Detergent-free extraction and purification of membrane proteins to enable structural and functional studies.

In modern times humanity has benefited from ever improving availability of healthcare and food. These improvements in our quality of life are built upon both scientific and technological innovations, many of which have been made possible by breakthroughs in biochemical sciences. This project aims to capitalise on a recent breakthrough in our laboratory to eventually develop safer and more effective crop protection (CP) compounds and therapeutic drugs. These will in turn increase crop yields, reducing food costs, and improve treatments for diseases, reducing side effects and increasing survival rates.
Our ability to develop such chemicals relies on the identification and study or proteins within the cell. More often than not these proteins are found bound to the outer surface of the cell, the membrane, which poses some significant technical difficulties when attempts are made to isolate and study their function. This is in stark contrast to their functional importance in cells, for example, 40% of CP compounds and therapeutic drugs work by interacting with proteins in the membrane.
One of the key bottlenecks that limits the study of many cell surface proteins occurs when scientists attempt to release these proteins from the cell surface. This process is essential for investigating the structure of the protein and how it works. The current method for releasing these proteins relies on breaking up the membrane using detergents (not dissimilar to detergents you would find in your kitchen cupboard). The problem with these detergents is that although they are very good at releasing proteins from the cell (we all know how good they are at releasing food, which is high in fat and protein, from a dirty saucepan) they are also very good at scrambling the protein so it no longer functions. This means that the majority of cell surface proteins cannot be released in an active form using these existing methods.
In our laboratory we have developed a revolutionary new method that doesn't require detergent to release the protein from the cell surface. Our method is much less harsh than the use of detergent, allowing the production of active proteins. The method is analogous to the way a cookie cutter cuts circular biscuits from a sheet of biscuit dough. We use a simple chemical in place of the biscuit cutter which is able to cut discs (biscuits) from the cell surface (biscuit dough). These discs contain the membrane protein in an active form that can be used in biochemical studies.
In this project we aim to make discs that contain proteins that are targets for both crop protection and drug discovery programs. Once we have made these proteins we then aim to develop methods that will allow the structure and function of these proteins to be determined more easily than before.
Data from these studies will then be used to improve our ability to develop new drugs and insecticides.

The rate at which new drug and crop protection (CP) compounds are being developed has declined (e.g. 50% drop in 10 years for pharmaceutics). To address this shortfall, there is a need for new approaches to the study of drug and CP targets. We aim to address one crucial bottle-neck that limits both discovery processes; the production and structural study of the main target class, membrane proteins (MP). MPs are the targets for more than 40% (by market value) of CP compounds and drugs. However, when compared to soluble proteins, the technical difficulties encountered during the production and subsequent study of MPs mean they still remain relatively enigmatic.

This project, a collaboration with a leading agribusiness, Syngenta, aims to improve the development of new CP compounds and therapeutics. The project applies a novel membrane extraction process using a co-polymer of styrene maleic acid (SMA) which encapsulates MPs in a nanoparticle. This allows the MPs to be produced quickly, reliably and in higher volumes than previously possible. We will encapsulate 4 proteins including two crop protection target proteins from Syngenta. We will then develop methods that allow x-ray crystallography, Cryo-electron microscopy, x-ray and neutron scattering studies of these proteins. The methods and data from the project will form the foundation for the widespread use of the SMA encapsulation system in both commercial and academic laboratories.

Success in this project will deliver methods that enhance:
1) Studies of protein structure-function relationships
2) In silico identification of potential lead compounds for the attenuation of protein function
3) Lead compound optimisation
4) Studies of native protein-lipid interactions

The overall aim of this project is use a reagent to facilitate the study of membrane proteins. If this aim is achieved it will trigger a cascade of impacts that begins with the scientists working on membrane proteins. Insights from these scientists will improve our understanding of membrane protein structure and function. This will, in turn, open new commercial opportunities which will benefit the population as a whole. In this section we aim to delineate these impacts.

PRIMARY IMPACT
Many of the details of the impact of the work in the proposal on the scientific community is given in the academic beneficiaries section. The inclusion of a Syngenta as a partner ensures that the impact will spread effectively across academic and commercial science. Our close collaboration with Syngenta will ensure that the outputs of the project will be, at least in part, tailored to the requirements of industry ensuring a seamless diffusion of the work into the commercial arena.

SECONDARY IMPACT
The SMALP method will increase the availability of pure active membrane proteins which will in turn increases the amount and quality of data on the structure and function of these proteins. This will improve the efficiency of both drug and CP discovery pipelines by increasing the availability of membrane proteins. This has a direct effect on the effectiveness of chemical library screening, while improved structural models produced using the methods developed in this application will allow rationale CP agent discovery to be used much more widely. Both of these benefits will improve the hit rate for CP agent discovery reducing costs and thereby the eventual price of the CP compound.

TERTIARY IMPACT
By addressing both agro-chemical and pharmaceutical targets in the project we aim to influence productivity of both sectors. This in turn will positively influence the provision of food and healthcare. An improvement in both areas has obvious benefits for society. In the case of food security, future predictions are stark. To feed the growing population, farmers will need to achieve at least a 70 % increase in food production by 2050. This will be a real challenge considering the megatrends of growing population, greater affluence, and increasing urbanization. Not only are more people demanding more food, but they want greater variety, including meat, dairy and vegetables. One aspect that has to be improved to meet this challenge is the development of CP agents. This project aims to enable the fundamental research that underpins this process.

IMPACT ON UKPLC
Given that these developments are going to be carried out by a UK research group using UK research infrastructure in collaboration with a UK company it is clear that success will have significant impact on UKplc. Central to this is an increase in competitiveness of the UK agrochemicals industry in the world market. In addition an improved availability of food and pharmaceutics directly benefits the population as a whole increasing productivity across the board. In 2011 TD became a BBSRC enterprise fellow perfectly placing him for exploitation of any other developments from the projects.

IMPACT ON PUBLIC SERVICE AND POLICY
The UK government has highlighted the food security and health as cornerstones of it's innovation goals (reflected in RCUK strategy). Success in this project will play a small but significant role in achieving these goals.

TIMESCALES FOR IMPACT
Our link with Syngenta alongside our on-going collaborations with the pharmaceutical sector make it likely that benefits will be felt within industry in 1-3 years and amongst the populace in 3-8 years.

IMPACT ON EMPLOYABILITY
It is implicit with the application that the people involved in the project will gain from exposure of cutting edge methods for the study of membrane proteins with obvious benefits should they enter the job market.