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Franzén, Lars-Gunnar
Publications (7 of 7) Show all publications
Farkas, D., Franzén, L.-G. & Hansson, Ö. (2011). Cloning, expression and purification of the luminal domain of spinach photosystem 1 subunit PsaF functional in binding to plastocyanin and with a disulfide bridge required for folding. Protein Expression and Purification, 78(2), 156-166
Open this publication in new window or tab >>Cloning, expression and purification of the luminal domain of spinach photosystem 1 subunit PsaF functional in binding to plastocyanin and with a disulfide bridge required for folding
2011 (English)In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 78, no 2, p. 156-166Article in journal (Refereed) Published
Abstract [en]

The photosystem 1 subunit PsaF is involved in the docking of the electron-donor proteins plastocyanin and cytochrome c6 in eukaryotic photosynthetic organisms. Here we report the expression, purification and basic characterization of the luminal domain of spinach PsaF, encompassing amino-acid residues 1-79. The recombinant protein was expressed in Escherichia coli BL21 (DE3) using a pET32 Xa/LIC thioredoxin fusion system. The thioredoxin fusion protein contained a His6 tag and was removed and separated from PsaF through proteolytic digestion by factor Xa followed by immobilized metal affinity chromatography. Further purification with size-exclusion chromatography resulted in a final yield of approximately 6 mg PsaF from one liter growth medium. The correct identity after the factor Xa treatment of PsaF was verified by FT-ICR mass spectrometry which also showed that the purified protein contains an intact disulfide bridge between Cys residues 6 and 38. Secondary structure and folding was further explored using far-UV CD spectroscopy indicating a α-helical content in agreement with the 3.3 Å-resolution crystal structure of photosystem I Ref. [5] and a helix-coil transition temperature of 29 °C. Thermofluorescence studies showed that the disulfide bridge is necessary to keep the overall fold of the protein and that hydrophobic regions become exposed at 50-65 °C depending on the ionic strength. The described expression and purification procedure can be used for isotopic labeling of the protein and 15N-HSQC NMR studies indicated a slow or intermediate exchange between different conformations of the prepared protein and that it belongs to the molten-globule structural family. Finally, by using a carboxyl- and amine-reactive zero-length crosslinker, we have shown that the recombinant protein binds to plastocyanin by a specific, native-like, electrostatic interaction, hence, confirming its functionality.

Place, publisher, year, edition, pages
San Diego: Academic Press, 2011
Keywords
NMR, Photosynthesis, Plastocyanin, Protein–protein interaction, PsaF, Thioredoxin
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:hh:diva-15882 (URN)10.1016/j.pep.2011.02.007 (DOI)000292020400007 ()21354471 (PubMedID)2-s2.0-79958155132 (Scopus ID)
Available from: 2011-08-10 Created: 2011-08-10 Last updated: 2018-03-22Bibliographically approved
Eriksson, K., Clarke, A., Franzén, L.-G., Kuylenstierna, M., Martinez, K. & Blanck, H. (2009). Community-Level Analysis of psbA Gene Sequences and Irgarol Tolerance in Marine Periphyton. Applied and Environmental Microbiology, 75(4), 897-906
Open this publication in new window or tab >>Community-Level Analysis of psbA Gene Sequences and Irgarol Tolerance in Marine Periphyton
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2009 (English)In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 75, no 4, p. 897-906Article in journal (Refereed) Published
Abstract [en]

This study analyzes psbA gene sequences, predicted D1 protein sequences, species relative abundance, and pollution-induced community tolerance in marine periphyton communities exposed to the antifouling compound Irgarol 1051. The mechanism of action of Irgarol is the inhibition of photosynthetic electron transport at photosystem II by binding to the D1 protein. The metagenome of the communities was used to produce clone libraries containing fragments of the psbA gene encoding the D1 protein. Community tolerance was quantified with a short-term test for the inhibition of photosynthesis. The communities were established in a continuous flow of natural seawater through microcosms with or without added Irgarol. The selection pressure from Irgarol resulted in an altered species composition and an inducted community tolerance to Irgarol. Moreover, there was a very high diversity in the psbA gene sequences in the periphyton, and the composition of psbA and D1 fragments within the communities was dramatically altered by increased Irgarol exposure. Even though tolerance to this type of compound in land plants often depends on a single amino acid substitution (Ser(264)-> Gly) in the D1 protein, this was not the case for marine periphyton species. Instead, the tolerance mechanism likely involves increased degradation of D1. When we compared sequences from low and high Irgarol exposure, differences in nonconserved amino acids were found only in the so-called PEST region of D1, which is involved in regulating its degradation. Our results suggest that environmental contamination with Irgarol has led to selection for high-turnover D1 proteins in marine periphyton communities at the west coast of Sweden.

