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dc.contributor.advisorWilbanks, Sigurd
dc.contributor.authorPearl, Esther
dc.date.available2015-02-20T04:07:26Z
dc.date.copyright2006
dc.identifier.citationPearl, E. (2006). Requirements for splicing by Cne PRP8, a novel intein from Cryptococcus neoformans (Thesis, Doctor of Philosophy). Retrieved from http://hdl.handle.net/10523/5469en
dc.identifier.urihttp://hdl.handle.net/10523/5469
dc.descriptionThe research in this thesis has led to the following publications, produced by the author of this thesis: Pearl, Esther J., Joel D.a. Tyndall, Russell T.m. Poulter, and Sigurd M. Wilbanks. "Sequence Requirements for Splicing by the Cne PRP8 Intein." FEBS Letters 581.16 (2007): 3000-004. http://www.ncbi.nlm.nih.gov/pubmed/17544410 Pearl EJ, Bokor AA, Butler MI, Poulter RT, Wilbanks SM. “Preceding hydrophobic and beta-branched amino acids attenuate splicing by the CnePRP8 intein.” Biochim Biophys Acta 1774.8 (2007): 995-1001. http://www.ncbi.nlm.nih.gov/pubmed/17604706en_NZ
dc.description.abstractInteins are autocatalytic protein domains that splice out of the nascent polypeptide shortly after translation, requiring no co-factors to facilitate splicing. There is an intein coding sequence within the Prp8 gene of Cryptococcus neoformans, a human pathogen that causes cryptococcosis in immunocompromised people. The intein, Cne PRP8, is a drug target as Prp8 is a central component of the spliceosome and thus believed to be essential to the fungus. Improved knowledge of the intein and its requirements for splicing can contribute to design of a screening system and the search for an inhibitor of intein splicing. Purification of Cne PRP8 for crystallisation was performed using either an N- or a C-terminal His·Tag ®, where theN-terminal His·Tag® was removed by 3C protease prior to crystallisation trials. C-terminally His·Tagged® Cne PRP8 formed the largest crystals. The crystals were triangular plates with stepped faces. A 2.8 Å data set was collected with an Rmerge of 0.151 and a mosaicity of 2.1°. A smaller crystal gave a 3.6 Å data set with an Rmerge of 0.085 and a mosaicity of 1.5°. Molecular replacement was not sufficient to solve the structure, likely because the data were weak and the molecules in the asymmetric unit too numerous. Purified Cne PRP8 was additionally shown by circular dichroism to lack regular secondary structure, suggesting that regions of Cne PRP8 could be natively unstructured. Cne PRP8 was expressed as a fusion protein between Haemophilus influenzae trigger factor (HiTF) and a chitin binding domain (CBD). Antibodies to the different parts of the fusion protein facilitated the observation of splicing ability by western blotting. From this it was determined that Cne PRP8 is capable of splicing in a foreign protein context. Context is important, with maximum splicing occurring when Cne PRP8 has two native N-terminal extein residues and one native C-terminal extein residue. The first residue and the last two residues of Cne PRP8 are essential for splicing; additionally the conserved threonine (T62) and histidine (H65) were shown to be catalytically important. Also required for splicing are arginine 154, tyrosine 162, and aspartate 166. Leucine 161 undergoes ~50% splicing when mutated to alanine, and tryptophan 151 undergoes limited C-terminal cleavage, but no splicing, when mutated to alanine. Tryptophan 151 was identified as a potentially crucial residue, which may function to prevent C-terminal cleavage before the N-terminal rearrangements have taken place. Overall it appears that Cne PRP8 residues that are more diverged from the general intein consensus are less essential for splicing. Wild type Cne PRP8 is insensitive to zinc inhibition in vivo. It is also unresponsive to cadmium, calcium, cobalt, lithium, magnesium, manganese and nickel. However, a partially splicing-deficient mutant exhibited further inhibition in response to zinc and cadmium. This mutant also showed a limited increase in splicing efficiency in response to temperatures lower than 37°C. This study has identified critical residues, in addition to those at the splice junctions necessary for catalysis, which participate in splicing intermediates.en_NZ
dc.format.mimetypeapplication/pdf
dc.language.isoenen_NZ
dc.titleRequirements for splicing by Cne PRP8, a novel intein from Cryptococcus neoformansen_NZ
dc.typeThesisen_NZ
dc.date.updated2015-02-20T04:06:38Z
thesis.degree.disciplineBiochemistryen_NZ
thesis.degree.nameDoctor of Philosophyen_NZ
thesis.degree.grantorUniversity of Otagoen_NZ
thesis.degree.levelPhDen_NZ
otago.openaccessOpenen_NZ
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