Structural studies on yeast eIF5A using biomolecular NMR and molecular dynamics
- Authors: Sigauke, Lester Takunda
- Date: 2015
- Subjects: Molecular dynamics , Reverse transcriptase , HIV (Viruses) , HIV infections , Eukaryotic cells , Yeast
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4547 , http://hdl.handle.net/10962/d1017927
- Description: Eukaryotic initiation factor 5A, eIF5A, is a ubiquitous eukaryotic protein that has been shown to influence the translation initiation of a specific subset of mRNAs. It is the only protein known to undergo hypusination in a two-step post translational modification process involving deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) enzymes. Hypusination has been shown to influence translation of HIV-1 and HTLV-1 nuclear export signals, while the involvement of active hypusinated eIF5A in induction of IRES mediated processes that initiate pro-apoptotic process have inspired studies into the manipulation of eIF5A in anti-cancer and anti-diabetic therapies. eIF5A oligomerisation in eukaryotic systems has been shown to be influenced by hypusination and the mechanism of dimerisation is RNA dependent. Nuclear magnetic resonance spectroscopy approaches were proposed to solve the structure of the hypusinated eIF5A in solution in order to understand the influence of hypusination on the monomeric arrangement which enhances dimerisation and activates the protein. Cleavage of the 18 kDa protein monomer by introduction of thrombin cleavage site within the flexible domain was thought to give rise to 10 kDa fragments accessible to a 600 MHz NMR spectrometer. Heteronuclear single quantum correlation experiments of the mutated isotopically labelled protein expressed in E. coli showed that the eIF5A protein with a thrombin cleavage insert, eIF5AThr (eIF5A subscript Thr), was unfolded. In silico investigations of the behaviour of eIF5A and eIF5AThr (eIF5A subscript Thr) models in solution using molecular dynamics showed that the mutated model had different solution dynamics to the native model. Chemical shift predictors were used to extract atomic resolution data of solution dynamics and the introduction of rigidity in the flexible loop region of eIF5A affected solution behaviour consistent with lack of in vivo function of eIF5AThr (eIF5A subscript Thr) in yeast. Residual dipolar coupling and T₁ relaxation times were calculated in anticipation of the extraction of experimental data from RDC and relaxation dispersion experiments based on HSQC measurable restraints.
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- Date Issued: 2015
The effect of hydrostatic carbon dioxide pressure and extracellular ethanol on the performance of the yeast strain Saccharomyces cerevisiae during fermentation
- Authors: Longden, Nicholas Guy
- Date: 1993
- Subjects: Brewing -- Microbiology , Yeast , Fermentation , Saccharomyces cerevisiae
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4044 , http://hdl.handle.net/10962/d1004105 , Brewing -- Microbiology , Yeast , Fermentation , Saccharomyces cerevisiae
- Description: The brewing industry constantly experiences problems in trying to maintain the quality of beer produced. Unfavourable conditions during fermentation may alter the performance of the yeast strain Saccharomyces cerevisiae, resulting in a "poor" end-product. It has been established that high concentrations of extracellular ethanol, when added to the fermentation medium inhibit yeast activity. It has been recently suggested that increased carbon dioxide pressure could inactivate the yeast activity adding to further brewing problems. The aim of this study was to investigate the effect of extracellular carbon dioxide pressure and ethanol addition, on yeast performance when added to a fermentation medium, and to establish whether an inhibitory relationship existed between ethanol and carbon dioxide pressure, when combined and added to the fermentation medium. Dissolved C0₂ in the medium, medium pH and substrate utilisation were analysed daily during a fermentation, as were membrane fatty acid composition. These parameters were used to assess the effect of ethanol and carbon dioxide on the yeast performance and consequently the final end-product. Supplementing the medium with extracellular ethanol, even as low as 5%, was shown to inhibit yeast performance during fermentation. This effect was even more marked as the ethanol concentration was increased, with almost total inhibition of yeast activity occuring after the addition of 15% ethanol (v/v). A similar effect was observed when elevated C0₂ pressures were applied to the medium, and although low C0₂ pressures initially induced the synthesis of saturated yeast membrane fatty acids, elevated C0₂ pressures (greater than 1,0 atm.) was shown to follow a similar inhibitory trend, if not as dramatic, as ethanol. A combination of both ethanol and C0₂ pressure showed a further increase in the level of yeast inhibition, although the low C0₂ pressure appeared to initially inhibit the toxicity of ethanol on the yeast. Increasing the levels of the C0₂/ethanol treatment (1,0 atm.), showed a synergistic effect on yeast performance. The results of this study indicate that both extracellular ethanol and carbon dioxide do appear to inhibit yeast performance and affect membrane fatty acid composition of the cells by inhibiting the synthesis of the respective fatty acid. This affect has a significant bearing on the general metabolism of the yeast cell.
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- Date Issued: 1993