THE RESOLUTION OF CHLOROPHYLL a/b BINDING PROTEINS BY A PREPARATIVE METHOD BASED ON FLAT BED ISOELECTRIC FOCUSING

We describe a new fractionation method for intrinsic membrane proteins based on flat bed isoelectric focusing (IEF) in granulated gel. The characteristics of the separation in the presence of the non-ionic detergent dodecylmaltoside are considered. The method has been applied to the fractionation of chlorophyll a/b binding proteins from chloroplast grana membranes. Several Light Harvesting Complexes II (LHC II) have been resolved showing differences in their polypeptide composition. Probing with monoclonal and polyclonal antibodies showed that polypeptides belonging to different [EF fractions with the same mobility in denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis, are immunologically distinct polypeptides. This is the first report of the presence in the thylakoid membrane of a number of LHCII polypeptides that may reflect the genetic complexity of the Cab genes. Moreover preparative amounts have been obtained of the minor chlorophyll a/b proteins CP 29, CP 26 and CP 24 that have been recently described. The analysis of a currently used LHCII preparation by the present method shows that this fraction is actually contaminated by two minor chlorophyll a/b proteins.     

STRUCTURAL RELATIONSHIPS OF THE PHOTOSYSTEM I AND PHOTOSYSTEM II CHLOROPHYLL a/b AND a/c LIGHT-HARVESTING APOPROTEINS OF PLANTS AND ALGAE

Polyclonal antibodies against four different apoproteins of either the chlorophyll (Chl) a/b light-harvesting antenna of photosystem I or II, or a chlorophyll-protein complex homologous to CP26 from Chlamydomonas reinhardtii, crossreact with11–13 thylakoid proteins of Chlamydomonas, Euglena gracilis and higher plants. The number of antigenically-related proteins correlates with the quantity of light-harvesting chlorophyll-protein complex (LHC) gene types that have been sequenced in higher plants. The antibodies also react specifically with Chi a/c-binding proteins of three diatoms and Coccolithophora sp. as determined by immunoblot and Ouchterlony assays. Four to six crossreacting proteins are observed in each chromophyte species and a functional role for some can be deduced by antibody reactivity. It appears that despite major differences in the structures of their pigment ligands, at least some domains of Chl-binding LHC apoproteins have been conserved during their evolution, possibly functioning in protein: protein, as opposed to pigment: protein, interactions in photosynthetic membranes.     

Structure and dynamics of photosystem II light-harvesting complex revealed by high-resolution FTICR mass spectrometric proteome analysis

Structure and dynamics of membrane-bound light-harvesting pigment-protein complexes (LHCs), which collect and transmit light energy for photosynthesis and thereby play an essential role in the regulation of photosynthesis and photoprotection, were identified and characterized using high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). LHCs from photosystem II (LHCII) were isolated from the thylakoid membrane of Arabidopsis thaliana leaves after light stress treatment using sucrose density gradient centrifugation, and separated by gel-filtration into LHCII subcomplexes. Using reversed-phase high-performance liquid chromatography and two-dimensional (2D) gel electrophoresis, the LHCII proteins, Lhcb1-6 and fibrillins, were efficiently separated and identified by FTICR-MS. Some of the LHCII subcomplexes were shown to migrate from photosystem II to photosystem I as a result of short-term adaptation to changes in light intensity. In the mobile LHCII subcomplexes, decreased levels of fibrillins and a modified composition of LHCII protein isoforms were identified compared to the tightly bound LHCII subcomplexes. In addition, FTICR-MS analysis revealed several oxidative modifications of LHCII proteins. A number of protein spots in 2D gels were found to contain a mixture of proteins, illustrating the feasibility of high-resolution mass spectrometry to identify proteins that remain unseparated in 2D gels even upon extended pH gradients.     

