Engineered Affimers for cryo-EM studies of unsolved protein structures

低温电子显微镜(Cryo-EM)使结构生物学发生了革命性的变化，使我们能够看到具有挑战性的大分子组件的原子结构，包括淀粉样纤维、SARS-CoV-2和细胞中的蛋白质结构。然而，常规使用冷冻EM来确定小蛋白质的结构仍然存在挑战。像激酶这样的小蛋白是药物发现的极佳靶点。然而，由于它们的体积很小，冷冻-EM并不是特别有用的研究这类蛋白质的结构与配体。在这个项目中，我们将设计非抗体结合蛋白仿射器来开发一种新的工具包和方法来解决这些挑战。仿射器是利兹诞生的一项技术，已成功地用于可视化蛋白质和调节其功能。我们寻求将仿射器的应用扩展到低温EM。该项目特别侧重于开发一种普遍适用于重组蛋白质的基于仿射分子的冷冻-EM方法学。作为一个模型靶蛋白，我们将使用一个40 kDa的有丝分裂激酶Aurora-A，其冷冻-EM结构尚未确定。Aurora-A是有丝分裂的重要调节者，也是抗癌药物发现的一个引人注目的靶点。因此，该项目将对基础生物学和人类健康方面的重大挑战做出贡献。

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.