Label-free nanopore sensing of single actin dynamics and drug binding in real-time

肌动蛋白是真核细胞内动态过程的核心关键蛋白。迄今为止，探索肌动蛋白分子状态的方法仅限于从结构方法中获得的见解，这些方法仅提供蛋白质折叠的快照，或基于溶液的单分子方法，这些方法需要化学修饰，从而影响肌动蛋白单体的热稳定性。近年来，纳米孔传感的发展已经允许研究天然蛋白质。这种无标记方法非常适合研究需要专门缓冲液和辅因子的蛋白质，如肌动蛋白。该项目是基于使用固态纳米孔，以确定肌动蛋白在大分子水平（丝状或球状）和其单体形式结合抑制剂的状态。我们将使用该技术来获得尿素依赖性和电压依赖性的过渡状态，关键的是，我们观察一个展开过程中，瞬时肌动蛋白寡聚体的亚群。此外，我们计划使用药物结合或顺性生长事件实时计算化学计量，从而提出了一个模型的蛋白质动力学使用未经修改的，天然的肌动蛋白分子。溶液中肌动蛋白的这种动态读数展示了纳米移液管在未来应用中的能力，例如更深入地了解蛋白质折叠景观和药物发现。

Actin is a key protein at the heart of dynamic processes within the eukaryotic cell. To date, methods exploring the molecular state of actin have been limited to insights gained from structural approaches, which provide only a snapshot of protein folding, or solution-based single-molecule methods that require chemical modifications compromising actin monomer thermostability. In recent years, the development of nanopore sensing has permitted investigation of native proteins. This label-free method is ideally suited to study proteins such as actin that require specialised buffers and cofactors. This project is based around the use of solid-state nanopores, to determine the state of actin at the macromolecular level (filamentous or globular) and in its monomeric form bound to inhibitors. We will use the technology to access urea-dependent and voltage-dependent transitional states and, critically, we to observe an unfolding process within which sub-populations of transient actin oligomers. Furthermore, we plan to use drug-binding or filament-growth events in real-time to calculate stoichiometries and thus propose a model for protein dynamics using unmodified, native actin molecules. This dynamic readout of actin in solution demonstrates the power of nanopipettes for future applications such as gaining a more in-depth understanding of protein folding landscapes and for drug discovery.