Place, publisher, year, edition, pages
Washington, D.C.: American Society for Microbiology, 2009
Keywords
PICT, Ecotoxicogenomics, D1 protein, PEST region, Antifouling, Amination, Amines, Amino acids, Biochemistry, Cloning, Degradation, Gene encoding, Genes, Organic acids, Photosynthesis, Seawater, Marine pollution
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:hh:diva-2970 (URN)10.1128/AEM.01830-08 (DOI)000263119100002 ()19088321 (PubMedID)2-s2.0-59949087740 (Scopus ID)2082/3373 (Local ID)2082/3373 (Archive number)2082/3373 (OAI)
Available from: 2009-09-17 Created: 2009-09-17 Last updated: 2018-03-23Bibliographically approved
Cardol, P., Figueroa, F., Remacle, C., Franzén, L.-G. & González-Halphen, D. (2009). Oxidative Phosphorylation: Building blocks and related components. In: Elizabeth H. Harris, David B. Stern & George B. Witman (Ed.), The Chlamydomonas Sourcebook: Organellar and Metabolic Processes, Volume 2, Second Edition (pp. 469-502). Oxford: Academic Press
Open this publication in new window or tab >>Oxidative Phosphorylation: Building blocks and related components
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2009 (English)In: The Chlamydomonas Sourcebook: Organellar and Metabolic Processes, Volume 2, Second Edition / [ed] Elizabeth H. Harris, David B. Stern & George B. Witman, Oxford: Academic Press, 2009, p. 469-502Chapter in book (Other academic)
Abstract [en]

This chapter summarizes the knowledge of the oxidative phosphorylation (OXPHOS) constituents of Chlamydomonas and of the components involved in their biogenesis and addresses alternate dehydrogenases and oxidases which are particular to photosynthetic organisms, and several other mitochondrial components related to OXPHOS. Reference to the components of Polytomella sp., a colorless alga closely related to Chlamydomonas is clearly made. The main complexes involved in electron transport seem to share a similar number of subunits, and many of the algal polypeptides have plant homologues. Some differences are apparent, such as the presence of a fragmented COX2 subunit, which seems to be unique to chlorophyte algae. OXPHOS is defined as an electron transfer chain driven by substrate oxidation that is coupled to the synthesis of ATP through an electrochemical transmembrane gradient. The characterization of Arabidopsis mitochondrial components through proteomic approaches has advanced significantly. As a unicellular organism, Chlamydomonas offers the unique opportunity to study organelle-organelle interactions, particularly between mitochondria and chloroplasts. It has become evident that crosstalk between these organelles takes place, mainly through intracellular metabolite pools. © 2009 Elsevier Inc. All rights reserved.

Place, publisher, year, edition, pages
Oxford: Academic Press, 2009
Keywords
Chlamydomonas, mitochondrion, proteins
National Category
Microbiology
Identifiers
urn:nbn:se:hh:diva-2969 (URN)10.1016/B978-0-12-370873-1.00021-6 (DOI)2-s2.0-84878398933 (Scopus ID)2082/3372 (Local ID)0-12-370875-3 (ISBN)2082/3372 (Archive number)2082/3372 (OAI)
Note

Funding: Work in our laboratories is supported by grants 56619 from CONACyT (Mexico), IN217108 from DGAPA, UNAM (Mexico) and by grants from the Belgian FRFC (2.4587.04 and 2.4582.05) C.R and from the Belgian FNRS (F.4735.06) to P.C.