Monoclonal antibodies as a tool for structure-function studies of the MDR1-P-glycoprotein

P-glycoprotein is considered one of the most important member of the rapidly growing superfamily of integral proteins known as the ATP-binding cassette (ABC) which in human also include several other multidrug resistance membrane proteins (i.e., MRP), the product of the cystic fibrosis gene, the TAP-1/TAP2 peptide transporters encoded by the major histocompatibility complex genes and the gene encoding for breast cancer resistance protein (BCRP) also known as MXR1 (mitoxantrone resistance protein). Many monoclonal antibodies (MAbs) reacting with distinct P-glycoprotein domains have been isolated and used to study the molecular organization and cellular functions of this ABC protein. MAbs have been used for multidrug resistance (mdr) gene cloning, delineation of the secondary and tertiary structure of P-glycoprotein and molecular analysis of the mechanisms involved in substrate recognition and transport. The immunodetection of the distinct products of the mdr gene family in normal and malignant cells and tissues has greatly contributed to the understanding of the physiological role of P-glycoprotein and its possible involvement in the refractory of tumors to chemotherapy. The present article deals with the immunological methods used for the structure-function studies of the P-glycoprotein. After introducing the basic structural features of this ABC transporter, the antibody based-approach is discussed with aiming to furnishing methodological perspectives for further investigations of the physiological role of P-glycoprotein and the multidrug resistance phenomenon.     

Synthesis and Functional Reconstitution of Light‐Harvesting Complex II into Polymeric Membrane Architectures

One of most important processes in nature is the harvesting and dissipation of solar energy with the help of light‐harvesting complex II (LHCII). This protein, along with its associated pigments, is the main solar‐energy collector in higher plants. We aimed to generate stable, highly controllable, and sustainable polymer‐based membrane systems containing LHCII–pigment complexes ready for light harvesting. LHCII was produced by cell‐free protein synthesis based on wheat‐germ extract, and the successful integration of LHCII and its pigments into different membrane architectures was monitored. The unidirectionality of LHCII insertion was investigated by protease digestion assays. Fluorescence measurements indicated chlorophyll integration in the presence of LHCII in spherical as well as planar bilayer architectures. Surface plasmon enhanced fluorescence spectroscopy (SPFS) was used to reveal energy transfer from chlorophyll b to chlorophyll a, which indicates native folding of the LHCII proteins.     

Gene-protein database of Escherichia coli K-12: edition 3

The first two editions of the E. coli Gene-Protein Index were published to provide identifications of protein spots resolved by two-dimensional gel electrophoresis as the products of known genes. This third edition has been expanded to include information about genes and proteins gained directly from two-dimensional gel analysis--including information about protein spots not yet characterized genetically or biochemically--and is therefore more properly called a cellular protein database. An alpha-numeric designation has been uniquely assigned to each of the 616 polypeptide spots in the current database. To this, information is linked about the polypeptide's identification (protein name, gene name, Enzyme Commission--EC number), location on reference gels (x-y coordinates), genetics (Genbank code, DNA sequence reference), biochemistry (molecular weight, isoelectric point), and physiology (steady state level of the protein as a function of media and temperature, membership in various regulons and stimulons).     

Two-dimensional gel protein database of Saccharomyces cerevisiae (update 1999).

By proving the opportunity to visualize several hundred proteins at a time, two-dimensional (2-D) gel electrophoresis is an important tool for proteome research. In order to take advantage of the full potential of this technique for yeast studies, we have undertaken a systematic identification of yeast proteins resolved by this technique. We report here the identification of 92 novel protein spots on the yeast 2-D protein map. These identifications extend the number of protein spots identified on our yeast reference map to 401. These spots correspond to the products of 279 different genes. They have been essentially identified by three methods: gene overexpression, amino acid composition and mass spectrometry. These data can be accessed on the Yeast Protein Map server (htpp://www.ibgc.u-bordeaux2.fr/YPM).     

Heterogeneity of the main light-harvesting chlorophyll a/b-protein complex of photosystem II (LHCII) at the level of trimeric subunits.

