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获得的见解，这些方法只能提供蛋白质折叠的快照，或者基于溶液的单分子方法，需要化学修饰，损害肌动蛋白单体的热稳定性。近年来，纳米孔传感技术的发展使研究天然蛋白质成为可能。这种无标签的方法非常适合研究蛋白质，如肌动蛋白，需要专门的缓冲液和辅助因子。这个项目是基于固体纳米孔的使用，以确定肌动蛋白的状态在大分子水平（丝状或球状），并在其单体形式结合抑制剂。我们将使用该技术来获取尿素依赖和电压依赖的过渡状态，关键的是，我们将观察一个展开过程，在这个过程中，瞬态肌动蛋白低聚物的亚群。此外，我们计划利用实时的药物结合或纤维生长事件来计算化学计量学，从而提出一个使用未经修饰的天然肌动蛋白分子的蛋白质动力学模型。这种溶液中肌动蛋白的动态读数显示了纳米吸管在未来应用中的强大功能，例如获得对蛋白质折叠景观的更深入了解和药物发现。

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.