Correlative smFRET/electrophysiology for long-term studies of protein dynamics

The conformational dynamics of proteins are essential for their proper functioning, and impaired dynamics can lead to disease.

However, it remains difficult to experimentally determine the multitude of conformations adopted by proteins, their lifetimes and the transitions between them.

Single-molecule FRET (smFRET) is probably the most suitable technique available due to its ability to operate under ambient conditions while avoiding ensemble averaging.

Yet the technique is limited by the short observation times (a few seconds) and the difficulty of measuring more than one molecular distance at the same time.The goal of this proposal is to develop an innovative correlative approach that can extend smFRET observations to much longer timescales, while also providing a way to integrate multiple measurements into a coherent molecular picture.

I will do so by combining smFRET measurements with nanopore-based electrophysiological experiments that have a high temporal resolution and are also sensitive to conformational changes.

Our corelative system will thus combine the speed and the sensitivity of electrical readout with the direct observation of molecular distances using smFRET. We will apply this methodology to map conformational dynamics of the biologically-important Ras proteins. We expect that this project will allow us to develop our innovative methodology and claim the novelty of the concept.

This new methodology should open entirely new opportunities to monitor fast and long-term conformational dynamics of proteins or assembly processes of macromolecules.