To study organization of the main light-harvesting chlorophyll a/b-protein complex of photosystem II (LHCII) from spinach thylakoid membranes at the level of trimeric subcomplexes, we have applied non-denaturing isoelectric focusing (ndIEF) in vertical, slab polyacrylamide gels. When analyzed by two consecutive ndIEF/electroelution runs, spinach BBY membrane preparations (PSII(alpha)-enriched, stacked thylakoid membranes) were resolved into nine fractions of 100% purity, labelled 1-9 in order of decreasing pI values. Seven of these fractions (3-9) were shown by absorption spectroscopy to stand for LHCII subcomplexes. The subcomplexes were established - by monitoring their circular dichroism spectra and comparing them to the spectra of native LHCII trimers and monomers - to be structurally intact trimers. The analysis of polypeptide composition of the subcomplexes in terms of apparent molecular masses and Lhcb genes' products led us to the conclusion that each of the subcomplexes might be a mixed population of closely similar individual trimers, comprising of permutations of Lhcb1 and Lhcb2 (subcomplexes 3-7) or Lhcb1, Lhcb2 and Lhcb3 (subcomplexes 8 and 9).     

PHTHALOCYANINE AND NAPHTHALOCYANINE PHOTOSENSITIZED OXIDATION OF 2'-DEOXYGUANOSINE: DISTINCT TYPE I AND TYPE II PRODUCTS

Abstract
The photodynamic properties of the di-and tetrasulfonated zinc and aluminium phthalocyanines and a tetrasulfonated aluminium napththalocyanine were studied using 2'-deoxyguanosine as a DNA model compound. The major photooxidation products of this nucleoside were identified and classified according to their formation through a radical mechanism (type I) or a singlet oxygen mediated mechanism (type II). The major type I product was obtained and identified as 2,2-diamino [(2-deoxy-β- d - erythro pentofuranosyl)-4-amino]-5( 2H )-oxazolone. Two major type II products were characterized as the 4R* and 4S* diastereomers of 9-(2-deoxy-β- d - erythro pentofuranosyl)-7,8-dihydro-4-hydroxy-8-oxoguanine. In addition a third product, also resulting from a type II photooxidation, was identified as 8-oxo-7,8-dihydro-2'-deoxyguanosine. Quantification of these products provided a means to estimate the contribution of type I and type II pathways during the phthalocyanine and naphthalocyanine mediated photooxidation of 2'-deoxyguanosine, confirming the major role of singlet oxygen in these processes.     

PHTHALOCYANINE AND NAPHTHALOCYANINE PHOTOSENSITIZED OXIDATION OF 2'-DEOXYGUANOSINE

Abstract— The photodynamic properties of the di-and tetrasulfonated zinc and aluminium phthalocyanines and a tetrasulfonated aluminium napththalocyanine were studied using 2'-deoxyguanosine as a DNA model compound. The major photooxidation products of this nucleoside were identified and classified according to their formation through a radical mechanism (type I) or a singlet oxygen mediated mechanism (type II). The major type I product was obtained and identified as 2,2-diamino [(2-deoxy-β- d - erythro pentofuranosyl)-4-amino]-5( 2H )-oxazolone. Two major type II products were characterized as the 4R* and 4S* diastereomers of 9-(2-deoxy-β- d - erythro pentofuranosyl)-7,8-dihydro-4-hydroxy-8-oxoguanine. In addition a third product, also resulting from a type II photooxidation, was identified as 8-oxo-7,8-dihydro-2'-deoxyguanosine. Quantification of these products provided a means to estimate the contribution of type I and type II pathways during the phthalocyanine and naphthalocyanine mediated photooxidation of 2'-deoxyguanosine, confirming the major role of singlet oxygen in these processes.     

The acclimative response of the main light-harvesting chlorophyll a/b-protein complex of photosystem II (LHCII) to elevated irradiances at the level of trimeric subunits

