Purification, Characterisation and Crystallisation From a Range of Rhodospirillineae Pigment-Protein Complexes

Botany, Molecular biology

Gall, Andrew

University:University of Glasgow

关键词: Botany, Molecular biology;   

Others  :  http://theses.gla.ac.uk/76294/1/13834026.pdf

来源: University of Glasgow

【 摘 要 】

A range of photosynthetic integral membrane pigment-protein complexes were purified from several species of purple bacteria. The light-harvesting complexes that were isolated were the RC-LHI conjugate (also called the core conjugate) and the B800-850 peripheral light-harvesting complex (LHII). Uniform growth and protein purification protocols were established for each species. The stability of the RC-LHI conjugates was species dependent. The most stable cores were obtained from Rp. acidophila, Rp. cryptolactis and Rp. palustris. The least stable RC-LHI conjugates were from R. centenum and Rh. sphxieroides. Biochemical analysis of the RC-LHI conjugate foimd that all the species investigated had a similar RC:BChla ratio of approximately -1:30. An experimentally determined extinction coefficient for BChl a at 880nm (which is used to calculate the RC:BChl a ratios) for each species was obtained and found to lie within the range of 100-120 mM cm-1. When the RC:BChl a ratios were recalculated using the experimentally determined extinction coefficients the average RC:BChl a ratio increased to -1:34. The purified membrane proteins were then screened to see if suitable crystals could be obtained for structural analysis. Two Dimensional and Three Dimensional crystallisation techniques were utilised and the results compared. 2D arrays of the RC-LHI conjugate from Rp. palustris and Rp. acidophila were obtained. Initial image analysis the Rp. acidophila RC-LHI conjugate suggests a hexagonal lattice with a centre-to-centre distance of approximately 115+/-5A, gamma =120