High-throughput low-volume crystallisation facility

This is an equipment proposal, to enable ground-breaking research. Almost all of our understanding of how proteins and nucleic acids such as DNA work has come from structural biology. This requires growing crystals of these macromolecules, as in the pioneering work of Max Perutz, who won a Nobel prize in 1962 for solving the structure of the oxygen-carrying protein of the blood, haemoglobin. The hardest things to crystallise are the proteins that sit in the membranes that surround living cells, because they are not soluble in water - but these proteins are the targets for 50% of all drugs. They are also the ones that turn sunlight into energy, conduct nerve impulses and transport nutrients of all kinds into cells.

Over the last ten years, there has been a revolution in methods for crystallising proteins, especially membrane proteins, and our proposal is to equip the University of Leeds, and thus other local universities (Sheffield, Huddersfield, Newcastle and Manchester) with this cutting-edge equipment. The equipment has three components: (1) characterisation equipment (SEC-MALLS, LCP-FRAP), which will help us determine if the protein is likely to be crystallisable in the conditions being used; (2) a crystallisation robot so that we can use 20-50 times less protein than before ("drops" of 20-50 nl, rather than 1 ul) in each crystallisation trial; and (3) robotic imagers both at 4 C and room temperature. As we will be doing tens of thousands of trials, robotic imagers make visualising the experiments much easier than having to look at each experiment one by one under a microscope. In addition, the crystallisation robot can make "lipidic cubic phase" (LCP) drops, which corresponds to squeezing out 50 nl of toothpaste at a time. LCP has in particular revolutionised the crystallisation of membrane proteins but, like toothpaste, it is opaque. Consequently, we are also buying a "SONICC" imager, which will enable us to see very small protein crystals in the opaque LCP.

The post-genomic era has provided unimagined insights into the chemistry and regulatory mechanisms underlying life, and structural biology has been an very important part of this. Despite successes with water-soluble proteins, major challenges remain, particularly for membrane proteins and large mammalian/eukaryotic protein complexes, which this equipment will address. The structural work at the Astbury Centre for Structural Molecular Biology is centred around four major overlapping theme areas: (1) Membrane proteins; (2) large complexes; (3) pathogen-host interactions; and (4) design of small molecules (i.e. drugs).

Examples of projects where we expect breakthroughs are: how do the ion-channels involved in sensing pain, temperature or taste work? How do viruses that contain RNA, like the common cold or smallpox, package the RNA inside themselves? This is required for the virus to be infective. How do large molecular machines, like the vacuolar ATPase, work and how are they regulated? These are important in trypanosomal parasites that cause major diseases in both animals and humans. Can we understand better how some plants resist the toxicity of metals such as aluminium, and can we therefore enhance this ability in major crops? This will help make crops grow better, with less use of fertilisers, in acidic soils. Finally, bacteria that accumulate on surfaces form biofilms - around teeth, around prosthetic implants, on the surfaces of ships, with adverse consequences. Understanding how this happens and preventing it requires understanding the structures of the proteins involved.

Our goal is to add a modern crystallisation section to our structural pipeline. We lack the critical equipment components.
The main technical objectives are thus to:

(1) Acquire a size-exclusion chromatography setup with multiangle laser light scattering (MALLS), refractive index and dynamic light scattering detectors. With this, we wil be able to determine parameters critical for crystallisation such as: the absolute molecular mass of the peak on the column as a function of time, the protein:detergent ratio in the peak, and if it is monodisperse or not.
(2) Acquire crystallisation robotics including lipidic cubic phase technology (LCP), which has become an essential component of membrane protein crystallography.
(3) Acquire robotic imaging at 20 and 4 C, so that we can routinely scan 96-well crystallisation plates and deliver those images to internal and external users. This should include a SONICC-TPEF imager, which allows the detection of very small (micron-sized) protein crystals in the opaque LCP matrix.
(4) Acquire a lipidic cubic phase-fluorescence recovery after photobleaching (LCP-FRAP) setup to determine the mobility of proteins in the LCP matrix. Proteins that are not mobile do not crystallise.

These new tools, which form a consistent whole that exists nowhere in the North of England, will eliminate the major bottleneck in our current structure solution pipeline. The improvements will thus both enhance the productivity of current users and increase the number of in-house and external users.

Our scientific goals will be to use the new equipment to characterise and crystallise a variety of high-value targets, including TRP and Kv channels, vacuolar ATPases and pyrophosphatases, nucleoside transporters and RNA-virus protein complexes.

Our milestones will be purchase, delivery and installation, use by internal users, and use by external users. We will have a workshop to introduce users to the new equipment and techniques.

This is an equipment grant for enabling technology to complete the structural biology pipeline at the University of Leeds from protein production to x-ray diffraction and structure solution by providing the vital missing intermediate step: modern characterisation, crystallization and imaging equipment. The academic impact is thus on solving breakthrough structures. Who are the other beneficiaries?

SMEs, big pharmaceutical and agribusiness companies will benefit from this research. We already work with a number of such companies, including MedImmune, GlaxoSmithKline, AstraZeneca and Aptamer Solutions, and structures of important agricultural or pharmaceutical targets will be of direct benefit to them as they will provide the basis for design of new small molecules (e.g. antibacterials, antifungals). The research will also lead to better understanding of how proteins work, which is of great importance to SMEs in synthetic biology and in metabolic engineering. We have in the past and will in the future patent our discoveries, so the work will lead to improving the economic competitiveness of the EU in general and the UK in particular. The timeframe for drug development is 5-15 years.

A number of the structures are of relevance for public health and animal welfare, and so for international policy and policy makers. This is particularly true for those like the vacuolar ATPase and pyrophosphatase, which are important in trypanosomal diseases such as sleeping sickness. Trypanosomiasis reduces human and animal productivity across large swathes of sub-Saharan Africa. New approaches and drugs will thus improve the quality of life in the developing world in particular. Some of the work has benefits for government agencies and regulators because it will lead to new ways of determining the effects of food additives and drugs: the effects can be studied on the target molecules, rather than via animal testing. The timeframe for this is the next 5-10 years.

There are three immediate benefits to this proposal. First, the immediate research environment in the North of England benefits hugely, as the complete structural biology pipeline proposed does not exist here. It thus meets the goals of the research councils to coordinate and minimize duplication, and it provides important regional support so that Leeds and the North maintain their traditional strength in structural biology, rather than have it all concentrated within 80 miles of Whitehall.

Second, all of the investigators, and the University of Leeds, are committed to outreach to the local and UK community in a variety of ways: inviting schoolchildren in to the University, having them do summer projects in laboratories, giving talks and radio interviews, and acting as a consultants for outreach efforts aimed at teenagers.

Finally, a major transferrable benefit of all academic research is the people trained during the project. The scientists using the equipment acquired through this grant will acquire professional skills that they can use in research-based biotechnological industry. In addition the University of Leeds has an extensive career development program that will provide transferrable skills. These trained people as they move to other institutions in academia, in government and in industry, will affect the larger society positively in all the ways described above.