The changes in structural organization of the major light-harvesting chlorophyll a/b–protein complex of photosystem II (LHC II) at the level of trimeric subcomplexes were studied in spinach plants grown under low light conditions (50 μmol quanta m⁻² s⁻¹) and then acclimated to elevated irradiances. By monitoring photochemical quenching of fluorescence yield (q_P), photosystem II (PS II) functional status was assessed in leaves of plants acclimated to a range of elevated irradiances. Three separate acclimative irradiances were selected for the experiments, reflecting: limiting light conditions (150 μmol quanta m⁻² s⁻¹), near to the inflexion point on the irradiance curve conditions (300 μmol quanta m⁻² s⁻¹) and an excessive light, causing a moderate stress in the form of down regulation of PS II (450 μmol quanta m⁻² s⁻¹). An immunoblot analysis showed that there was a clear decline in an abundance on chlorophyll basis of Lhcb1-3 apoproteins as an acclimative irradiance increased from 50 to 450 μmol quanta m⁻² s⁻¹, with Lhcb1 decreasing to a lesser extent than Lhcb2 and Lhcb3 (only at excessive irradiance). When analyzed by non-denaturing isoelectric focusing BBY membrane fragments (PSII-enriched, stacked thylakoid membranes) isolated from low light-grown plants were resolved into nine fractions, seven of which (labelled 3–9) were established by us previously [Jackowski and Pielucha, J. Photochem. Photobiol. B: Biol. 64 (2001) 45] to be LHC II subcomplexes representing mixed populations of closely similar trimers, comprising permutations of Lhcb1 and Lhcb2 (subcomplexes 3–7) or Lhcb1-3 (subcomplexes 8 and 9). A heterogeneity with regard to accumulation behaviour of LHC II subcomplexes in response to elevated irradiances was revealed. The subcomplexes 5 and 6 were accumulating at similar level, regardless of the light irradiance experienced. Another group consisting of the subcomplexes 3 and 4 (the most basic ones) showed a progressive increase in relative abundance with increasing an irradiance intensity whereas the subcomplexes 7–9 (the most acidic ones) exhibited a progressive decline in their relative abundance during an acclimation of spinach plants to elevated irradiances thus they may collectively represent an elevated irradiance-responsive subunit of LHCII.     

Protein Differentiation: Emergence of Novel Proteins during Evolution

The almost limitless number of proteins can be reduced to approximately 200 basic structures, i.e. geometries of polypeptide chains; these form, the set from which the tracable protein differentiation started. Protein differentiation begins with a gene duplication. One of the gene products is conserved since it must continue to fulfill the original function. The other gene product is conserved as soon as it has taken over a new function. Comparing amino acid sequences of equivalent proteins in two species yields the evolutionary distance between these species, which allows dating of their common ancestor (“molecular palaeontology”). These comparisons are now applied to the geometries of nonequivalent proteins, elucidating the pathways of protein differentiation.     

Cloning and characterization of a phosphopantetheinyl transferase from Streptomyces verticillus ATCC15003, the producer of the hybrid peptide-polyketide antitumor drug bleomycin.

BACKGROUND: Phosphopantetheinyl transferases (PPTases) catalyze the posttranslational modification of carrier proteins by the covalent attachment of the 4'-phosphopantetheine (P-pant) moiety of coenzyme A to a conserved serine residue, a reaction absolutely required for the biosynthesis of natural products including fatty acids, polyketides, and nonribosomal peptides. PPTases have been classified according to their carrier protein specificity. In organisms containing multiple P-pant-requiring pathways, each pathway has been suggested to have its own PPTase activity. However, sequence analysis of the bleomycin biosynthetic gene cluster in Streptomyces verticillus ATCC15003 failed to reveal an associated PPTase gene. RESULTS: A general approach for cloning PPTase genes by PCR was developed and applied to the cloning of the svp gene from S. verticillus. The svp gene is mapped to an independent locus not clustered with any of the known NRPS or PKS clusters. The Svp protein was overproduced in Escherichia coli, purified to homogeneity, and shown to be a monomer in solution. Svp is a PPTase capable of modifying both type I and type II acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs) from either S. verticillus or other Streptomyces species. As compared to Sfp, the only 'promiscuous' PPTase known previously, Svp displays a similar catalytic efficiency (k(cat)/K(m)) for the BlmI PCP but a 346-fold increase in catalytic efficiency for the TcmM ACP. CONCLUSIONS: PPTases have recently been re-classified on a structural basis into two subfamilies: ACPS-type and Sfp-type. The development of a PCR method for cloning Sfp-type PPTases from actinomycetes, the recognition of the Sfp-type PPTases to be associated with secondary metabolism with a relaxed carrier protein specificity, and the availability of Svp, in addition to Sfp, should facilitate future endeavors in engineered biosynthesis of peptide, polyketide, and, in particular, hybrid peptide-polyketide natural products.     

