| 1. |
- Geijer, Paulina, et al.
(author)
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Simultaneous detection of spin-coupled and decoupled QA- EPR-signals in Photosystem II complexes isolated with isoelectric focusing
- 1998
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In: Photosynthesis Research. - 0166-8595. ; 58:3, s. 231-243
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Journal article (peer-reviewed)abstract
- The Photosystem II multisubunit protein complex can be extracted from thylakoid membranes with non-ionic detergents and subjected to various spectroscopical and biochemical investigations. This paper shows that after extraction with dodecyl-beta-D-maltoside, several Photosystem II complexes could be resolved by isoelectric focusing. Structurally, the various Photosystem II complexes differed from each other in polypeptide composition, especially with regard to the chlorophyll a/b-binding proteins, which gave rise to differing isoelectric points. Functionally, the various Photosystem II complexes differed from each other on the acceptor side, as judged by acceptor side-dependent electron transfer and electron paramagnetic resonance (EPR). The Q_{A}^- Fe2+-signal (g = 1.84), arising from Q_{A}^- spin-coupled to the acceptor-side iron, and a radical signal arising from decoupled Q_{A}^- (g = 2.0045) could be detected simultaneously in some of the Photosystem II complexes, and the amount of each of the two signals were inversely related. The results are discussed in relation to previously known heterogeneities in Photosystem II.
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| 2. |
- Gustavsson, Niklas, et al.
(author)
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A peptide methionine sulfoxide reductase highly expressed in photosynthetic tissue in Arabidopsis thaliana can protect the chaperone-like activity of a chloroplast-localized small heat shock protein.
- 2002
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In: Plant Journal. - 1365-313X. ; 29:5, s. 545-553
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Journal article (peer-reviewed)abstract
- The oxidation of methionine residues in proteins to methionine sulfoxides occurs frequently and protein repair by reduction of the methionine sulfoxides is mediated by an enzyme, peptide methionine sulfoxide reductase (PMSR, EC 1.8.4.6), universally present in the genomes of all so far sequenced organisms. Recently, five PMSR-like genes were identified in Arabidopsis thaliana, including one plastidic isoform, chloroplast localised plastidial peptide methionine sulfoxide reductase (pPMSR) that was chloroplast-localized and highly expressed in actively photosynthesizing tissue (Sadanandom A et al., 2000). However, no endogenous substrate to the pPMSR was identified. Here we report that a set of highly conserved methionine residues in Hsp21, a chloroplast-localized small heat shock protein, can become sulfoxidized and thereafter reduced back to methionines by this pPMSR. The pPMSR activity was evaluated using recombinantly expressed pPMSR and Hsp21 from Arabidopsis thaliana and a direct detection of methionine sulfoxides in Hsp21 by mass spectrometry. The pPMSR-catalyzed reduction of Hsp21 methionine sulfoxides occurred on a minute time-scale, was ultimately DTT-dependent and led to recovery of Hsp21 conformation and chaperone-like activity, both of which are lost upon methionine sulfoxidation (Härndahl et al., 2001). These data indicate that one important function of pPMSR may be to prevent inactivation of Hsp21 by methionine sulfoxidation, since small heat shock proteins are crucial for cellular resistance to oxidative stress.
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| 3. |
- Gustavsson, Niklas, et al.
(author)
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Methionine sulfoxidation of the chloroplast small heat shock protein and conformational changes in the oligomer
- 1999
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In: Protein Science. - : Wiley. - 1469-896X .- 0961-8368. ; 8:11, s. 2506-2512
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Journal article (peer-reviewed)abstract
- The small heat shock proteins (sHsps), which counteract heat and oxidative stress in an unknown way, belong to a protein family of sHsps and alpha-crystallins whose members form large oligomeric complexes. The chloroplast-localized sHsp, Hsp21, contains a conserved methionine- rich sequence, predicted to form an amphipatic helix with the methionines situated along one of its sides. Here, we report how methionine sulfoxidation was detected by mass spectrometry in proteolytically cleaved peptides that were produced from recombinant Arabidopsis thaliana Hsp21, which had been treated with varying concentrations of hydrogen peroxide. Sulfoxidation of the methionine residues in the conserved amphipatic helix coincided with a significant conformational change in the Hsp21 protein oligomer.
