Characterisation and Crystallisation of a Bacterial Light-Harvesting Complex

Guthrie, Neil (1992) Characterisation and Crystallisation of a Bacterial Light-Harvesting Complex. PhD thesis, University of Glasgow.

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Abstract

The B800-820 light harvesting antenna complex, an integral membrane protein, from Rhodopseudomonas acidophila strain 7750 has been characterised. The antenna complex is comprised of four low molecular weight pigment binding apoproteins, namely the B800-820alpha1, B800-820alpha2, B800-820beta1 and B800-820beta2. The apparent molecular weights of bands corresponding to these apoproteins have been determined using tricine SDS polyacrylamide gel electrophoresis as 2550 daltons, 3350 daltons, 6300 daltons and 6600 daltons. Differences in the true molecular weight (Mr) deduced from the amino acid sequence and these apparent molecular weights, make it impossible to determine which is the alpha1, beta1 or beta2 aprotein. Electrospray mass spectrometry indicates that the unsequenced alpha2 may be the heaviest of these four apoproteins. The stoichiometric ratio of bacteriochlorophyll a: carotenoid has been determined as 2:1, which confirms work by Evans in 1989. Polyacrylamide gel electrophoresis shows that the complex exists as an oligomeric assembly with an apparent molecular weight of 92,000 daltons. The integrity of the antenna complex has been maintained after exchange of lauryl dimethyl N-amine oxide for any of the following detergents: heptyl glucoside, beta octyl-glucoside, nonyl glucoside, lauryl maltoside, heptyl thioglucoside, N-dodecyl-N, N-dimethyl-3-amrnonio-1-propane sulfonate, CHAPS, CHAPSO and MEGA8. This integrity was demonstrated by examination of the near infrared spectra and circular dichroism studies. Ultraviolet circular dichroism demonstrated the presence of alpha-helix which is thought to span the lipid bilayer. Infrared circular dichroism showed that the absorption of both carotenoid and bacteriochlorophyll pigments in the antenna complex are influenced by their surrounding environment, with possible excitonic coupling between bacteriochlorophyll molecules. Microcrystals of the antenna complex could be grown in a variety of different detergents in the presence of a small amphiphilic molecule. Crystals of the antenna complex could be grown using beta-octyl glucoside and N-dodecyl-N, N-dimethyl-3-ammonio-1-propane sulfonate, in the presence of the small amphiphile benzamidine hydrochloride. These diffracted X-rays to a resolution of 10A and 15A respectively. Crystallisation using beta-octyl glucoside was then optimised with respect to different physical parameters. This led to an increase in the crystal size, with a decrease in crystallisation time, and also an improvement in the resolution of X-ray diffraction (by approximately 5A). X-ray diffraction data were collected on two forms(#1 and #2) of tabular plate crystal, which both indexed as hexagonal cells with space group R12. The cell dimensions of crystal form #1 were a=b=120.01A, c=293.91A and those of crystal form #2 were a=b=121.81A, c=283.11A. The crystal density in both forms was determined to be 1.17g/cm3. Assuming a molecular weight of 83.5 kDa in the asymmetric unit, this indicates a Vn, of 2.47A3/dalton for crystal form #1 and 2.42A3/dalton for crystal form #2. This gives a solvent composition of 49.2% and 50.8% for crystal form #1 and #2. Using the percentage solvent, the density was back calcu-lated as 1.17g/cm3. This indicates that the B800-820 antenna complex has an asymmetric unit consisting of a trimer of tetramers. This is consistent with the experimentally derived stoichiometric ratio of bacteriochlorophyll a: carotenoid (2:1). Analysis of the Rotation function indicates the presence of a local two fold symmetry axis parallel to the z axis. It is possible there is also a local three fold axis parallel, or at a few degrees to the z axis. The z=0 section of the Patterson map shows regular ordering of distinct sets of peaks, with interatomic vector lengths consistent with the distances between a helices arranged as a trimer of tetramers. On the basis of the biochemical and X-ray data, a model of the asymmetric unit was proposed which consisted of a trimer of molecular assemblies, with each assembly consisting of alpha1alpha2beta1beta2 proteins, six bacteriochlorophyll and three carotenoid molecules. The close similarity in the unit cell and space group of these protein crystals and crystals of the B800-850 antenna complex suggests a similar asymmetric unit in both cases.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Richard Cogdell
Keywords: Biochemistry
Date of Award: 1992
Depositing User: Enlighten Team
Unique ID: glathesis:1992-75840
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 17:55
Last Modified: 19 Nov 2019 17:55
URI: http://theses.gla.ac.uk/id/eprint/75840

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