence in pea chloroplasts on a picosecond pulse fluorometer.

Picosecond fluorescence kinetics of pea chloroplasts have been investigated at room temperature using a pulse fluorometer with a resolution time of 10-11 s. Fluorescence has been excited by both a ruby and neodymium-glass mode-locked laser and has been reocrded within the 650 to 800 nm spectral region. We have found three-component kinetics of fluorescence from pea chloroplasts with lifetimes of 80, 300 and 4500 ps, respectively. The observed time dependency of the fluorescence of different components on the functional state of the photosynthetic mechanism as well as their spectra enabled us to conclude that Photosystem I fluoresces with a lifetime of 80 ps (tauI) and Photosystem II fluoresces with a lifetime of 300 ps (tauII). Fluorescence with a lifetime of 4500 ps (tauIII) may be interpreted as originating from chlorophill monomeric forms which are not involved in photosynthesis. It was determined that the rise time of Photosystem I and Photosystem II fluorescence after 530 nm photoexcitation is 200 ps, which corrsponds to the time of energy migration to them from carotenoids.     

Molecular weight determination of an active photosystem I preparation from a thermophilic cyanobacterium, Synechococcus elongatus

An active photosystem I (PSI) complex was isolated from the thermophilic cyanobacterium Synechococcus elongatus by a procedure consisting of three steps: First, extraction of photosystem II from the thylakoids by a sulfobetaine detergent yields PSI-enriched membranes. Second, the latter are treated with Triton X-100 to extract PSI particles, which are further purified by preparative isoelectric focusing. Third, anion-exchange chromatography is used to remove contaminating phycobilisome polypeptides. The purified particles show three major bands in sodium dodecyl sulfate gel electrophoresis of apparent molecular mass of 110, 15, and 10 kDa. Charge separation was monitored by the kinetics of flash-induced absorption changes at 820 nm. A chlorophyll/P700 ratio of 60 was found. When the particles are stored at 4 degrees C, charge separation was stable for weeks. The molecular mass of the PSI particles, determined by measurement of zero-angle neutron scattering intensity, was 217,000 Da. The PSI particles thus consist of one heterodimer of the 60-80-kDa polypeptides and presumably one copy of the 15- and 10-kDa polypeptides, respectively.     

Ascorbate as a substrate for photoproduction of hydrogen by photosystem I of chloroplasts

The photoproduction of hydrogen by 2-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-inhibited chloroplasts from ascorbate under anaerobic conditions was studied in the pH range 5.0 to 7.5 using methyl viologen (MV), N,N,N′,N′-tetramethyl-P-phenylenediamine (TMPD), and excess hydrogenase from Desulfovibrio desulfuricans. (a) At neutral and basic pHs, the photoreduction of MV, which reacted back with photoxidized ascorbate (dehydroascorbate [DHASC]), and the rates of H₂ photoproduction were very low. The slow H₂ photoproduction was explained by the reversible reduction of MV by the photoproduced H₂ (catalyzed by hydrogenase) and its reoxidation by DHASC resulting in H₂ uptake. (b) At pH 5.2, relatively high initial rates of H₂ photoproduction were obtained, which were comparable to the rates of O₂ consumption at pH 5.2 by photosystem I (catalyzed by photoreduced MV). However, accumulation of photoreduced MV under anaerobic conditions was not detected. In the presence of high concentrations of protons, the H₂ uptake by DHASC was very slow because the equilibrium concentration of H₂-reduced MV was very small, thus allowing H₂ evolution mediated by photoreduced MV to compete with the back reaction with DHASC. (c) The continuous accumulation of DHASC, which was generated together with H₂, gradually slowed the H₂ evolution until it stopped after about 3 hours. At high concentrations, DHASC was able to compete with the coupling of photoreduced MV to hydrogenase and H₂ evolution. (d) Dithiothreitol (DTT) reduced the DHASC and consequently competed with the back reaction of the photoreduced and H₂-reduced MV with DHASC. DTT thus prolonged the time period of H₂ photoproduction from ascorbate and abolished the dependence of its rate on pH in the range of 5.2 to 7.5 (e) A study of H₂ uptake by chemically oxidized ascorbate (in the dark) showed that MV and hydrogenase were both required to catalyze electron transfer from H₂ to DHASC. TMPD prevented this H₂ consumption by DHASC (in a chloroplast reaction mixture containing MV and hydrogenase). Illumination restored the H₂ uptake presumably by generating reduced MV which activated the hydrogenase.