Single-molecule FRET combined with electrokinetic trapping reveals real-time enzyme kinetics of individual F-ATP synthases

Single-molecule Forster resonance energy transfer (smFRET) is a key technique to observe conformational changes in molecular motors and to access the details of single-molecule static and dynamic disorder during catalytic processes. However, studying freely diffusing molecules in solution is limited to a few tens of milliseconds, while surface attachment often bears the risk to restrict their natural motion. In this paper we combine smFRET and electrokinetic trapping (ABEL trap) to non-invasively hold single FOF1-ATP synthases for up to 3 s within the detection volume, thereby extending the observation time by a factor of 10 as compared to Brownian diffusion without surface attachment. In addition, we are able to monitor complete reaction cycles and to selectively trap active molecules based on their smFRET signal, thus speeding up the data acquisition process. We demonstrate the capability of our method to study the dynamics of single molecules by recording the ATP-hydrolysis driven rotation of individual FOF1-ATP synthase molecules over numerous reaction cycles and extract their kinetic rates. We argue that our method is not limited to motor proteins. Instead, it can be applied to monitor conformational changes with millisecond time resolution for a wide range of enzymes, thereby making it a versatile tool for studying protein dynamics.

Automated summary

In this study, we combined smFRET and electrokinetic trapping to non-invasively hold single FOF1-ATP synthases. The observation time was extended by a factor of 10 compared to Brownian diffusion without surface attachment. Our method allows for monitoring complete reaction cycles and extracting kinetic rates, making it a versatile tool for studying protein dynamics, not limited to motor proteins.