| 1. |
- Edvardsson, Anna, et al.
(författare)
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The major peptidyl-prolyl isomerase activity in thylakoid lumen of plant chloroplasts belongs to a novel cyclophilin TLP20
- 2003
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Ingår i: FEBS Letters. - 0014-5793. ; 542:1-3, s. 137-141
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Tidskriftsartikel (refereegranskat)abstract
- Fractionation of proteins from the thylakoid lumen of spinach chloroplasts combined with peptidyl-prolyl cis/trans isomerase (PPIase) measurements revealed a major isomerase activity that was ascribed to a novel enzyme TLP20 ( hylakoid umen PIase of kDa). TLP20 was inhibited by cyclosporin A and mass spectrometric sequencing of tryptic peptides confirmed its classification as a cyclophilin. Genes encoding similar putative thylakoid cyclophilins with a unique insert of three amino acids NPV in their N-termini were found in chromosome 5 of both Arabidopsis and rice. TLP20 is suggested to be the major PPIase and protein folding catalyst in the thylakoid lumen of plant chloroplasts.
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| 2. |
- Eshaghi, Said, 1972-
(författare)
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Photosystem II : organization, function and regulation
- 2001
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- What makes plants, green algae and cyanobacteria so remarkable is their ability to convert light energy to chemical energy and at the same time evolve oxygen. In order to do so, they have developed a complex molecular machinery involving pigment and co-factor binding multi-protein complexes. Photosystem II is one of the protein complexes responsible for trapping photons and carrying out this energy conversion in a process where water is oxidized and molecular oxygen is released.In this thesis, I have studied the organizational, functional and regulatory aspects of photosystem II. The results from these investigations can be summarized as follows: (i) A simple method was developed to isolate a highly pure, intact and active photosystem II complex containing the light-harvesting LHCII directly from thylakoid membranes. (ii) This so-called photosystem II supercomplex has properties similar to the membrane fragments enriched in photosystem II (BBY), but contain less of other non-photosystem II proteins, hence providing a better experimental alternative to these membrane fragments. (iii) A CP47 containing photosystem II reaction center with neither extrinsic proteins nor CP43 could be photoactivated to evolve oxygen, demonstrating that the latter proteins are not required for water oxidation. (iv) The re-association of the cyclophilin-like protein (TLP40) with the lumenal surface of the thylakoid membranes, which regulates dephosphorylation and turnover of the photosystem II reaction center protein, is dependent on both temperature and pH. (v) The association between TLP40 and the photosynthetic membranes may also involve the cytochrome b6/f complex.Finally, a model is presented discussing the possibilities that a GTP-33-kDa complex could regulate the interaction between TLP40 and the thylakoid membranes in light.
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| 3. |
- Lundbäck, Anna-Karin, et al.
(författare)
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Exploring the activity of tobacco etch virus protease in detergent solutions
- 2008
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 382:1, s. 69-71
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Tidskriftsartikel (refereegranskat)abstract
- Tobacco etch virus (TEV) protease is generally used to remove affinity tags from target proteins. It has been reported that some detergents inhibit the activity of this protease, and therefore should be avoided when removing affinity tags from membrane proteins. The aim of this study was to explore and evaluate this further. Hence, affinity tag removal with TEV protease was tested from three membrane proteins (a Pgp synthase and two CorA homologs) in the presence of 16 different detergents commonly used in membrane protein purification and crystallization. We observed that in the presence of the same detergent (Triton X-100), TEV protease could remove the affinity tag completely from one protein (CorA) but not from another protein (Pgp synthase). There was also a large variation in yield of cleaved membrane protein in different detergents, which probably depends on features of the protein-detergent complex. These observations show that, contrary to an earlier report, detergents do not inhibit the enzymatic activity of the TEV protease.
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| 4. |
- Lundin, Björn, et al.
(författare)
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Subsequent events to GTP binding by the plant PsbO protein: structural changes, GTP hydrolysis and dissociation from the photosystem II complex
- 2007
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Ingår i: Biochimica et Biophysica Acta - Bioenergetics. - Amsterdam : Elsevier BV. - 0005-2728 .- 1879-2650. ; 1767:6, s. 500-508
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Tidskriftsartikel (refereegranskat)abstract
- Besides an essential role in optimizing water oxidation in photosystem II (PSII), it has been reported that the spinach PsbO protein binds GTP [C. Spetea, T. Hundal, B. Lundin, M. Heddad, I. Adamska, B. Andersson, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 1409–1414]. Here we predict four GTP-binding domains in the structure of spinach PsbO, all localized in the β-barrel domain of the protein, as judged from comparison with the 3D-structure of the cyanobacterial counterpart. These domains are not conserved in the sequences of the cyanobacterial or green algae PsbO proteins. MgGTP induces specific changes in the structure of the PsbO protein in solution, as detected by circular dichroism and intrinsic fluorescence spectroscopy. Spinach PsbO has a low intrinsic GTPase activity, which is enhanced fifteen-fold when the protein is associated with the PSII complex in its dimeric form. GTP stimulates the dissociation of PsbO from PSII under light conditions known to also release Mn2+ and Ca2+ ions from the oxygen-evolving complex and to induce degradation of the PSII reaction centre D1 protein. We propose the occurrence in higher plants of a PsbO-mediated GTPase activity associated with PSII, which has consequences for the function of the oxygen-evolving complex and D1 protein turnover.
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| 5. |
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| 6. |
- Molina, Daniel Martinez, et al.
