The interaction between different domains of staphylococcal protein A and human polyclonal IgG, IgA, IgM and F(ab')2: separation of affinity from specificity.

Binding properties of staphylococcal protein A (SpA) to different human immunoglobulins have been investigated. In this analysis, intact SpA as well as SpA-derived fragments containing one to five IgG-binding domains of different compositions, were used. The affinity binding constants of the different proteins to human polyclonal IgG, IgA, IgM and F(ab')2-fragments as well as their binding capacity to the immunoglobulin molecules were determined. The results show that although all the proteins bound to IgG, regardless of size or composition, the binding strength differed significantly. Proteins containing five domains have a stronger affinity for IgG than those containing one or two. There were no marked differences in binding strength between different domains. However, the binding ability to IgA and IgM showed a marked difference between the various SpA-derived proteins of different compositions. This discrepancy was correlated to differences in their relative binding properties to isolated F(ab')2-fragments of IgG. Hence, we conclude that the binding affinity is mainly affected by the number of domains, whereas the binding specificity is to a large extent determined by which domains are selected.

The single-copy gene psbS codes for a phylogenetically intriguing 22 kDa polypeptide of photosystem II.

Recombinant phages that encode the complete precursor polypeptide for the 22 kDa polypeptide associated with photosystem II have been serologically selected from two lambda gt11 expression libraries made from polyadenylated RNA of spinach seedlings. The cDNAs hybridize to a 1.3 kb RNA species. The precursor protein is comprised of 274 amino acid residues and carries an N-terminal transit peptide of probably 69 amino acid residues. The mature protein exhibits four predicted transmembrane segments and is shown to be an integral component of photosystem II originating in a single-copy gene. The unique characteristics of this protein are: (i) it is the result of a gene-internal duplication of an ancestor with two membrane spans, (ii) a striking resemblance to LHC I/II, CP24/CP29 apoproteins, and ELIPs, although it does not bind chlorophyll and is present in cyanobacteria, and, as these proteins, (iii) it integrates into the membrane with uncleaved routing signals that display remarkable resemblance to patterns found in bipartite transit peptides.

Differential biogenesis of photosystem-II in mesophyll and bundle-sheath cells of 'malic' enzyme NADP(+)-type C4 plants. A comparative protein and RNA analysis.

We have investigated the photosystem-II organization in differentiating-bundle-sheath cells of the three malate dehydrogenase (oxaloacetate decarboxylating) (NADP+)-type C4 species maize, Sorghum and Pennisetum. Using a set of nine different antisera raised against individual subunits of photosystem-II, we demonstrate that photosystem-II components constitute a substantial part of the thylakoid membranes of young bundle-sheath chloroplasts. The abundance of subunits of the photosystem-II core, i.e. the 47-and 43-kDa chlorophyll-a-binding proteins, polypeptides D1 and D2, cytochrome b559, and the 34-kDa polypeptide, varies with the developmental state of the plant. However, the levels of the 23-kDa, 16-kDa and 10-kDa extrinsic polypeptides of the water-oxidation complex are drastically reduced in bundle-sheath chloroplasts of all three species analyzed, regardless of their state of differentiation. The reduction in protein abundance is also reflected at the transcript level: only traces of the nuclear-encoded mRNAs are found in differentiating bundle-sheath cells of Sorghum, suggesting that the transcription of these genes has been switched off. Our data are compatible with the idea that the water-oxidation complex is a prime site for initiating or maintaining the process leading to photosystem-II depletion during differentiation of bundle-sheath cells.

Nucleotide sequences of cDNAs encoding the entire precursor polypeptides for subunits II and III of the photosystem I reaction center from spinach.

Several cDNA clones encoding the complete subunit II and III precursor polypeptides of the photosystem I reaction center were isolated from two spinach lambda gt1 1 expression libraries by immunoscreening and homologous hybridization. The identity of the recombinant cDNAs was verified by an N-terminal amino acid sequence of 14 and 20 residues for the respective mature spinach proteins. The ca. 880 nucleotide long sequence and derived amino acid sequence for subunit II predict a precursor of 23.2 kDa (212 residues) and a positively charged, mature protein of 17.9 kDa (162 residues). The corresponding data for subunit III are ca. 710 nucleotides (cDNA), 13.4 kDa (125 residues, precursor polypeptide) and, again, a positively charged, mature protein of 9.7 kDa (91 residues). Secondary structure predictions indicate that both subunits are extramembraneous components of photosystem I. Subunit II is probably located on the matrix-side, subunit III in the lumen of stroma lamellae which is consistent both with biochemical findings and the proposed roles of these proteins in the electron transition from and to photosystem I, respectively. Major transcripts of 1.1 kb (subunit II) and 0.8 kb (subunit III) have been observed by RNA-DNA hybridization.

