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Title: The light harvesting 2 antenna complex of Rhodobacter sphaeroides
Author: Timney, John Anthony
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2007
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This thesis presents an analysis of the contribution that the polypeptide Puc2B makes to the binding of BChl(s) within the purified LH2 complexes. Mass spectroscopy established for the first time that Puc2B is incorperated within the LH2 in native complexes. The effect the second puc operon has on the ability of the cell to adapt to high light conditions was measured; it was shown that the puc2BA gene pair is crucial in modulating levels of LH2 complex assembly under these conditions. Using AFM, initial attempts were made to analyse the effects of the second puc operon upon the membrane architecture. Images of Rba. sphaeroides 2.4.1. ,12BA membranes were obtained, with large scale hexagonally packed arrays of LH2 observed for the first time. The development of a purification protocol for a LH2 complex possessing only PuclB as its ~-polypeptide (LH2-l B lA) is described. The crystallisation of LH2 complexes purified using this protocol is detailed, along with the unsuccessful attempts to obtain structural data from the resulting crystals by X-ray crystallography. A previously undescribed protein, RSP6l24, was identified during the purification of LH2-lB 1 A. A bio-informatic analysis of this protein is presented. Although its role within the organism is still unclear, there is evidence which suggests that strong electrostatic interactions between RSP6l24 and the extrinsic regions of the LH2 complex may exist. Contamination of the purified LH2-l B 1 A sample with RSP6124 provides an alternative explanation for current failures to obtain crystals of sufficient quality to yield high resolution diffraction data. Two new deletion strains of Rba. sphaeroides 2.4.1. that lack the ability to assemble any LH2, LH 1 or RC complex have been created. Assembly levels of the LH2 complex in the deletion strains lacking both puc operons were shown to be markedly lower than in a deletion strain which carries puc2BA on its genome. The spectroscopic characteristics of LH2 complexes comprising exclusively Puc 1 B, Puc2B or a mixture of both were analysed while bound within the native membrane. Puc2B is shown to be integral to B800 BChl binding within the LH2 complex of Rba. sphaeroides 2.4.1. A hypothesis to account for the reduction of B800 BChl binding in the absence of Puc2B is proposed. The final chapter describes the specific attachment of purified LH2 complexes from Rba. sphaeroides to patterned self assembled monolayers on the micron scale. Surface plasmon resonance studies were used to select SAMs possessing tail groups with specific chemical properties. Two surfaces that have contrasting attractive and repUlsive responses to membrane protein adsorption have been identified. Confocal microscopy was used to demonstrate the functionality of the purified LH2 complexes whilst they are covalently attached to the surface.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available