(författare)
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Expression and purification of the recombinant membrane protein YidC : A case studyfor increased solubility and stability
- 2008
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Ingår i: Protein Expression and Purification. - : Elsevier BV. - 1046-5928 .- 1096-0279. ; 62:1, s. 49-52
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Tidskriftsartikel (refereegranskat)abstract
- YidC is an inner membrane protein from Escherichia coli and is an essential component in insertion, trans- location and assembly of membrane proteins in the membranes. Previous purification attempts resulted in heavy aggregates and precipitated protein at later stages of purification. Here we present a rapid and straightforward stability screening strategy based on gel filtration chromatography, which requires as little as 10 lg of protein and takes less than 15 min to perform. With this technique, we could rapidly screen several buffers in order to identify an optimum condition that stabilizes purified YidC. After optimization we could obtain several milligrams of purified YidC that could be easily prepared at high con- centrations and that was stable for weeks at +4 C. The isolated protein is thus well suited for structural studies.
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| 7. |
- Niegowski, Damian, 1978-, et al.
(författare)
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A simple strategy towards membrane protein purification and crystallization
- 2006
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Ingår i: International Journal of Biological Macromolecules. - : Elsevier BV. - 0141-8130 .- 1879-0003. ; 39:1-3, s. 83-87
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Tidskriftsartikel (refereegranskat)abstract
- A simple and cost-efficient detergent screening strategy has been developed, by which a number of detergents were screened for their efficiency to extract and purify the recombinant ammonium/ammonia channel, AmtB, from Escherichia coli, hence selecting the most efficient detergents prior to large-scale protein production and crystallization. The method requires 1 ml cell culture and is a combination of immobilized metal ion affinity chromatography and filtration steps in 96-well plates. Large-scale protein purification and subsequent crystallization screening resulted in AmtB crystals diffracting to low resolution with three detergents. This strategy allows exclusion of detergents with the lowest probability in yielding protein crystals and selecting those with higher probability, hence, reducing the number of detergents to be screened prior to large-scale membrane protein purification and perhaps also crystallization.
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| 8. |
- Niegowski, Damian, 1978-, et al.
(författare)
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Crystal structure of a divalent metal ion transporter CorA at 2.9 angstrom resolution
- 2006
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 313:5785, s. 354-357
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Tidskriftsartikel (refereegranskat)abstract
- CorA family members are ubiquitously distributed transporters of divalent metal cations and are considered to be the primary Mg2 transporter of Bacteria and Archaea. We have determined a 2.9 angstrom resolution structure of CorA from Thermotoga maritima that reveals a pentameric cone– shaped protein. Two potential regulatory metal binding sites are found in the N-terminal domain that bind both Mg2+ and Co2+. The structure of CorA supports an efflux system involving dehydration and rehydration of divalent metal ions potentially mediated by a ring of conserved aspartate residues at the cytoplasmic entrance and a carbonyl funnel at the periplasmic side of the pore.
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| 9. |
- Palombo, Isolde, 1984-
(författare)
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The social life of a membrane protein; It's complex
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Membrane proteins are key players in many biological processes. Since most membrane proteins are assembled into oligomeric complexes it is important to understand how they interact with each other. Unfortunately however, the assembly process (i.e. their social life) remains poorly understood. In the work presented in this thesis I have investigated when and how membrane proteins assemble with each other and their cofactors to form functional units. We have shown that that cofactor insertion in the hetero-tetrameric cytochrome bo3 occurs at an early state in the assembly process. We also found that the pentameric CorA magnesium ion channel is stabilised by different interactions depending on the magnesium ion concentration in the cell. These studies indicate that the assembly of a functional unit is a dynamic process, which is a result of many different forces. By studying the assembly of membrane proteins we have obtained a deeper insight into their function, which cannot be explained by static crystal structures.
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| 10. |
- Xia, Yu, et al.
(författare)
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Co(2+) Selectivity of Thermotoga maritima CorA and Its Inability to Regulate Mg(2+) Homeostasis Present a New Class of CorA Proteins
- 2011
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 286:18
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Tidskriftsartikel (refereegranskat)abstract
- CorA is a family of divalent cation transporters ubiquitously present in bacteria and archaea. Although CorA can transport both Mg(2+) and Co(2+) almost equally well, its main role has been suggested to be that of primary Mg(2+) transporter of prokaryotes and hence the regulator of Mg(2+) homeostasis. The reason is that the affinity of CorA for Co(2+) is relatively low and thus considered non-physiological. Here, we show that Thermotoga maritima CorA (TmCorA) is incapable of regulating the Mg(2+) homeostasis and therefore cannot be the primary Mg(2+) transporter of T. maritima. Further, our in vivo experiments confirm that TmCorA is a highly selective Co(2+) transporter, as it selects Co(2+) over Mg(2+) at > 100 times lower concentrations. In addition, we present data that show TmCorA to be extremely thermostable in the presence of Co(2+). Mg(2+) could not stabilize the protein to the same extent, even at high concentrations. We also show that addition of Co(2+), but not Mg(2+), specifically induces structural changes to the protein. Altogether, these data show that TmCorA has the role of being the transporter of Co(2+) but not Mg(2+). The physiological relevance and requirements of Co(2+) in T. maritima is discussed and highlighted. We suggest that CorA may have different roles in different organisms. Such functional diversity is presumably a reflection of minor, but important structural differences within the CorA family that regulate the gating, substrate selection, and transport.
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