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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe here a new procedure permitting rapid (12-13 h) isolation of a pure oxygen-evolving photosystem II (PSII) core complex from the cyanobacterium Synechocystis PCC 6803. This procedure involves dodecyl maltoside extraction of thylakoid membranes followed by single-step column chromatography using a weak anion-exchanger. SDS-PAGE and immunoblotting show that the complex consists of five intrinsic membrane proteins (CP47, CP43, D1, D1, and cyt b559), one extrinsic protein (MSP), and one unknown protein with a molecular mass of approximately 26 kDa. A chemical and functional analysis, normalized to 2 molecules of pheophytin a, indicates that this PSII core complex contains 1 photoactive plastoquinone, QA, 4 manganese atoms, 38 chlorophyll a molecules, 1 cytochrome b559, 2 plastoquinone-9, and 9-10 beta-carotenes. The complex exhibits high rates of oxygen evolution, typically 2400-2600 mumol of O2 (mg of Chl)-1 h-1 in the presence of 2,5-dichlorobenzoquinone as an artificial electron acceptor with a pH optimum of 6.5. A strong light minus dark multiline EPR signal, arising from the S2 state of the oxygen-evolving complex (OEC), is observed at 10 K following illumination at 198 K. The determination of the absolute oxygen yield per saturating microsecond flash indicates that essentially all of the PSII centers contain functional oxygen-evolving complexes. This point is further supported by the absence of photoaccumulation, upon room temperature illumination, of the immediate oxidant of the OEC, redox-active tyrosine, YZ.. On the basis of EPR spectra, oxidized minus reduced difference spectra, and SDS-PAGE, the preparation contains on a per mole basis with PSII only trace amounts of PSI (approximately 0.04), cytochrome b6/f complex (< or = 0.01), and ATPase (< or = 0.05). All of these results indicate that this PSII preparation is to date the most highly purified oxygen-evolving core complex from Synechocystis 6803 that retains all of the reaction centers active for oxygen evolution. As Synechocystis 6803 is being used extensively for site-directed mutagenesis of PSII, this preparation is particularly valuable for spectroscopic and biochemical analyses of PSII from wild-type and from site-directed mutants.
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PMID:Biochemical and spectroscopic characterization of a new oxygen-evolving photosystem II core complex from the cyanobacterium Synechocystis PCC 6803. 816 15

Under native conditions, apocytochrome b(5) exhibits a stable core and a disordered heme-binding region that refolds upon association with the cofactor. The termini of this flexible region are in close proximity, suggesting that loop closure may contribute to the thermodynamic properties of the apocytochrome. A chimeric protein containing 43 residues encompassing the cytochrome loop was constructed using the cyanobacterial photosystem I accessory protein E (PsaE) from Synechococcus sp. PCC 7002 as a structured scaffold. PsaE has the topology of an SH3 domain, and the insertion was engineered to replace its 14-residue CD loop. NMR and optical spectroscopies showed that the hybrid protein (named EbE1) was folded under native conditions and that it retained the characteristics of an SH3 domain. NMR spectroscopy revealed that structural and dynamic differences were confined near the site of loop insertion. Variable-temperature 1D NMR spectra of EbE1 confirmed the presence of a kinetic unfolding barrier. Thermal and chemical denaturations of PsaE and EbE1 demonstrated cooperative, two-state transitions; the stability of the PsaE scaffold was found only moderately compromised by the insertion, with a DeltaT(m) of 8.3 degrees C, a DeltaC(m) of 1.5 M urea, and a DeltaDeltaG degrees of 4.2 kJ/mole. The data implied that the penalty for constraining the ends of the inserted region was lower than the approximately 6.4 kJ/mole calculated for a self-avoiding chain. Extrapolation of these results to cytochrome b(5) suggested that the intrinsic stability of the folded portion of the apoprotein reflected only a small detrimental contribution from the large heme-binding domain.
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PMID:Insertion of the cytochrome b5 heme-binding loop into an SH3 domain. Effects on structure and stability, and clues about the cytochrome's architecture. 1545 37

The open reading frames (ORFs) encoding two potential protein-serine/threonine phosphatases from the cyanobacterium Synechocystis sp. strain PCC 6803 were cloned and their protein products expressed in Escherichia coli cells. The product of ORF sll1033, SynPPM3, is a homologue of the PPM family of protein-serine/threonine phosphatases found in all eukaryotes as well as many members of the Bacteria. Surprisingly, the recombinant protein phosphatase dephosphorylated phosphotyrosine- as well as phosphoserine-containing proteins in vitro. While kinetic analyses indicate that the enzyme was more efficient at dephosphorylating the latter, replacement of Asp608 by asparagine enhanced activity toward a phosphotyrosine-containing protein fourfold. The product of ORF sll1387, SynPPP1, is the sole homolog of the PPP family of protein phosphatases encoded by the genome of Synechocystis sp. strain PCC 6803. Like many other bacterial PPPs, the enzyme dephosphorylated phosphoserine- and phosphotyrosine-containing proteins with comparable efficiencies. However, while previously described PPPs from prokaryotic organisms required the addition of exogenous metal ion cofactors, such as Mg2+ or Mn2+, for activity, recombinantly produced SynPPP1 displayed near-maximal activity in the absence of added metals. Inductively coupled plasma mass spectrometry indicated that recombinant SynPPP1 contained significant quantities, 0.32 to 0.44 mol/mole total, of Mg and Mn. In this respect, the cyanobacterial enzyme resembled eukaryotic members of the PPP family, which are metalloproteins. mRNA encoding SynPPP1 or SynPPM3 could be detected in cells grown under many, but not all, environmental conditions.
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PMID:The protein phosphatases of Synechocystis sp. strain PCC 6803: open reading frames sll1033 and sll1387 encode enzymes that exhibit both protein-serine and protein-tyrosine phosphatase activity in vitro. 1610 28