'Gel-to-Grid' cryoEM of membrane proteins using SMALP technology

Since the development of the microscope in the middle of the 17th century biologists have marvelled at how structures that are often many times smaller than the width of a human hair underpin the marvel of life. Understanding the structures of these tiny yet exquisitely formed particles has allowed us to unlock the secrets of nature. In turn these insights have allowed us to make some of the most important advances in our understanding of disease in humans and animals. For example, our understanding of the structure of viruses has allowed us to develop more effective vaccines while structures of proteins have revolutionised the speed with which we develop drugs. Understanding these nano-scale structures also allows us to answer some of the most important questions in science from how genetic material replicated to how muscles work.

These insights have been enabled by regular breakthroughs in the methods used to visualise these small particles. Each breakthrough allows more detail to be seen more rapidly for more and more challenging samples. Just consider, in 300 years we have been able to move from visualising cells to a position where scientists can routinely see the arrangement of individual atoms that make up the cells. In our laboratories in Birmingham and Warwick we have been working on a method that will allow us to visualise the atomic arrangement of some of the most important proteins in biology. These proteins live in the membrane that surrounds cells and carry out some many essential jobs that allow cells to survive. They allow nutrients into the cell and allow waste products to leave, they also mediate the movement of signals into and out of the cell. These functions make them exceptionally interesting to researchers, but perhaps more importantly they are also the most important targets for developing new therapeutics for animal and human health.

Unfortunately these proteins have proved to be exceptionally difficult to study and have confounded a large number of scientists over the past 50 years. In 2009 we found an entirely new way of making these proteins by using a simple polymer more commonly found in car dashboards and paint. This method "wraps" the protein in a polymer "belt" that keeps it stable allowing us to study it. In this project we am to develop a new method that will make it easier to transfer these polymer belted proteins into microscopes to allow them to be imaged. We hope that the method will make this process much easier than current methods and will allow more images to be taken of important membrane proteins. This should allow us the make a more complete "atomic picture" of the biological world greatly aiding the development of new therapeutics.

Membrane protein structural biology has remained frustratingly challenging with only 1 atomic structure of a membrane protein being produced for every 50 soluble protein structures. This is in a background where more than 50% of therapeutics are targeted at a membrane protein. The lack of progress in membrane protein biology has resulted from a number of technical challenges in the production and structure solution process. Over the last 10 years these technical challenges have been addressed one by one. In 2009 we solved one of these technical challenges by developing the first detergent free method for extracting membrane proteins for purification. The method uses a low cost polymer based on Styrene Maleic Acid (SMA) that can insert into membranes and excise a 10 nm diameter disc (SMALP) that contains a membrane protein solvated by its local lipid environment. We have shown that the method can successfully extract a range of active, stabilised membrane proteins from a range of membranes. We have used these SMALP solubilised membrane proteins in single molecule electron microscopy studies to produce structures of these proteins.

In the last 5 years atomic resolution EM has shown that atomic resolution structures can be produced from < 1,000,000 particles. Our SMALP method has the potential to complement this new method but our current protocols are suited to proteins that can be expressed at a reasonably high level. In this project we aim to exploit a recent observation that SMALP proteins can be separated intact on native PAGE. We have also shown that the resulting SMALP-rich band can be directly transferred to an EM grid and used for imaging. We will explore whether this technique can be extended to drastically reduce the amount of membrane required to generate a structure by EM thereby removing one of the remaining technical technical challenges that have limited our knowledge of membrane protein structure.

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 are given in the academic beneficiaries section. In addition an improved knowledge of membrane protein structure and function is of significant benefit to industry. Membrane proteins are significant targets for pharmaceutical and agrochemical industries. Our historically close collaboration with these industries 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 commercial discovery pipelines by increasing the availability of structural information on membrane protein. The improved structural models produced using the methods developed in this application will allow rational discovery approaches to be used much more widely. Both of these benefits will improve the hit rate for agent discovery reducing costs and thereby the eventual price of the compound.

TERTIARY IMPACT
By addressing both agrochemical 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 new 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 and Pharmaceutical 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.

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 close on-going collaborations with industrial partners 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 in 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.