Title:
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Diatom silicon transporters : from protein function to biomimetic silica synthesis
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Biomineralisation is the synthesis of inorganic materials in biological systems.
Many biominerals - such as bone! teeth, and shells - are high-performance
composites synthesised with extreme precision under physiological conditions.
Understanding biomineralisation is expected to inspire 'green' methods for the
manufacture of novel materials.
Diatoms are eukaryotic algae that mineralise an external cell wall, or frustule,
composed of hydrated silica. Silicification depends upon the uptake of soluble
silicon (silicic acid) from the local environment by specific silicic acid transport
proteins (SITs). This unusual family of integral membrane proteins are relatively
uncharacterised. This project aimed to express and purify 5113 from the diatom
Thalassiosira pseudonana (TpSIT3) for further characterisation in vitro, and to
explore whether synthetic SIT3 proteoliposomes could be used as a model
mineralisation system with potential applications in nanotechnology.
TpSIT3 was successfully overexpressed in yeast and purified in the solubilising
detergents Fos-choline 12 and octyl glucoside. The purified protein was
successfully reconstituted into synthetic liposomes and silicic acid uptake was
assessed using two fluorescent assays including a novel method which utilised
zinc silicate fluorescence. This method was used to determine that silicic acid
transport by TpSIT3 displayed Michaelis-Menten kinetics with a Km of 6.1 ± 2.7
μM, similar to silicic acid uptake studies in diatom cultures. The structure and
function of a silicifying cationic peptide were also characterised for the first time.
Peptide-mediated silicification only proceeded at ≥2 mM silicic acid when pH was
>6.4 and peptide concentration was ≥2.5 mM. These results underpinned efforts
to synthesise silica within the int erior lumen of peptide-loaded SIT
proteoliposomes. Preliminary electron microscopy and elemental analysis
suggested that such an approach was feasible. This thesis thus establishes a
series of novel methods that can be used to study silicic acid transport and silica
mineralisation in vitro
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