Structural studies on purple non-sulphur bacterial pigment-protein complexes

Mitchell, Iain Andrew (2003) Structural studies on purple non-sulphur bacterial pigment-protein complexes. MSc(R) thesis, University of Glasgow.

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The B800-820 light harvesting complexes from the purple non-sulphur bacteria Rhodopseudomonas acidophila strains 7050 and 7750 are integral membrane pigment-protein complexes. They have been studied using protein crystallography and mass spectrometry in an attempt to determine the high-resolution structure and to characterise the polypeptides.The growth of the bacteria and the subsequent purification of the B800-820 complex from each of the strains of bacteria are described. Also described is the growth of crystals of both complexes, which were then exposed to x-rays in order to test their diffraction quality. No crystals were observed to diffract x-rays beyond the 3.0A resolution currently available for the LH3 complex from strain 7050 (McLuskey, 1999) and none were observed to diffract to high resolution for the LH3 complex from strain 7750. MALDI-TOF mass spectrometry was employed to identify any contaminating polypeptides in the purified complexes. The polypeptides and contaminants were proposed to be identifiable by comparison of theoretical masses. Fragmentation studies were carried out by the enzymatic digest of the whole complex by different proteinases. The differences in masses of the fragments produced were then used in an attempt to assign partial sequences to the polypeptides. The results from the mass spectrometry are described, showing that several polypeptides are present in sample previously thought to contain only one pair. X-ray crystallography was used to determine the structure of a reaction centre mutant from Rhodobacter sphaeroides R26-1 at 2.8A resolution. This mutant is carotenoidless, and this work was carried out in order to prove, crystallographically, the presence of a reconstituted carotenoid. The carotenoid was reconstituted chemically, with one purified from another strain of bacteria. The structure was solved using molecular replacement, with the search model being a high-resolution reaction centre structure containing a carotenoid. Several rounds of refinement were carried out with the carotenoid omitted from the search model in order to minimise model bias. The carotenoid, spheroidene, is found in the same binding pocket, and in the 15-cis confirmation as in the wild type structure (within resolution limits). A comparison of the solved reaction centre with the structure used as the search model is given.

Item Type: Thesis (MSc(R))
Qualification Level: Masters
Additional Information: Adviser: Neil Issacs
Keywords: Biochemistry, Microbiology
Date of Award: 2003
Depositing User: Enlighten Team
Unique ID: glathesis:2003-74225
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 23 Sep 2019 15:33
Last Modified: 23 Sep 2019 15:33

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