Ray, Nicola; (2001) Genetic manipulation of photosystem two polypeptides in Chlamydomonas reinhardtii. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Photosystem two (PSII) catalyses the light-driven oxidation of water to molecular oxygen and protons in oxygenic photosynthesis. In order to elucidate the structure and function of PSII a multidisciplinary approach has been underway over recent years which has included biochemical, biophysical, molecular, genetic and crystallographic studies of PSII from a number of different organisms. The molecular approach can provide an insight into the function of particular polypeptides and their amino acid residues by carrying out deletions of polypeptides or altering specific amino acids thought to be of importance in the function of PSII. Chlamydomonas reinhardtii is amenable to genetic manipulation and is a model system for the study of PSII. Site-directed mutants have been created in an attempt to elucidate the role of amino acid residues important in the mechanism of water oxidation. Unfortunately, biophysical analysis of PSII in C. reinhardtii has been hindered by the poor range of highly active PSII preparations compared to those available from higher plants. This thesis describes the construction of a novel, stable mutant of C reinhardtii in which the PsbH polypeptide, a component of PSII, is tagged with six histidine residues at the carboxyl terminal end. This tag facilitates a rapid PSII purification procedure using nickel column affinity chromatography. The C. reinhardtii PSII is free from Photosystem I (PSI) contamination and is suitable for biophysical background allowing detailed studies of PSII by techniques that were previously difficult or impossible to utilise. The construction of several PSII mutants created in the his-tagged background are described and their analysis presented. Both the D1 and D2 polypeptides were mutated allowing us to investigate the mechanism of water oxidation. In addition to analysis of PSII site-directed mutants further attempts at epitope tagging the C. reinhardtii PsbH polypeptide are presented. Epitope tagging has become a standard method which enables rapid and effective characterisation, purification and in vivo localisation of the protein products of tagged genes. This work presents attempts at tagging the PsbH polypeptide with both six and ten histidine residues, a haemagglutinin tag and also with the mature pea plastocyanin sequence.

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