A new tool for investigating biological interactions based on nanoparticle interactions

Proteins are large molecules which are critical for control over almost all biological processes. Unfortunately due to their size and complexity proteins often have a highly complex mode of operation with interactions often involving more than one partner and often simultaneously or in sequence with allosteric effects (effects on binding efficiencies due to one partner binding ahead of another) all too common. Methods to study the binding and interaction with proteins and their partners are constantly being developed but are often hampered by either a lack of sensitivity, the ability to work with proteins in their native state or the ability to look at the effect of allostery on these different interactions. In this proposal we aim to develop a new tool to allow investigation of protein interactions and in particular allostery by developing a new nanoparticle based methodology. Metal nanoparticles have unique optical properties. They can be investigated through their ability to scatter light very efficiently but also through their ability to enhance Raman scattering. In this proposal we propose to make use of the fact that when nanoparticles come close together and interact with each other, they change their optical signature and also they increase their ability to enhance Raman scattering. By functionalising specific nanoparticles with unique probe molecules and introducing them to proteins, we will investigate the interaction with these probe molecules and the target protein. Should there be more than one binding site for different molecules on a protein, the nanoparticles will be pulled close together through these binding interactions. This will cause a change in the properties of the nanoparticles which we can measure and due the vibrational fingerprint produced by the interactions and the enhanced Raman scattering, we will be able to identify which particular probes have interacted in a complex mixture. We can test the allosteric effects of interactions by changing the order of addition and also by using the same probe molecules but without attachment to the nanoparticles. This will allow us to develop a new tool to investigate protein interactions and also allow us to test these interactions by introducing molecules which can disrupt these binding events.

The tool proposed here involves the plasmonic coupling of metal nanoparticles brought close together through the biological interaction of a probe species and the target protein. Batches of nanoparticles will be functionalised with a specific probe molecule such as a peptide to mimic a binding site along with a specific Raman active molecule. These metal nanoparticle conjugates will have unique Raman codes although these signatures will be low in intensity due to the monodispersed nature and non-interacting properties of these particles. The conjugates will be designed to interact with a target protein and this target protein will have potentially more than one binding site to enable more than one type of nanoparticle to interact. When these nanoparticle conjugates are exposed to the protein of interest, a binding event will take place and the nanoparticles will be pulled close together allowing their plasmon bands to interact, changing their optical signature and also enhancing the Raman scattering of the reporter molecules of each of the particles. This event will allow us to identify which probe molecules have interacted with the protein and due to the extended distance range of plasmonic couplings (up to the diameter of the NP i.e. ~40 nm!), will be greater than that currently achievable using fluorescence techniques. By changing the order of addition, we will be able to also investigate the effect that one conjugate has on the binding affinity of other conjugates i.e. allostery. This is a highly significant aspect of protein interactions as very often binding at one part of the protein will change the conformation and binding affinity at another site. At present it is very hard to assess the effect of allostery on proteins and this new tool offers the opportunity to achieve this in a rapid, highly sensitive and solution based method.

The main beneficiaries from this research will be initially academic beneficiaries as stated in the previous section. However, as the tool emerges in terms of its utility there will be additional beneficiaries arising from the commercial sector. There are opportunities for those who are involved in the synthesis of the nanoparticles and also modified peptides of which the research grouping has good links. The tool is designed to be deployed in investigating new biological interactions and their relevance to potential sites of disruption. This of course has implications for drug screening down the line and we can foresee beneficiaries in the pharmaceutical and drug discovery sectors. Ultimately, this could lead on to benefit to individuals as specific proteins related to disease states are identified in terms of their interactions and sites of drug targeting developed. This is much longer term but the tool proposed in this application would enable this sort of benefit to arise.

The research proposed would potentially contribute to the nation's health and wealth. For example, the healthcare aspects would arise from the discovery of a new potential site for drug targeting where a protein was key to a particular disease pathway. This is much further down the line however it should be borne in mind that the development of this tool would act as an enabling step to move into these sorts of specific sectors. Obviously for research to move from being academic based to wider spread uptake requires investment but with investment comes profitable return. As such, we again envisage there being opportunities for economic growth from the outcome of this proposal which is related to the wellbeing of the nation's health. We feel that this is a very important step forward in biological capability with far reaching benefits to both the academic and industrial sectors with knock on effects into the general taxpaying population. A realistic timescale for these benefits to be realised is measured in years, although this is a one-year proposal. The applicants have a strong track record of entrepreneurial activities arising from their research and there is an ethos within the research group that fosters this activity through direct mentoring but also the availability of specific courses run by the Hunter Centre for Entrepreneurial Research at Strathclyde and also opportunities for further engagement with prospective industrial and academic partners.