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| 4. |
- Gustavsson, Niklas, et al.
(author)
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Substitution of conserved methionines by leucines in chloroplast small heat shock protein results in loss of redox-response but retained chaperone-like activity
- 2001
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In: Protein Science. - 1469-896X. ; 10:9, s. 1785-1793
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Journal article (peer-reviewed)abstract
- During evolution of land plants, a specific motif occurred in the N-terminal domain of the chloroplast-localized small heat shock protein, Hsp21: a sequence with highly conserved methionines, which is predicted to form an amphipathic -helix with the methionines situated along one side. The functional role of these conserved methionines is not understood. We have found previously that treatment, which causes methionine sulfoxidation in Hsp21, also leads to structural changes and loss of chaperone-like activity. Here, mutants of Arabidopsis thaliana Hsp21 protein were created by site-directed mutagenesis, whereby conserved methionines were substituted by oxidation-resistant leucines. Mutants lacking the only cysteine in Hsp21 were also created. Protein analyses by nondenaturing electrophoresis, size exclusion chromatography, and circular dichroism proved that sulfoxidation of the four highly conserved methionines (M49, M52, M55, and M59) is responsible for the oxidation-induced conformational changes in the Hsp21 oligomer. In contrast, the chaperone-like activity was not ultimately dependent on the methionines, because it was retained after methionine-to-leucine substitution. The functional role of the conserved methionines in Hsp21 may be to offer a possibility for redox control of chaperone-like activity and oligomeric structure dynamics.
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| 5. |
- Härndahl, Ulrika, et al.
(author)
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The chaperone-like activity of a small heat shock protein is lost after sulfoxidation of conserved methionines in a surface-exposed amphipathic α-helix
- 2001
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In: BBA - Protein Structure and Molecular Enzymology. - 0167-4838. ; 1545:1-2, s. 227-237
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Journal article (peer-reviewed)abstract
- The small heat shock proteins (sHsps) possess a chaperone-like activity which prevents aggregation of other proteins during transient heat or oxidative stress. The sHsps bind, onto their surface, molten globule forms of other proteins, thereby keeping them in a refolding competent state. In Hsp21, a chloroplast-located sHsp in all higher plants, there is a highly conserved region forming an amphipathic α-helix with several methionines on the hydrophobic side according to secondary structure prediction. This paper describes how sulfoxidation of the methionines in this amphipathic α-helix caused conformational changes and a reduction in the Hsp21 oligomer size, and a complete loss of the chaperone-like activity. Concomitantly, there was a loss of an outer-surface located α-helix as determined by limited proteolysis and circular dichroism spectroscopy. The present data indicate that the methionine-rich amphipathic α-helix, a motif of unknown physiological significance which evolved during the land plant evolution, is crucial for binding of substrate proteins and has rendered the chaperone-like activity of Hsp21 very dependent on the chloroplast redox state.
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| 6. |
- Härndahl, Ulrika, et al.
