Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.617457
Title: The structural characterisation of two DNA protectants during stress : the tandem RRM domains of mouse TDP-43 and E. coli DPS
Author: Austin, James
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2013
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Abstract:
TAR DNA Binding protein (TDP-43) is a member of the heterogeneous nuclear ribonucleoprotein family with crucial splicing, transport and regulatory function of genetic material inside mammalian cells. Unfortunately, TDP-43 positive cytoplasmic aggregates occurring with post-translational modifications are a common hallmark in neurodegenerative diseases observed in Alzheimer’s, Parkinson’s, amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration (FTLD) diseases. Mutations in the TARDBP gene responsible for encoding TDP-43, have been directly correlated with onset of ALS and FLTD. Disease models describing TDP-43 proteinopathy suggests onset may derive through either cytoplasmic mis-localisation or a loss of nuclear function but it is unclear if or how disease associated point-mutations contribute to these observations. In order to determine the effects these mutations have on the protein, a fragment containing the tandem RRM domains (residues 101-265), responsible for the proteins nucleic acid binding function was tested. Using small angle X-ray scattering, circular dichroism, isothermal titration calorimetry and thermal assay methodology it was demonstrated that initial structures of all variants are similar but mutations (D169G and K263E) confer resistance to thermal denaturation by up to 4.9 ± 0.6˚C. This stability positively correlated with an increase in half-life when tested in the full-length variant using a neuron cell model suggesting that protein turn-over is a contributing disease factor. This study was also concerned with solving an X-ray crystallographic DNA binding complex structure for E.coli DPS and mapping interactions with neighbouring DPS complexes. These mechanisms are important in DPS function to protect nucleic acids during prokaryote stress. DPS is conserved in almost all prokaryotes however not all species can interact with DNA. Using X-ray crystallography, a model of E.coli DPS was built to 2.8 Å resolution from DNA containing samples showing both DNA and N-terminal residues were absent. Stabilising polar interactions were shown to form between neighbouring dodecamer structures involving T12, R18, D20, N99, S100, S106 and K134. Polar contacts are observed in all compared crystallographic structures from different species but the residues involved are poorly conserved, despite strong similarities between sequence and structure. This suggests that these contacts may contribute to stabilising the DNA-DPS complexes but form indiscriminately between exposed polar residues available on the dodecamer surface. These interactions are likely to contribute to the thermal stability of DNA-DPS complexes to aid in the proteins protective function.
Supervisor: Hasnain, Samar; Antonyuk, Svetlana Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.617457  DOI: Not available
Keywords: QP Physiology
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