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Title: Investigating the structures of evolutionarily conserved cytoskeletal proteins
Author: Höng, J.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2007
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The parasitic protist Giardia intestinalis, considered to be one of the most ancient eukaryotic organisms, contains a typical eukaryotic cytoskeleton composed of microtubules (αβ-tubulin), microfilaments (actin) and intermediate filaments. Proteins associated with the microtubules include kinesins and additional members of the tubulin superfamily. δ-Tubulin is a member of the tubulin superfamily. The methylotropic yeast P. pastoris was identified as the most suitable host for recombinant expression of G. intestinalis δ-tubulin. High-resolution crystal structures of G. intestinalis wild-type kinesin-2 GiKIN2a motor domain with docked neck linker and its hydrolysis deficient mutant GiKIN2aT104N, expressed in and purified from E. coli, were solved in complex with ADP and Mg2+ at 1.6 Å and 1.8 Å resolutions, respectively. They represent the first high-resolution structures of a kinesin-2 motor domain. They confirm that the structural fold of the kinesin motor domain is remarkably conserved and also provide insights into the nucleotide coordination within its active site. Furthermore, in vivo experiments confirmed the role of G. intestinalis kinesin-2 in intraflagellar transport. Previously, structural and functional homologues of eukaryotic cytoskeletal proteins have been identified in prokaryotes. In magnetotactic bacteria, magnetosomes are held in place by cytoskeletal filaments formed by a protein, MamK, that is predicted to be actin-like. Magnetospirillum magnetotacticum MamK was expressed, purified and crystallised with apparent space group P321 and P422. However, these crystals appear to be perfectly twinned and thus determination of MamK structure has not yet been possible. Electron microscopy analysis of filamentous sheets, assembled from MamK in the presence of AMPPCP nucleotide, showed a longitudinal repeat of 53 Å, characteristic for actin-like protofilaments.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available