To increase the time resolution for a visualization of short rotary steps with dwell times <5 ms, we chose the ‘sliding time window FRET analysis' ( Margittai et al, 2003) and used 50 photons (sum of both FRET donor and acceptor) to calculate one FRET distance data point per 100 μs. Characteristic confocal FRET data from active, single F oF 1-ATP synthases are shown in Figure 2A–D. Enzymes were reconstituted singly into liposomes, and ATP synthesis was initiated by mixing two buffers to generate the pH difference and electrical potential across the membrane ( Steigmiller et al, 2008). ATP synthesis activity of this mutant enzyme (27 ATP s −1 at 25☌) was slightly reduced by 15% compared with the wild-type enzyme (31 ATP s −1 at 25☌). Alexa568-maleimide was used as a FRET acceptor and was covalently bound to a cysteine (E2C mutation) of one c subunit. To deconvolute the sequential stepping of proton-driven c-ring rotation, we fused EGFP as FRET donor to the C terminus of the a subunit ( Duser et al, 2008). Here, we develop this approach further to unravel the individual step size of c-ring rotation and the rotor compliance in the proton-driven F o motor of F oF 1-ATP synthase.ĭetecting proton-driven c-ring rotation in single F oF 1-ATP synthases Single-molecule FRET is measured in a confocal microscope setup, in which freely diffusing liposomes generate bursts of photons while traversing the laser focus/detection volume, respectively ( Figure 1B). The distance between dyes is calculated from the time trajectory of the FRET efficiency E FRET= I A/( I A+ I D) according to the Förster theory ( Forster, 1948). On subunit rotation, the proximity of the fluorophores varies with time and anti-correlated changes in the relative fluorescence intensities I D and I A of FRET donor and acceptor dye are observed. One fluorophore is attached to a rotor subunit and the second label to a stator subunit in E. We have developed a single-molecule fluorescence resonance energy transfer (FRET) approach for load-free detection of subunit rotation during ATP synthesis in real time ( Borsch et al, 2002 Diez et al, 2004 Zimmermann et al, 2005 Duser et al, 2008). However, individual steps during ATP synthesis could not be determined. To enable ATP synthesis by ion gradients across the membrane, ion-impermeable lipid vesicles and small markers without viscous drag limitations were used ( Kaim et al, 2002). Using video microscopy of a large fluorescent pointer ( Sambongi et al, 1999 Sielaff et al, 2008) (μm-long actin filaments or polystyrene double-beads), 120° rotations were reported for surface-attached enzymes in the presence of detergent or embedded in lipid bilayer fragments ( Nishio et al, 2002 Ueno et al, 2005). In the upper panel, the fluorescence intensity of the directly excited Alexa568 (pulsed interleaved excitation with 561 nm, see text) of the same F oF 1-ATP synthase is shown (black trace) confirming the existence of both markers on the enzyme.Įvidence for c-ring rotation in single F oF 1 was reported for the reverse chemical reaction ATP hydrolysis. In the lower panel, fluorescence intensities of FRET donor EGFP (green trace) and acceptor Alexa568 (red trace) are shown excited with 488 nm. ( C) Photon burst of a single FRET-labelled F oF 1-ATP synthase. coli F oF 1-ATP synthase is reconstituted and diffuses freely through the dual laser foci of the duty cycle-optimized alternating laser excitation scheme. Rotation of c results in stepwise distance changes to EGFP. coli F oF 1-ATP synthase with EGFP (green fused to the C terminus of subunit a, orange) and Alexa568 (red, at residue E2C) at one of the c subunits (blue). Single-molecule FRET approach to detect the 36° step size of the rotary c subunits in F oF 1-ATP synthase during ATP synthesis.
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