Investigations into the structure and subunit dispersal of the pea plant (Pisum sativum) chloroplast chaperonin Ch-cpn60
Investigations into the structure and subunit dispersal of the pea plant (Pisum sativum) chloroplast chaperonin Ch-cpn60
Preparations of the plastid chaperonin Cpn60 from pea leaves (Pisum sativum) contain equal amounts of two homologous subunit types, Cpn60α and Cpn60β. The organization of these subunits within the tetradecameric chaperonin complex and the functional significance of their distribution are unknown.
In this thesis I describe the preparation of both subunit types by overexpression in E. coli and confirm that while Cpn60β can assemble as an homooligomer, Cpn60α remains monomeric. The structural basis of this divergent behaviour was analysed by homology modelling with the X-ray structure of GroEL. This revealed a selection of sequence differences in Cpn60α that would be expected to destabilize intersubunit interactions.
A method was developed to crosslink adjacent plastid Cpn60 subunits with a bifunctional reagent, SPDP. By reacting with a single cpn60β Cys 519 residue and a lysine 22 residue, ~6Δ apart on an adjacent α or β subunit, the method has the potential to report a nearest-neighbour subunit identity in mixed complexes.
Rhombohedral crystals (R32, a=b=108A, c=154A γ=120o) were grown from the cpn60α preparation and X-ray analysis attempted to discover a structural basis for the behaviour of the protein. The asymmetric unit of the crystal was too small to accommodate an entire subunit, suggesting the proteolysis had occurred during crystallization. A weak molecular replacement solution employing the E. coli apical domain as the search model indicated that the crystal contained a portion of the intermediate domain also. In parallel to the molecular replacement experiments, a set of Seleno-methionine experiments were attempted with the intention of carrying out MAD structural analysis of the cpn60α subunit, via the bioincorporation of the SeMet analog. However, the lack of SeMet incorporated product potency, due to the toxicity of the SeMet to the cpn60α protein, was to become a major hindrance to completing this set of experiments.
University of Southampton
Robinson, Terence
62dd7e9c-ba0b-4724-a940-3f9d5e05b405
2003
Robinson, Terence
62dd7e9c-ba0b-4724-a940-3f9d5e05b405
Robinson, Terence
(2003)
Investigations into the structure and subunit dispersal of the pea plant (Pisum sativum) chloroplast chaperonin Ch-cpn60.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
Preparations of the plastid chaperonin Cpn60 from pea leaves (Pisum sativum) contain equal amounts of two homologous subunit types, Cpn60α and Cpn60β. The organization of these subunits within the tetradecameric chaperonin complex and the functional significance of their distribution are unknown.
In this thesis I describe the preparation of both subunit types by overexpression in E. coli and confirm that while Cpn60β can assemble as an homooligomer, Cpn60α remains monomeric. The structural basis of this divergent behaviour was analysed by homology modelling with the X-ray structure of GroEL. This revealed a selection of sequence differences in Cpn60α that would be expected to destabilize intersubunit interactions.
A method was developed to crosslink adjacent plastid Cpn60 subunits with a bifunctional reagent, SPDP. By reacting with a single cpn60β Cys 519 residue and a lysine 22 residue, ~6Δ apart on an adjacent α or β subunit, the method has the potential to report a nearest-neighbour subunit identity in mixed complexes.
Rhombohedral crystals (R32, a=b=108A, c=154A γ=120o) were grown from the cpn60α preparation and X-ray analysis attempted to discover a structural basis for the behaviour of the protein. The asymmetric unit of the crystal was too small to accommodate an entire subunit, suggesting the proteolysis had occurred during crystallization. A weak molecular replacement solution employing the E. coli apical domain as the search model indicated that the crystal contained a portion of the intermediate domain also. In parallel to the molecular replacement experiments, a set of Seleno-methionine experiments were attempted with the intention of carrying out MAD structural analysis of the cpn60α subunit, via the bioincorporation of the SeMet analog. However, the lack of SeMet incorporated product potency, due to the toxicity of the SeMet to the cpn60α protein, was to become a major hindrance to completing this set of experiments.
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Published date: 2003
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Local EPrints ID: 465055
URI: http://eprints.soton.ac.uk/id/eprint/465055
PURE UUID: aee2177d-ea79-43dc-b182-b33126abb66b
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Date deposited: 05 Jul 2022 00:19
Last modified: 16 Mar 2024 19:55
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Author:
Terence Robinson
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