Metal ligand-induced alterations in the surface structures of lactoferrin and transferrin probed by interaction with immobilized copper(II) ions

We have evaluated immobilized Cu(II) ions as a potential site-directed molecular probe to monitor ligand-induced alterations in protein surface structures. Metal ion-induced alterations in the surface structures of different lactoferrins (human and porcine), transferrins (human and rabbit), and ovotransferrin (chicken) were examined. Although these 78,000-dalton glycoproteins are related gene products with similar overall structure and function, they differ greatly in the number and distribution of surface-exposed electron-donor groups thought to interact with Cu(II) ions. Each of these proteins interacted with immobilized Cu(II) ions through sites which are distinct from the two specific high affinity metal binding sites identified for iron. In both the presence and absence of bound iron, transferrins interacted more strongly with the immobilized Cu(II) ions than did lactoferrins; ovotransferrin interacted only weakly. Although iron binding increased the affinities of lactoferrins for immobilized Cu(II), iron binding decreased the affinities of transferrins and ovotransferrin for immobilized Cu(II) ions. Iron-saturated and iron-free lactoferrins were resolved by pH gradient elution, but only in the presence of 3 M urea; they were not resolved by imidazole affinity elution. Conversely, the iron-saturated and iron-free forms of transferrin were only separated by imidazole affinity elution. Urea did not influence the resolution of apo and holo ovotransferrins by imidazole. The differential effects of urea and imidazole suggest the participation of different types of surface electron-donor groups. The progressive site-specific modification of surface-exposed histidyl residues by carboxyethylation revealed several lactoferrin forms of intermediate affinity for immobilized iminodiacetate-Cu(II) ions. In summary, independent of species, the affinity for immobilized Cu(II) ions increased as follows: iron-saturated ovotransferrin less than metal-free ovotransferrin less than apolactoferrin less than hololactoferrin much less than diferric or holotransferrin less than monoferric transferrin less than apotransferrin. We have demonstrated the use of immobilized Cu(II) ions to distinguish and to monitor ligand-induced alterations in protein surface structure. The results are discussed in relation to protein surface-exposed areas of electron-donor groups.     

Electrophoretic mapping of highly homologous keratins: A novel marker peptide approach

Identification of the intermediate filament proteins (IFPs) in the wool proteome has formerly been hampered by limited sequence information, the high degree of IFP homology and their close proximity on 2‐DE maps. This has been partially rectified by the recent acquisition of four new Type I and two Type II wool IFP sequences. Among closely migrating proteins, such as IFP clusters in a 2‐DE map, proteins with higher sequence coverage will be assigned higher scores, but the identification of unique peptides in such tight clusters may distinguish these closely migrating proteins. Two approaches were adopted for the study of wool IFPs. In the first, searches were conducted for peptides known to be unique to each member of the family in each spot. In the second, MALDI imaging was employed to examine peptides bound to a PVDF membrane from a poorly resolved part of the Type I IFP region of the 2‐DE map. As a result, a distinct picture has emerged of the distribution of the six Type I and four Type II IFPs across the 2‐DE wool protein map.     

Spectroscopic characterization of a high-potential lipo-cupredoxin found in Streptomyces coelicolor

For many streptomycetes, a distinct dependence on the "bioavailability" of copper ions for their morphological development has been reported. Analysis of the Streptomyces coelicolor genome reveals a number of gene products encoding for putative copper-binding proteins. One of these appears as an unusual copper-binding protein with a lipoprotein signal sequence and a cupredoxin-like domain harboring a putative Type-1 copper-binding motif. Cloning of this gene from S. coelicolor and subsequent heterologous expression in Escherichia coli has allowed for a thorough spectroscopic interrogation of this putative copper-binding protein. Optical and electron paramagnetic resonance spectroscopies have confirmed the presence of a "classic" Type-1 copper site with the axial ligand to the copper a methionine. Paramagnetic NMR spectroscopy on both the native Cu(II) form and Co(II)-substituted protein has yielded active-site structural information, which on comparison with that of other cupredoxin active sites reveals metal-ligand interactions most similar to the "classic" Type-1 copper site found in the amicyanin family of cupredoxins. Despite this high structural similarity, the Cu(II)/(I) midpoint potential of the S. coelicolor protein is an unprecedented +605 mV vs normal hydrogen electrode at neutral pH (amicyanin approximately +250 mV), with no active-site protonation of the N-terminal His ligand observed. Suggestions for the physiological role/function of this high-potential cupredoxin are discussed.     