Available from: 2009-09-17 Created: 2009-09-17 Last updated: 2018-07-12Bibliographically approved
Figueroa-Martinez, F., Funes, S., Franzén, L.-G. & González-Halphen, D. (2008). Reconstructing the mitochondrial protein import machinery of Chlamydomonas reinhardtii. Genetics, 179(1), 149-155
Open this publication in new window or tab >>Reconstructing the mitochondrial protein import machinery of Chlamydomonas reinhardtii
2008 (English)In: Genetics, ISSN 0016-6731, E-ISSN 1943-2631, Vol. 179, no 1, p. 149-155Article in journal (Refereed) Published
Abstract [en]

In Chlamydomonas reinhardtii several nucleus-encoded proteins that participate in the mitochondrial oxidative phosphorylation are targeted to the organelle by unusually long mitochondrial targeting sequences. Here, we explored the components of the mitochondrial import machinery of the green alga. We mined the algal genome, searching for yeast and plant homologs, and reconstructed the mitochondrial import machinery. All the main translocation components were identified in Chlamydomonas as well as in Arabidopsis thaliana and in the recently sequenced moss Physcomitrella patens. Some of these components appear to be duplicated, as is the case of Tim22. In contrast, several yeast components that have relatively large hydrophilic regions exposed to the cytosol or to the intermembrane space seem to be absent in land plants and green algae. If present at all, these components of plants and algae may differ significantly from their yeast counterparts. We propose that long mitochondrial targeting sequences in some Chlamydomonas mitochondrial protein precursors are involved in preventing the aggregation of the hydrophobic proteins they carry.

Place, publisher, year, edition, pages
Austin, Tex.: The Genetics Society, 2008
Keywords
Chlamydomonas, Mitochondrial protein, Cytochrome-C-Oxidase, Intracellular gene-transfer, Iron-sulfur protein, Membrane-proteins, Intermembrance space, Yeast mitochondria, Export machinery, Outer-membrane, Inner membrane, CDNA sequence
National Category
Genetics
Identifiers
urn:nbn:se:hh:diva-1879 (URN)10.1534/genetics.108.087965 (DOI)000256312000014 ()18493047 (PubMedID)2-s2.0-52049084400 (Scopus ID)2082/2274 (Local ID)2082/2274 (Archive number)2082/2274 (OAI)
Available from: 2008-09-10 Created: 2008-09-10 Last updated: 2018-03-23Bibliographically approved
Funes, S., Franzén, L.-G. & González-Halphen, D. (2007). Chlamydomonas reinhardtii: the model of choice to study mitochondria from unicellular photosynthetic organisms.. Methods in Molecular Biology, 372, 137-149
Open this publication in new window or tab >>Chlamydomonas reinhardtii: the model of choice to study mitochondria from unicellular photosynthetic organisms.
2007 (English)In: Methods in Molecular Biology, ISSN 1064-3745, Vol. 372, p. 137-149Article in journal (Other academic) Published
Abstract [en]

Chlamydomonas reinhardtii is a model organism to study photosynthesis, cellular division, flagellar biogenesis, and, more recently, mitochondrial function. It has distinct advantages in comparison to higher plants because it is unicellular, haploid, and amenable to tetrad analysis, and its three genomes are subject to specific transformation. It also has the possibility to grow either photoautotrophically or heterotrophically on acetate, making the assembly of the photosynthetic machinery not essential for cell viability. Methods developed allow the isolation of C. reinhardtii mitochondria free of thylakoid contaminants. We review the general procedures used for the biochemical characterization of mitochondria from this green alga.

Place, publisher, year, edition, pages
Clifton, NJ: Humana Press, 2007
Keywords
Mitochondria, Blue native polyacrylamide gel electrophoresis, Chlamydomonas reinhardtii, Green alga, Import - Competent mitochondria, OXPHOS
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:hh:diva-1880 (URN)10.1007/978-1-59745-365-3_10 (DOI)18314723 (PubMedID)2-s2.0-40949153878 (Scopus ID)2082/2275 (Local ID)978-1-58829-667-2 (ISBN)978-1-59745-365-3 (ISBN)2082/2275 (Archive number)2082/2275 (OAI)
Note

Ingår i: MitochondriaPractical Protocols, ISBN: 978-1-58829-667-2 (Print) 978-1-59745-365-3 (Online)

Available from: 2008-09-11 Created: 2008-09-11 Last updated: 2018-03-23Bibliographically approved
Atteia, A., van Lis, R., Wetterskog, D., Gutiérrez-Cirlos, E.-B., Ongay-Larios, L., Franzén, L.-G. & González-Halphen, D. (2003). Structure, organization and expression of the genes encoding mitochondrial cytochrome c1 and the Rieske iron-sulfur protein in Chlamydomonas reinhardtii. Molecular Genetics and Genomics, 268(5), 637-644
Open this publication in new window or tab >>Structure, organization and expression of the genes encoding mitochondrial cytochrome c1 and the Rieske iron-sulfur protein in Chlamydomonas reinhardtii
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2003 (English)In: Molecular Genetics and Genomics, ISSN 1617-4615, E-ISSN 1617-4623, Vol. 268, no 5, p. 637-644Article in journal (Refereed) Published
Abstract [en]

