Detecting intramolecular conformational dynamics of single molecules in short distance range with subnanometer...

Single molecule detection is useful for characterizing nanoscale objects such as biological macromolecules, nanoparticles and nanodevices with nanometer spatial resolution. Fluorescence resonance energy transfer (FRET) is widely used as a single-molecule assay to monitor intramolecular dynamics in the distance range of 3-8 nm. Here we demonstrate that self-quenching of two rhodamine derivatives can be used to detect small conformational dynamics corresponding to subnanometer distance changes in a FRET-insensitive short-range at the single molecule level. A ParM protein mutant labeled with two rhodamines works as a single molecule adenosine 5'-diphosphate (ADP) sensor that has 20 times brighter fluorescence signal in the ADP bound state than the unbound state. Single molecule time trajectories show discrete transitions between fluorescence on and off states that can be directly ascribed to ADP binding and dissociation events. The conformational changes observed with 20:1 contrast are only 0.5 nm in magnitude and are between crystallographic distances of 1.6 and 2.1 nm, demonstrating exquisite sensitivity to short distance scale changes. The systems also allowed us to gain information on the photophysics of self-quenching induced by rhodamine stacking: (1) photobleaching of either of the two rhodamines eliminates quenching of the other rhodamine fluorophore and (2) photobleaching from the highly quenched, stacked state is only 2-fold slower than from the unstacked state.