Nucleotide sequence of cDNA clones encoding the entire precursor polypeptides for subunits IV and V of the photosystem I reaction center from spinach.

Using lambda gt11 expression cloning and immunoscreening, cDNA-containing recombinant phages for subunits IV and V of the photosystem I reaction center were isolated, sequenced and used to probe Northern blots of polyadenylated RNA prepared from spinach seedlings. The mRNA sizes for both components are approximately 1000 and 850 nucleotides, respectively. The 968 nucleotide cDNA sequence and derived amino acid sequence for subunit IV predict a single open reading frame of 231 amino acid residues (25.4 kDa). Comparison with a 13-residue N-terminal amino acid sequence determined for subunit IV suggests a mature protein of 17.3 kDa (154 residues) and a transit sequence of 77 amino acids (8.1 kDa). The corresponding data for subunit V are 677 bp (cDNA), 167 residues for the precursor protein (18.2 kDa), 98 residues for the mature polypeptide (10.8 kDa) and 69 residues for the transit peptide (7.4 kDa). Secondary structure predictions indicate that both proteins possess greatly different transit sequences and that none is membrane-spanning.

Nucleotide sequence of cDNA clones encoding the complete precursor for the "10-kDa" polypeptide of photosystem II from spinach.

By screening lambda gt11 cDNA expression libraries prepared from poly(A+) RNA of illuminated (4 and 16 h) spinach seedlings (Spinacia oleracea var. Monatol), we have recovered phage containing inserts corresponding to the complete "10-kDa" precursor polypeptide associated with the water oxidizing photosystem II. The amino acid sequence deduced from the nucleotide sequence indicates a polypeptide of 140 amino acid residues (15.0 kDa). The mature protein is predicted to consist of 99 amino acid residues (10.8 kDa). The identity of the recombinant DNAs was established serologically and by determining an N-terminal sequence of 25 amino acid residues from the purified authentic protein. This sequence was found in the cDNA-derived amino acid sequence. In addition, a monospecific antiserum detected the appropriate polypeptides when copy-RNA was translated into protein which was imported into isolated unbroken chloroplasts. The transit sequence (41 amino acid residues; 4.2 kDa) is markedly dissimilar to those of the three extrinsic, luminal photosystem II proteins. Secondary structure prediction suggests a transmembrane arrangement of the 10-kDa polypeptide in the thylakoid membrane, with the N terminus in the thylakoid lumen and a 17-amino acid C-terminal sequence at the outside partitions. This is consistent with biochemical and functional data for the protein. A major transcript of 1.0 kilobases has been observed in polyadenylated RNA.

Isolation and characterization of the 10-kDa and 22-kDa polypeptides of higher plant photosystem 2.

Two polypeptides of 10 kDa and 22 kDa, shown to be components of the higher plant photosystem 2, were purified and examined. A NaCl/Triton X-100 treatment was designed, which released these two polypeptides from the thylakoid membrane, in concert with the extrinsic 16-kDa and 23-kDa proteins, concomitant with a loss in oxygen-evolution activity. After this treatment the oxygen-evolving activity of the photosystem 2 membranes devoid of the 10-kDa and the 22-kDa polypeptides could be restored with CaCl2, but not by readdition of the purified 23-kDa protein. This deficiency was caused by an inability of the 23-kDa protein to rebind to the photosystem 2 membranes. In analogy, the oxygen-evolution activity of a highly purified photosystem 2 core preparation, devoid of the 10-kDa and 22-kDa polypeptides, was stimulated by CaCl2, but not by the 23-kDa protein. We, therefore, suggest that the 10-kDa or the 22-kDa polypeptides provide a binding-site for the extrinsic 23-kDa protein to the thylakoid membrane. The 10-kDa and 22-kDa polypeptides were isolated through ion-exchange chromatography in the presence of detergents. They both displayed hydrophobic properties, verified by their low proportion of polar amino acid residues and their partition to the hydrophobic phase during Triton X-114 fractionation. The purified polypeptides did not contain metallic cofactors or substances with absorption in the visible region of the spectrum.

Reconstitution of oxygen evolution in high salt washed photosystem II particles.

Photosystem II thylakoid particles possessing high rates of oxygen evolution, were shown to have a very simple polypeptide composition. Upon washing of these particles with 250 mM NaCl the oxygen evolution was inhibited up to 80% concomitant with a release of two polypeptides of 23 and 16 kDa. Readdition of the pure 23 kDa protein to the depleted thylakoids under low ionic strength reconstituted more than half of the lost activity. No stimulation was obtained with the 16 kDa protein alone or in combination with glycerol. The results give further strong evidence that the 23 kDa protein is an essential component in the oxygen evolving complex. The possible involvement of other proteins in this complex is discussed in light of the demonstrated simple polypeptide pattern of the photosystem II particles.