Silicon transport proteins: from membrane protein biophysics to bionanoscience

Nanotechnology will change the world by ushering in a new era of high-performance materials with applications in healthcare, agriculture, energy, biotechnology, computing, environmental technology and more. One of the central pillars of this revolution will be organic and inorganic materials with dimensions on the order of 1-100 nanometres. These nano-sized particles, or nanoparticles, behave differently to bulk materials because of their high surface-to-volume ratio and size-dependent quantum effects. Many nanoparticles have novel or enhanced optical, physicochemical and magnetic properties and will either improve existing products or provide a platform for new technologies as sensors, optical tools, biomarkers, therapeutics, catalysts and electronic components. These advances will depend upon synthetic methods that can generate particles of a defined size, shape and composition that are stable and do not aggregate. However, fulfilling all of these criteria remains a challenging technical goal and new synthetic routes will be critical in realising the full promise of nanoparticle technology. Intriguingly, biology - which can be considered to be the living embodiment of functional nanotechnology - provides numerous examples of nanomaterials that are assembled very precisely from simple precursors under mild synthetic conditions. This leads to the hypothesis: can we learn new tricks from biology to develop fresh prospects in nanoparticle synthesis? The proposal will address this hypothesis by using silica transport proteins as novel devices in nanoscale fabrication. The silica transporters (SITs) are a unique family of integral membrane proteins that are able to bind a soluble form of silica, silicic acid, with high affinity and transport it across the cell membrane into the cell. This activity is used by microscopic single-celled organisms to help build 'glass houses' around themselves. However, the precise details of silica transport are currently not well understood. The proposal aims to (i) discover the molecular basis for silica recognition and transport by the SITs and (ii) apply that understanding in the synthesis of silica nanoparticles. The first of these aims will be addressed by in vitro studies of SIT structure and function. This classical biochemical approach will provide an unprecedented insight into the only high-affinity protein-silica interaction found in nature. The second aim will be met by reconstituting SIT proteins into spherical lipid vesicles and transporting silica into the vesicle interior, so that the vesicles become nanoscale reaction chambers for the preparation of silica nanoparticles. These nanoreactors will have the advantages of exquisite kinetic control over substrate delivery to the growing nanoparticle and stringent regulation of particle composition and the synthetic environment. This latter benefit will be particularly useful for the controlled synthesis of composite or functionalized nanoparticles. The proposal thus presents one of the first examples of combining membrane protein science with inorganic chemistry and offers new horizons in biological nanoscience. It will provide a platform for a range of further developments in this area.

This proposal will use natural silicon transport proteins (SITs) for the controlled delivery of silicon into proteoliposomes, which in turn provide a nanoreactor for synthesis of silicon-based nanoparticles. The SITs are the only proteins in nature that specifically bind to silicon. Silica is used extensively in materials science and nanotechnology and understanding this high-affinity organic/inorganic interface provides the opportunity to exploit this interaction in bionanoscience. Reconstituting silicon transport activity in proteoliposomes provides a novel route to the synthesis of silica nanoparticles and using these proteoliposomes as confined nanoreactors offers several advantages over current bulk approaches, including consistent particle size and dispersity. This method should be readily adapted to manufacture passivated functional core-shell nanoparticles. The composition and functionality of these nanoparticles will be analysed by standard chemical techniques including by electron microscopy. We will characterise the structure and function of a unique family of silicon transport proteins and investigate ways to exploit these protein in bionanoscience. Protein function will be characterised using a classical biophysical approach in vitro. In particular, silicon binding affinity, stoichiometry and transport kinetics will be determined with isothermal titration calorimetry, fluorescence methods, radiolabelled silicic acid assay and direct silica. Site-directed mutagenesis will be used to identify amino acid residues that are critical for binding and/or transport. This will provide a definitive description of silicon binding at the molecular level. Systems will then be developed for efficient and controlled transport of silicon into proteolipsomes to form nanoparticles under tunable, kinetic control, as well as coating functionalised nanoparticles enclosed within the proteoliposome with a protective silica shell.

Scientific discovery is central to the international competitiveness of the UK. This project will deliver an unprecedented advance in membrane protein science that will contribute substantially to the UK's global leadership in this area. This will impact significantly upon the public sector. The importance of developing new tools and fresh insight through interdisciplinary collaboration is closely aligned with current policy and priorities across the Research Councils, including the current BBSRC Strategic Research Priority of 'Nanoscience through engineering to application: bionanotechnology'. This proposal will provide a clear demonstration of the benefits of multidisciplinary research and as such will influence future policy and practice in this area. We will continue to capitalise on our track record in interdisciplinary science through the proposed collaborations in the UK and abroad. Additionally PJB will continue to make a significant contribution to the success of public sector stakeholders. She has recently sat on two BBSRC funding committees, several University RAE committees and is currently a member of University Council. A number of third sector organisations will also benefit. The outputs of this proposal will impact strongly upon the activities and policies of Learned Societies; PJB has until recently been a Council member of the Biochemical Society and maintains close links with the Society. This will contribute toward the strategic objectives and long-term goals of these organisations. The impact on public sector and third sector beneficiaries will be realised by communicating the outputs from this project through publication in high-impact peer-reviewed journals, at conference talks and through public engagement activities (see below). PJB is regularly invited to speak at major national and international meetings and the co-RI (PC) has also given a number of invited talks and poster presentations at conferences. This project will also impact upon the commercial sector since understanding the fundamental processes of biomineralization - and recapitulating them in vitro - has considerable commercial value. For example, several groups have taken direct inspiration from biosilicification to encapsulate enzymes in biomimetic silica supports, with dramatic improvements in enzyme stability. This also offers the potential for incorporating these enzymes into nanoscale devices. The technical outputs from this project will be disseminated periodically in peer-reviewed journals and at conferences that are accessible to any commercial beneficiaries. Any intellectual property of commercial value arising from this project will be identified, secured and potentially exploited through standard processes implemented by the office of Research and Enterprise Development at the University of Bristol. Developments in nanoscience are expected to usher in a new technological age with the value of the global nanotechnology market expected to top $2.6tn by 2012 (The Guardian, March 2009). This includes advances in pharmaceuticals and healthcare, communications, security, environmental technology, biotechnology and food. There is thus substantial long-term gain to society in funding research in this area. PJB and PC maintain public websites describing their research, and the potential impacts arising from it, in an accessible format. PJB is perpetually engaged in regular formal and informal public engagement activities; for example, through participating in the public outreach programme of Biochemistry department at Bristol. There are thus a number of routes by which these societal impacts could be communicated to the public.