Single-Molecule FRET for structural biology of active membrane proteins

Since 1996, single-molecule Förster resonance energy transfer (smFRET) has become a powerful spectroscopic and imaging method to study the conformational dynamics of biological nanomachines. We started smFRET measurements in 2000 to monitor subunit rotation and regulatory control mechanisms in individual FoF1-ATP synthases in liposomes. To counteract diffusive motion actively in real-time and to prolong observation times, we have built a fast anti-Brownian electrokinetic trap (ABEL trap) with a laser focus pattern and electrode feedback. Using the ABEL trap for smFRET we revealed fast subunit rotation, broad distributions of ATP hydrolysis rates, and changing speeds of a single enzyme at work.