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

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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.