Relationship between the organization of the PS II super complex and the functions of the photosynthetic apparatus

The chlorophyll fluorescence and the photosynthetic oxygen evolution (flash-induced oxygen yield patterns and oxygen bursts under continuous irradiation) were investigated in the thylakoid membranes with different stoichiometry and organization of the chlorophyll-protein complexes. Data show that the alteration in the organization of the photosystem II (PS II) super complex, i.e. the amount and the organization of the light-harvesting chlorophyll a/b protein complex (LHCII), which strongly modifies the electric properties of the membranes, influences both the energy redistribution between the two photosystems and the oxygen production reaction. The decrease of surface electric parameters (charge density and dipole moments), associated with increased degree of LHCII oligomerization, correlates with the strong reduction of the energy transfer from PS II to PSI. In the studied pea thylakoid membranes (wild types Borec, Auralia and their mutants Coeruleovireus 2/16, Costata2/133, Chlorotica XV/1422) with enhanced degree of oligomerization of LHCII was observed: (i) an increase of the S(0) populations of PS II in darkness; (ii) an increase of the misses; (iii) an alteration of the decay kinetics of the oxygen bursts under continuous irradiation. There is a strict correlation between the degree of LHCII oligomerization in the investigated pea mutants and the ratio of functionally active PS II alpha to PS II beta centers, while in thylakoid membranes without oligomeric structure of LHCII (Chlorina f2 barley mutant) the PS II alpha centers are not registered.     

Energy transfer between surface-immobilized light-harvesting chlorophyll a/b complex (LHCII) studied by surface plasmon field-enhanced fluorescence spectroscopy (SPFS)

The major light-harvesting chlorophyll a/b complex (LHCII) of the photosynthetic apparatus in green plants can be viewed as a protein scaffold binding and positioning a large number of pigment molecules that combines rapid and efficient excitation energy transfer with effective protection of its pigments from photobleaching. These properties make LHCII potentially interesting as a light harvester (or a model thereof) in photoelectronic applications. Most of such applications would require the LHCII to be immobilized on a solid surface. In a previous study we showed the immobilization of recombinant LHCII on functionalized gold surfaces via a 6-histidine tag (His tag) in the protein moiety. In this work the occurrence and efficiency of Fo?rster energy transfer between immobilized LHCII on a functionalized surface have been analyzed by surface plasmon field-enhanced fluorescence spectroscopy (SPFS). A near-infrared dye was attached to some but not all of the LHC complexes, serving as an energy acceptor to chlorophylls. Analysis of the energy transfer from chlorophylls to this acceptor dye yielded information about the extent of intercomplex energy transfer between immobilized LHCII.     

Genomically linked cellular protein databases derived from two-dimensional polyacrylamide gel electrophoresis

In its most useful form a cellular protein database should be genomically based, because it is the genome which determines both the total number of proteins a cell can make and the particular ones that will be made under any given condition. Such a database should trace each protein back to its structural gene, and should account for every structural gene of a cell. Recent advances in molecular biology greatly facilitate the construction of such gene-protein databases. The mapping of genes of unidentified proteins resolved from total cell extracts on two-dimensional gels can now be accomplished by largely biochemical methods, without the necessity of isolating mutants or performing genetic crosses. Other techniques permit one to search gels for the product of any newly discovered gene (or open reading frame) suspected of encoding a protein. Consequently, gene-protein indices can be built independently and simultaneously from either direction--deducing the genetic map from the protein pattern, or finding the protein pattern from information encoded in the genome. A database of this sort is being constructed for the bacterium, Escherichia coli. Given the current pace of DNA nucleotide sequencing, the development of total gene-protein indices for a variety of cells can be anticipated in the near future.