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Site-directed mutagenesis of the D2 protein in the green alga 'Chlamydomonas reinhardtii'

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Title: Site-directed mutagenesis of the D2 protein in the green alga 'Chlamydomonas reinhardtii'
Authors: Andronis, Christos
Item Type: Thesis or dissertation
Abstract: The D2 polypeptide together with the D1 polypeptide are key components of the photosystem II (PS II) complex which is involved in photosynthetic oxygen evolution. This thesis describes the use of site-directed mutagenesis to introduce specific amino acid changes in the D2 protein of Chlamydomonas reinhardtii. The mutations were constructed at positions D2-Leu205 and D2-Thr2. Plasmids carrying mutated copies of the psbD gene (which encodes D2) were introduced into the chloroplast genome of C. reinhardtii by means of a particle gun. In order to identify the transformants, a selectable marker encoding resistance to spectinomycin, was inserted either upstream or downstream of the psbD gene. Genetic characterisation of the mutants was carried out using Southern blotting, PCR and DNA sequencing. D2-Leu205 is thought to be analogous to residue Tyr210 of the M subunit of the reaction centre (RC) of the purple photosynthetic bacterium Rhodobacter sphaeroides. Previous mutagenesis experiments have shown that the replacement of M-Tyr210 by leucine reduces the rate of the primary charge separation in purple bacterial RCs. Urns, the mutation Leu205Tyr was constructed in order to study whether the rate of primary charge separation is increased in this mutant. The Leu205Tyr mutant could grow photoautotrophically at wild type rates. However, its light-saturated PS II activity was reduced to ~62% compared to the wild type. PS II RCs isolated from a "wild type” control transformant and from the Leu205Tyr mutant showed in ultrafast transient absorption measurements that the rate of formation of the radical pair was slowed down in the mutant (lifetime of 40±7 ps compared to 21±2 ps for the wild type PS II RCs). Also the quantum yield of charge separation was reduced by about 50%. Fluorescence and thermoluminescence measurements in whole cells were also consistent with a reduced quantum yield of charge separation in PS II. Mutations Thr2Ala and Thr2Ser were constructed in order to study the role of D2 phosphorylation in C. reinhardtii. Biochemical characterisation of the Thr2Ala and Thr2Ser mutants indicated that both strains could grow photoautotrophically at wild type rates. Moreover, oxygen evolution, fluorescence and thermoluminescence measurements suggested that these site-directed mutations have only minor effects on PS II function. In addition to these experiments, attempts were carried out to find out whether D2 can be phosphorylated in C. reinhardtii. These attempts included in vivo labelling of whole cells with [32P]-orthophosphate, in vitro labelling of thylakoid membranes with [y-32P]-ATP, pulse-chase labelling of cells with [uC]-acetate and the use of a monospecific antibody against phosphothreonine. The data obtained from these experiments did not provide any indication that D2 can be phosphorylated in C. reinhardtii.
Content Version: Open access
Date Awarded: 1996
URI: http://hdl.handle.net/10044/1/73208
Copyright Statement: Creative Commons Attribution NonCommercial NoDerivatives Licence
Supervisor: Nixon, Dr. Peter
Telfer, Dr. Alison
Sponsor/Funder: State Scholarship Foundation of Greece (I.K.Y.)
Department: Department of Biochemistry
Publisher: Imperial College London
Qualification Level: Doctoral
Qualification Name: Doctor of Philosophy (PhD)
Appears in Collections:University of London awarded theses - Imperial authors