Monitoring subunit rotation in single FRET-labeled F_oF₁-ATP synthase in an anti-Brownian electrokinetic trap

Abstract

F o F 1 -ATP synthase is the membrane protein catalyzing the synthesis of the 'biological energy currency' adenosine triphosphate (ATP). The enzyme uses internal subunit rotation for the mechanochemical conversion of a proton motive force to the chemical bond. We apply single-molecule Förster resonance energy transfer (FRET) to monitor subunit rotation in the two coupled motors F 1 and F o . Therefore, enzymes have to be isolated from the plasma membranes of Escherichia coli, fluorescently labeled and reconstituted into 120-nm sized lipid vesicles to yield proteoliposomes. These freely diffusing proteoliposomes occasionally traverse the confocal detection volume resulting in a burst of photons. Conformational dynamics of the enzyme are identified by sequential changes of FRET efficiencies within a single photon burst. The observation times can be extended by capturing single proteoliposomes in an anti-Brownian electrokinetic trap (ABELtrap, invented by A. E. Cohen and W. E. Moerner). Here we describe the preparation procedures of F o F 1 -ATP synthase and simulate FRET efficiency trajectories for 'trapped' proteoliposomes. Hidden Markov Models are applied at signal-to-background limits for identifying the dwells and substeps of the rotary enzyme when running at low ATP concentrations, excited by low laser power, and confined by the ABELtrap.

Observing single FoF1-ATP synthase at work using an improved fluorescent protein mNeonGreen as FRET donor

“…The quality of the enzyme was comparable to the purified FoF1-a-EGFP. However, the enzyme exhibited more impurities than purified 'wildtype' FoF1 ATP synthase 41 . The most important purification step of the latter protocol was the anion exchange chromatography ( Figure 3B).…”

“…The new mNeonGreen fusion construct showed the same reduced expression level as the EGFP fusion as indicated by the reduced bands of the  and β subunits. Our purification procedures for ATP synthesis-active FoF1-ATP synthase from Escherichia coli were based on wellestablished protocols 5,11,31,41 . However, the low expression level of the FoF1-a-mNeonGreen mutant made purification more difficult.…”

Observing conformations of single FoF1-ATP synthases in a fast anti-Brownian electrokinetic trap

“…Our previous versions of the ABELtrap, EMCCD-based 82 or AOBD-based 32,80,81 , respectively, did not reach residence times of more than 100 seconds for fluorescent nanobeads as we have achieved now. However, the actual versions of the ABELtraps in Stanford and in Harvard have been developed even further and have implemented sophisticated learning algorithms for the diffusion and electromobility properties of a trapped molecule.…”

“…Photons were recorded in parallel on two computers, i.e. by a FPGA card (PCIe-7852R, National Instruments) and by two synchronized TCSPC cards (SPC150 and SPC150N, respectively, Becker&Hickl, Germany), similar to our different ABELtrap setup published previously 32,80,81 . The FPGA Labview program from A.…”

Analyzing conformational dynamics of single P-glycoprotein transporters by Förster resonance energy transfer using hidden Markov models

“…We used low count rates (sum intensity limited to less than 20 cpms) and short dwell times of 10 ms or 20 ms, respectively, and included high background count rates as well as photobleaching of donor or acceptor [59, 60]. The HMM reassigned the FRET states and dwell times precisely.…”