Structure and function studies on the LH1-RC core complex from a range of photosynthetic purple bacteria

Law, Christopher J. (1998) Structure and function studies on the LH1-RC core complex from a range of photosynthetic purple bacteria. PhD thesis, University of Glasgow.

Abstract

The LH1-RC core complexes from a range of purple bacterial species were investigated. The complexes were isolated by solubilisation with the detergent LDAO, then purified using a combination of techniques such as sucrose gradient centrifugation, anion exchange and gel filtration chromatography. The integrity of the complexes during purification was monitored by means of absorption spectroscopy. This showed that the stability of the detergent solubilised cores was species dependent. The most stable cores were obtained from Rps. acidophila, Rps. cryptolactis, Rps. palustris and Chr. vinosum. The least stable core complexes were from Rv. gelatinosus and Rs. rubrum. The intactness and stability of the purified cores was further investigated by circular dichroism (CD) spectroscopy. This technique provided a much more sensitive spectral test for intactness than that offered by absorption spectroscopy. CD spectra in the NIR region confirmed that the cores from Rv. gelatinosus and Rs. rubrum denatured during purification. The purified core complexes were screened for their suitability for forming 3-D crystals using vapour diffusion methods. Core complex crystals did not form when LDAO was used as detergent or when ammonium sulphate and potassium phosphate were used as precipitants. However, when LDAO was exchanged for other detergents, such as cholate and heptyl thioglucoside, and PEG was used as a precipitant core complex crystals did form. The presence of MgCl2 and other small additives seemed an absolute requirement for crystal formation. The best crystals were obtained using core complex from Rps. acidophila strain 10050. Preliminary characterisation of one of these crystals showed it diffracted X-rays to a resolution of 7.6A. Data analysis suggested a space group with putative tetragonal P4 symmetry and unit cell dimensions of a=b=156.56A and c=181.11A, and alpha=beta=gamma=90.0°. Due to a problem of irreproducibility the crystallisation experiments failed to yield crystals of a sufficient quality to allow a structural determination of the LH1-RC core. The LH1 (B880) complex from Rh. marinum was also isolated and purified, and the effects of chemical oxidation on its absorption and fluorescence emission spectra was investigated. Mild chemical oxidation of the LH1 complex, by addition of 10mM potassium ferricyanide, caused a 2-3% bleaching of the 880nm Qy absorption band. In contrast, at the same ferricyanide concentration, fluorescence emission intensity of the complex was quenched by about 50%. This result demonstrated that oxidation of a very few bacteriochlorophyll molecules in the LH1 ring is enough to completely quench its fluorescence. This suggests the possibility of redox control of energy transfer. The antenna arrangement in the photosynthetic membrane of the 7750 strain of the purple bacterium Rps. acidophila was investigated by means of fluorescence induction spectroscopy. The membrane of this species is thought to be composed of LH1-RC core complexes which are surrounded by peripheral LH2 complexes. The sigmoidicity of fluorescence induction curves was used to probe the excitonic connectivity of RC's, and this information were used to gain information on the structural arrangement of the antennae. The data obtained excluded models of the Rps. acidophila photosynthetic unit (PSU) that assume aggregates of LH1-RC complexes or linear chains of LH1-RC complexes to which LH2 complexes are attached on the periphery. Rather, they support the model suggested by Papiz et al. (1996) in which peripheral LH2 rings tightly surround each core complex circumferentially.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: Q Science > QR Microbiology
• Colleges/Schools: College of Medical Veterinary and Life Sciences
• Supervisor's Name: Cogdell, Professor Richard
• Date of Award: 1998
• Depositing User: Enlighten Team
• Unique ID: glathesis:1998-71507
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 10 May 2019 14:27
• Last Modified: 18 Oct 2022 14:51
• URI: https://theses.gla.ac.uk/id/eprint/71507