Turning symmetric protein scaffolds into robust enablers of structural biology

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Images that show relevant biological molecules, their fine details and their interactions, help biochemical and pharmaceutical researchers in their experimental programmes. Structural knowledge of biological molecules has historically transformed our view of what they do and how they do it. Today, such knowledge is increasingly being used to drive the discovery of drugs and vaccines to combat diseases that range from bacterial infection to cancer. Two experimental techniques are key to producing these images. Macromolecular X-ray crystallography (MX) is the most mature method for determining structures of the molecules (usually proteins) that govern the operation of biological systems. However, as recognized by the award of the 2017 Nobel Prize in Chemistry, electron microscopy (EM) has recently come of age as a viable alternative for atomic resolution visualization of large macromolecules. Despite significant technical advances in recent years for both MX and EM, these two techniques do not yet provide a complete answer to observing all biological molecules in a timely fashion. EM is typically only used to study relatively large biological molecules and complexes. X-ray crystallography is less demanding in respect of size, but, requires the protein of interest to form crystals - billions of identical copies arrayed in a near-faultless three-dimensional grid. This can only be accomplished by trial and error, by testing a protein in thousands of different solutions to see if it can be persuaded to crystallise. Alas, this fails more often than not, and for many of the most therapeutically interesting, it is difficult if not impossible.Our approach to addressing this issue is to prepare nano-sized 3D scaffolds ("crysalins") on to which a protein of choice can be attached in a crystalline array. Whilst we have successfully produced the nano-scaffolds and attached examplar targets onto them, we remain one step away from this becoming a universal platform for structure determination. Currently the target proteins are attached only loosely to the scaffold and, because X-rays show you an average picture of the repeating unit of the array, any looseness "smears out" the image of the target protein. This programme of work will firm up the connection to ensure that each target molecule is attached to the scaffold in exactly the same position and orientation.An added benefit of developing rigid target-scaffold connections is that the building blocks that make up our scaffolds are large and symmetric and therefore perfectly suited to imaging on an electron microscope. Even small targets (of a size usually inaccessible to EM) that are rigidly and uniformly attached to such building blocks have been shown to be straightforwardly imaged by EM. We are therefore producing a building block that can either be used in isolation to facilitate electron microscopy or assembled into a lattice to enable crystallography.An area in which MX still reigns supreme is high-throughput structure determination, especially where one is looking at a single protein target binding to a number of different small chemicals. If suitable crystals of a protein are available, they can be soaked in solutions of these chemicals and imaged. Indeed, one can screen a large library of chemicals and trawl through the structural images to find candidates that might be refined as therapeutic drugs. The whole process of refinement can be done rationally, seeing how variants of the original chemical bind and how their shape and properties might be changed in order to improve them to enhance their effectiveness. This process is known as structure-based ligand design, SBLD. We will demonstrate the potential of crysalin technology for SBLD in conjuction with the "XChem" chemical screening platform and apply it to proteins that are potential therapeutic targets under study in the laboratories of the PIs.