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Studies on spinach plastocyanin and mutants: Expression in Escherichia coli, folding and function
Göteborgs Universitet.ORCID-id: 0000-0002-3053-4543
1999 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Photosynthesis is a process in which photons from sunlight excite chlorophylls in the thylakoid membranes of plants, algae and cyanobacteria. The photo-oxidised reaction centre chlorophyll P700 is re-reduced by an electron transferred from the soluble, blue copper protein plastocyanin. The oxidised plastocyanin dissociates and binds to the cytochrome b6f complex, where it accepts an electron and a new redox cycle can begin. Plastocyanin has three regions of importance for the interaction with its redox partners, a hydrophobic patch and two acidic (negatively charged) patches. Electrostatic interactions between opposite charges are important for the association and the specificity and stability of the formed complexes.

In this work the interactions with photosystem 1 and cytochrome c have been studied using mutants of plastocyanin. The mutations introduced in the small acidic patch and position 88 of plastocyanin had small effects on the binding to photosystem 1 as compared to the weak binding reported for mutants in the large acidic patch. The affinity was increased by the Glu60Gln, Glu60Lys and Asp61Lys mutations and a more efficient electron transfer was observed for the Gln88Lys mutation. The association between Pc mutated in the small acidic patch and cytochrome c was weakened and the rearrangement hindered by lysines in positions 59 and 60.

The development of an efficient expression system for spinach plastocyanin in the bacterium Escherichia coli made it possible to produce sufficient amounts of isotopically labelled plastocyanin for NMR experiments. This technique was used for solving the structure of the complex between plastocyanin and cytochrome f. The hydrophobic patch on plastocyanin binds to an area close to the heme on cytochrome f. Electrostatic interactions between opposite charges on the two proteins are also important. The short distance from the heme to the copper ligand His87 suggests an electron transfer from the heme via Tyr1 or Phe4 on cytochrome f.

The involvement of specific amino-acid residues in copper binding or folding of plastocyanin has also been examined by site-directed mutagenesis. The copper-binding histidines have been replaced by other amino acids, but no blue protein could be produced. The stability of the different redox forms of copper plastocyanin as well as the zinc protein has also been determined by guanidinium-induced unfolding.

Ort, förlag, år, upplaga, sidor
Göteborg: Göteborgs universitet , 1999. , s. 49
Nyckelord [en]
plastocyanin, electron transfer, copper, protein-protein interactions, protein expression, folding, site-directed mutagenesis, NMR
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
Naturvetenskap
Identifikatorer
URN: urn:nbn:se:his:diva-9088Libris ID: 16258942ISBN: 91-628-3406-1 OAI: oai:DiVA.org:his-9088DiVA, id: diva2:718088
Disputation
1999-05-21, Göteborg, 15:54 (Engelska)
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Handledare
Tillgänglig från: 2014-05-20 Skapad: 2014-05-19 Senast uppdaterad: 2017-11-27Bibliografiskt granskad

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Doctoral Theses from University of Gothenburg / Doktorsavhandlingar från Göteborgs universitet

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Ejdebäck, Mikael

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