(author)
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The Chloroplast Small Heat Shock Protein-Purification and Characterization of Pea Recombinant Protein
- 1998
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In: Protein Expression and Purification. - : Elsevier BV. - 1046-5928. ; 14:1, s. 87-96
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Journal article (peer-reviewed)abstract
- We report here on a procedure to obtain large amounts of a chloroplast-localized heat shock protein (HSP21) with unknown structure and function, by using anEscherichia coliexpression system for the pea (Pisum sativum) protein and a purification procedure based on perfusion ion-exchange chromatography. After initial precipitation steps, the sample was applied to cation- and anion-exchange on two columns connected in sequence, which allowed rapid purification of HSP21 in one equilibration and one elution step. The purified recombinant protein had an isoelectric point of 5.0 and appeared in assembled, oligomeric form (approximately 200 kDa) composed of 21-kDa monomers, similar to the native HSP21 protein as detected by immunoblotting in plants after heat-stress treatment. This chloroplast-localized heat shock protein belongs to a special group of small heat shock proteins (sHSPs), which share an evolutionary conserved C-terminal domain with the vertebrate eye lens @a-crystallin. The crystallins are known from both crystallographic and spectroscopic data to be all-@b proteins. In contrast, this paper presents circular dichroism spectroscopy data which shows that the purified recombinant HSP21 oligomer has a content of more than 30% @a-helical secondary structure.
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| 7. |
- Härndahl, Ulrika, et al.
(author)
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The chloroplast small heat shock protein undergoes oxidation-dependent conformational changes and may protect plants from oxidative stress
- 1999
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In: Cell Stress & Chaperones. - 1466-1268. ; 4:2, s. 129-138
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Journal article (peer-reviewed)abstract
- The nuclear-encoded chloroplast-localized Hsp21 is an oligomeric heat shock protein (Hsp), belonging to the protein family of small Hsps and @a-crystallins. We have investigated the effects of high temperature and oxidation treatments on the structural properties of Hsp21, both in purified recombinant form and in transgenicArabidopsis thalianaplants engineered to constitutively overexpress Hsp21. A conformational change was observed for the 300 kDa oligomeric Hsp21 protein during moderate heat stress (=<40oC) ofArabidopsisplants, as judged by a shift to lower mobility in non-denaturing electrophoresis. Similar changes in mobility were observed when purified recombinant Hsp21 protein was subjected to an oxidant. Exposure of Hsp21 protein to temperatures above 70oC led to irreversible aggregation, which was prevented in presence of the reductant dithiothreitol. The transgenic plants that constitutively overexpressed Hsp21 were more resistant to heat stress than were wildtype plants when the heat stress was imposed under high light conditions. These results suggest that the physiological role of Hsp21 involves a response to temperature-dependent oxidative stress.
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| 8. |
- Sundby, Cecilia, et al.
(author)
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Conserved methionines in chloroplasts
- 2005
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In: Biochimica et Biophysica Acta - Proteins and Proteomics. - : Elsevier BV. - 1570-9639. ; 1703:2, s. 191-202
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Research review (peer-reviewed)abstract
- Heat shock proteins counteract heat and oxidative stress. In chloroplasts, a small heat shock protein (Hsp21) contains a set of conserved methionines, which date back to early in the emergence of terrestrial plants. Methionines M49, M52, M55, M59, M62, M67 are located on one side of an amphipathic helix, which may fold back over two other conserved methionines (M97 and M101), to form a binding groove lined with methionines, for sequence-independent recognition of peptides with an overall hydrophobic character. The sHsps protect other proteins from aggregation by binding to their hydrophobic surfaces, which become exposed under stress. Data are presented showing that keeping the conserved methionines in Hsp21 in a reduced form is a prerequisite to maintain such binding. The chloroplast generates reactive oxygen species under both stress and unstressed conditions, but this organelle is also a highly reducing cellular compartment. Chloroplasts contain a specialized isoform of the enzyme, peptide methionine sulfoxide reductase, the expression of which is light-induced. Recombinant proteins were used to measure that this reductase can restore Hsp21 methionines after sulfoxidation. This paper also describes how methionine sulfoxidation-reduction can be directly assessed by mass spectrometry, how methionine-to-leucine substitution affects Hsp21, and discusses the possible role for an Hsp21 methionine sulfoxidation-reduction cycle in quenching reactive oxygen species. (C) 2004 Elsevier B.V. All rights reserved.