The sequence and organization of the Chlamydomonas reinhardtii genes encoding cytochrome c 1 ( Cyc1) and the Rieske-type iron-sulfur protein ( Isp), two key nucleus-encoded subunits of the mitochondrial cytochrome bc 1 complex, are presented. Southern hybridization analysis indicates that both Cyc1 and Isp are present as single-copy genes in C. reinhardtii. The Cyc1 gene spans 6404 bp and contains six introns, ranging from 178 to 1134 bp in size. The Isp gene spans 1238 bp and contains four smaller introns, ranging in length from 83 to 167 bp. In both genes, the intron/exon junctions follow the GT/AG rule. Internal conserved sequences were identified in only some of the introns in the Cyc1 gene. The levels of expression of Isp and Cyc1 genes are comparable in wild-type C. reinhardtii cells and in a mutant strain carrying a deletion in the mitochondrial gene for cytochrome b (dum-1). Nevertheless, no accumulation of the nucleus-encoded cytochrome c 1 or of core proteins I and II was observed in the membranes of the respiratory mutant. These data show that, in the green alga C. reinhardtii, the subunits of the cytochrome bc1 complex fail to assemble properly in the absence of cytochrome b.

Place, publisher, year, edition, pages
Springer Verlag, 2003
Keywords
Expression of nucleus-encoded genes, Introns, Internal conserved sequences, Mitochondrial targeting sequences, Assembly of protein complexes
National Category
Medical Genetics
Identifiers
urn:nbn:se:hh:diva-421 (URN)10.1007/s00438-002-0779-x (DOI)000181616000008 ()12589438 (PubMedID)2-s2.0-0037293953 (Scopus ID)2082/759 (Local ID)2082/759 (Archive number)2082/759 (OAI)
Available from: 2007-01-24 Created: 2007-01-24 Last updated: 2018-03-23Bibliographically approved
Stevens, D., Atteia, A., Franzén, L.-G. & Purton, S. (2001). Cycloheximide resistance conferred by novel mutations in ribosomal protein L41 of Chlamydomonas reinhardtii. Molecular General Genetics, 264(6), 790-795
Open this publication in new window or tab >>Cycloheximide resistance conferred by novel mutations in ribosomal protein L41 of Chlamydomonas reinhardtii
2001 (English)In: Molecular General Genetics, ISSN 0026-8925, E-ISSN 1432-1874, Vol. 264, no 6, p. 790-795Article in journal (Refereed) Published
Abstract [en]

Although most eukaryotic cells are sensitive to the 80S ribosome inhibitor cycloheximide (CYH), naturally occurring CYH resistance is widespread amongst yeast species. The primary determinant of resistance appears to be a single residue within ribosomal protein L41; resistance is acquired by the substitution of a conserved proline (P-56) by a glutamate residue. We have isolated the L41 gene (RPL41) from the green alga Chlamydomonas and investigated the molecular basis of CYH resistance in various mutant strains. In both the wild-type strain and the mutant act-1, a proline is found at the key position in L41.; However, analysis of six independently isolated act-2 mutants reveals that all have point mutations that replace the proline with either leucine or serine. Of the two changes, the leucine mutation confers significantly higher levels of CYH resistance. This work identifies the ACT-2 locus as RPL41 and provides a possible dominant marker for nuclear transformation of C. reinhardtii.

Place, publisher, year, edition, pages
Berlin: Springer-Verlag, 2001
Keywords
Chlamydomonas, Cycloheximide resistance, Ribosomal protein L41, Intron
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:hh:diva-1878 (URN)10.1007/s004380000368 (DOI)000167341100007 ()11254126 (PubMedID)2-s2.0-0035089265 (Scopus ID)2082/2273 (Local ID)2082/2273 (Archive number)2082/2273 (OAI)
Available from: 2008-09-10 Created: 2008-09-10 Last updated: 2018-03-23Bibliographically approved
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