title: Expression, purification, and reconstitution of a diatom silicon transporter.
abstract: The synthesis and manipulation of silicon materials on the nanoscale are core themes in nanotechnology research. Inspiration is increasingly being taken from the natural world because the biological mineralization of silicon results in precisely controlled, complex silica structures with dimensions from the millimeter to the nanometer. One fascinating example of silicon biomineralization occurs in the diatoms, unicellular algae that sheath themselves in an ornate silica-based cell wall. To harvest silicon from the environment, diatoms have developed a unique family of integral membrane proteins that bind to a soluble form of silica, silicic acid, and transport it across the cell membrane to the cell interior. These are the first proteins shown to directly interact with silicon, but the current understanding of these specific silicon transport proteins is limited by the lack of in vitro studies of structure and function. We report here the recombinant expression, purification, and reconstitution of a silicon transporter from the model diatom Thalassiosira pseudonana. After using GFP fusions to optimize expression and purification protocols, a His(10)-tagged construct was expressed in Saccharomyces cerevisiae, solubilized in the detergent Fos-choline-12, and purified by affinity chromatography. Size-exclusion chromatography and particle sizing by dynamic light scattering showed that the protein was purified as a homotetramer, although nonspecific oligomerization occurred at high protein concentrations. Circular dichroism measurements confirmed sequence-based predictions that silicon transporters are α-helical membrane proteins. Silicic acid transport could be established in reconstituted proteoliposomes, and silicon uptake was found to be dependent upon an applied sodium gradient. Transport data across different substrate concentrations were best fit to the sigmoidal Hill equation, with a K(0.5) of 19.4 ± 1.3 μM and a cooperativity coefficient of 1.6. Sodium binding was noncooperative with a K(m)(app) of 1.7 ± 1.0 mM, suggesting a transport silicic acid:Na(+) stoichiometry of 2:1. These results provide the basis for a full understanding of both silicon transport in the diatom and protein-silicon interactions in general.
authors.affiliation: School of Biochemistry, Medical Sciences Building, University of Bristol, University Walk, Bristol BS8 1TD, UK. p.curnow@bristol.ac.uk
authors.forename: Paul
authors.initials: P
authors.lastname: Curnow
authors.forename: Laura
authors.initials: L
authors.lastname: Senior
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Knight
authors.forename: Kimberlee
authors.initials: K
authors.lastname: Thamatrakoln
authors.forename: Mark
authors.initials: M
authors.lastname: Hildebrand
authors.forename: Paula J
authors.initials: PJ
authors.lastname: Booth
mesh_terms: D001692:Biological Transport; D002473:Cell Wall / Q000737:chemistry; D003902:Detergents / Q000494:pharmacology; D017377:Diatoms / Q000737:chemistry*; D026901:Membrane Transport Proteins / Q000302:isolation & purification / Q000378:metabolism*; D036103:Nanotechnology / Q000379:methods; D011510:Proteolipids / Q000187:drug effects / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification; D012824:Silicic Acid / Q000378:metabolism*; D012825:Silicon / Q000378:metabolism; D012995:Solubility
chemical_list: D003902:Detergents; D026901:Membrane Transport Proteins; D011510:Proteolipids; D011994:Recombinant Proteins; C020485:proteoliposomes; D012824:Silicic Acid; D012825:Silicon