High-throughput low-volume crystallisation facility

点击翻译按钮获取中文摘要

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.