Improving FRET‐Based Monitoring of Single Chemomechanical Rotary Motors at Work

Abstract

Protein dynamics are observable in real time using internal distance measurements of reference positions. Based on Förster resonance energy transfer (FRET) as the distance ruler between two fluorescent markers, investigations of single molecules one at a time have modified our views of protein conformational changes. X-ray crystallography provides trapped conformations at atomic resolution and NMR can add information about flexible parts of proteins, but the pathways between these structures including unknown conformations are often not directly accessible. Especially for membrane enzymes comprising multiple subunits, limited structural information requires alternative experimental methods to follow conformational pathways. Herein we summarize the knowledge gathered from single-molecule FRET studies of the membrane-embedded rotary nanomotor F(o)F(1)-ATP synthase. In addition, new ideas and concepts to shift and extend the current limitations of the confocal FRET detection approach using freely diffusing, liposome-reconstituted, individual enzymes are discussed: nanodiamonds as new fluorophores, optimized laser excitation schemes, Hidden Markov Models for software-based FRET trajectory analysis, and application of the anti-Brownian electrokinetic trap (ABELtrap) to keep the single enzyme in focus.

Three-color Förster resonance energy transfer within single F[sub O]F[sub 1]-ATP synthases: monitoring elastic deformations of the rotary double motor in real time

“…The photophysics of the three fluorophores has to be optimized, 43 such as changing EGFP to Alexa488 as the primary FRET donor using a SNAP-tag fusion on the static subunit a. The second FRET donor should be changed to a rhodamine, with red-shifted absorbance and fluorescence, such as Atto565.…”

“…39 In single molecule detection, increasing the excitation power causes increased photobleaching probabilities and populates the non-fluorescent dark triplet state of the fluorophore. 43 Therefore, peak powers of pulsed lasers must be low, but repetition rates can be high. Switching a cw-laser on and off is limited by the switching times of the AOM and, for the off-switching, by the fluorescence lifetime of the dye.…”

Biophysical Characterization of a Thermoalkaliphilic Molecular Motor with a High Stepping Torque Gives Insight into Evolutionary ATP Synthase Adaptation

“…Indeed, the rotation of F 1 ATPases has been visualized using optical microscopy by attaching a probe to the ␥-or ⑀-subunit in the relative area of where the F 0 domain would be located had it been attached (23,28,29). As this method of study has developed, the type of probe utilized has diversified from actin filaments (23,30) into gold nanoparticles/nanorods (31,32), magnetic beads (33,34), polystyrene beads (35,36), and fluorescent molecules (37)(38)(39). The two most heavily studied bacterial F 1 ATPases come from the thermophile Bacillus PS3 (TF 1 ) and the mesophile Escherichia coli (EF 1 ).…”

Subunit rotation in a single F o F 1 -ATP synthase in a living bacterium monitored by FRET

“…FRET-labeled F o F 1 -ATP synthases were identified by photon bursts which exceeded the minimum count rate threshold for the FRET acceptor test. The mean burst duration was about 30 ms as expected for freely diffusing liposomes without trapping [42] , but also bursts with several hundred millisecond durations were found. FRET efficiency (E FRET ) levels within these bursts were assigned manually.…”