Engineered Affimers for cryo-EM studies of unsolved protein structures

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Cryogenic electron microscopy (cryo-EM) has revolutionised structural biology and has enabled us to see atomic structures of challenging macromolecular assemblies, including amyloid fibres, SARS-CoV-2 and protein structures in cells. However, challenges remain to determine structures of small proteins using cryo-EM routinely. Small proteins such as kinases are excellent targets for drug discovery. However, due to their small size, cryo-EM is not particularly useful to study structures of such proteins with ligands. In this project, we will engineer non-antibody-binding proteins Affimers to develop a new toolkit and methodology that solves these challenges. Affimers are a Leeds-born technology and have been successfully used to visualise proteins and regulate their functions. We seek to extend the application of Affimers to cryo-EM. The project particularly focuses on developing an Affimer-based cryo-EM methodology, universally applicable to recombinant proteins. As a model target protein, we will use a 40 kDa mitotic kinase Aurora-A, of which cryo-EM structure has been undetermined. Aurora-A is an essential regulator of mitosis and a compelling target for cancer drug discovery. Thus, the project will contribute to a major challenge in fundamental biology and human health.