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| 9. |
- Zheng, Bo, 1973-
(author)
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Characterisation of the Clp Proteins in Arabidopsis thaliana
- 2003
-
Doctoral thesis (other academic/artistic)abstract
- Unlike in the greenhouse, plants need to cope with many environmental stresses under natural conditions. Among these conditions are drought, waterlogging, excessive or too little light, high or low temperatures, UV irradiation, high soil salinity, and nutrient deficiency. These stress factors can affect many biological processes, and severely retard the growth and development of higher plants, resulting in massive losses of crop yield and wood production. Plants have developed many protective mechanisms to survive and acclimate to stresses, such as the rapid induction of specific molecular chaperones and proteases at the molecular level. Molecular chaperones mediate the correct folding and assembly of polypeptides, as well as repair damaged protein structures caused by stress, while proteases remove otherwise non-functional and potentially cytotoxic proteins. The Clp/Hsp100 family is a new group of chaperones that consists of both constitutive and stress-inducible members. Besides being important chaperones, many Clp/Hsp100 also participate in protein degradation by associating with the proteolytic subunit ClpP to form the Clp protease complex. Higher plants have the greatest number and complexity of Clp proteins than any other group of organisms, and more than 20 different Clp isomers in plants have been identified (Paper I). Because of this diversity, we have adopted a functional genomics approach to characterise all Clp proteins in the model plant Arabidopsis thaliana. Our ongoing research strategy combines genetic, biochemical and molecular approaches. Central to these has been the preparation of transgenic lines for each of the chloroplast Clp isomers. These transgenic lines will be analysed to understand the function and regulation of each chloroplast Clp protein for plant growth and development.In Paper II, an Arabidopsis thaliana cDNA was isolated that encodes a homologue of bacterial ClpX. Specific polyclonal antibodies were made and used to localise the ClpX homologue to plant mitochondria, consistent with that predicted by computer analysis of the putative transit peptide. In addition to ClpX, a nuclear-encoded ClpP protein, termed ClpP2, was identified from the numerous ClpP isomers in Arabidopsis and was also located in mitochondria. Relatively unchanged levels of transcripts for both clpX and clpP2 genes were detected in various tissues and under different growth conditions. Using β-casein as a substrate, plant mitochondria possessed an ATP-stimulated, serine-type proteolytic activity that could be strongly inhibited by antibodies specific for ClpX or ClpP2, suggesting an active ClpXP protease.In Paper III, four nuclear-encoded Clp isomers were identified in Arabidopsis thaliana: ClpC1 and ClpP3-5. All four proteins are localized within the stroma of chloroplasts, along with the previously identified ClpD, ClpP1 and ClpP6 proteins. Potential differential regulation among these Clp proteins was analysed at both the mRNA and protein level. A comparison between different tissues showed increasing amounts of all plastid Clp proteins from roots to stems to leaves. The increases in protein were mirrored at the mRNA level for most ClpP isomers but not for ClpC1, ClpC2 and ClpD and ClpP5, which exhibited little change in transcript levels. Potential stress induction was also tested for all chloroplast Clp proteins by a series of brief and prolonged stress conditions. The results reveal that these proteins, rather than being rapidly induced stress proteins, are primarily constitutive proteins that may also be involved in plant acclimation to different physiological conditions. In Paper IV, antisense repression transgenic lines of clpP4 were prepared and then later characterised. Within the various lines screened, up to 90% of ClpP4 protein content was specifically repressed, which also led to the down-regulation of ClpP3 and ClpP5 protein contents. The repression of clpP4 mRNA retarded the development of chloroplasts and the differentiation of leaf mesophyll cells, resulting in chlorotic phenotypes. The chlorosis was more severe in young than in mature leaves due likely to the developmental expression pattern of the ClpP4 protein. Chlorotic plants eventually turned green upon aging, accompanied by a recovery in the amount of the ClpP4 protein. The greening process could be affected by the light quantity, either by altering the photoperiod or light intensity.
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