Fast ATP-dependent Subunit Rotation in Reconstituted F_oF₁-ATP Synthase Trapped in Solution

Abstract

FoF1-ATP synthases are the ubiquitous membrane enzymes which catalyze ATP synthesis or ATP hydrolysis in reverse, respectively. Enzyme kinetics are controlled by internal subunit rotation, by substrate and product concentrations, by mechanical inhibitory mechanisms, but also by the electrochemical potential of protons across the membrane. By utilizing an Anti-Brownian electrokinetic trap (ABEL trap), single-molecule Forster resonance energy transfer (smFRET)-based subunit rotation monitoring was prolonged from milliseconds to seconds. The extended observation times for single proteoliposomes in solution allowed to observe fluctuating rotation rates of individual enzymes and to map the broad distributions of ATP-dependent catalytic rates of FoF1-ATP synthase. The buildup of an electrochemical potential of protons was confirmed to limit the maximum rate of ATP hydrolysis. In the presence of ionophores and uncouplers the fastest subunit rotation speeds measured in single reconstituted FoF1-ATP synthases were 180 full rounds per second, i.e. much faster than measured by biochemical ensemble averaging, but not as fast as the maximum rotational speed reported previously for isolated single F1 fragments without coupling to the membrane-embedded Fo domain of the enzyme.

Monitoring oligomerization dynamics of individual human neurotensin receptors 1 in living cells and in SMALP nanodiscs

“…After a 100-to 150-fold dilution to adjust fluorescent nanoparticle concentrations between 20 pM and 100 pM, the fluorescence brightness of individual SMALPs were recorded on a confocal ABEL trap one at a time. ABEL trapping of single biomolecules had been used for counting bound ATP molecules by stepwise photobleaching 50 , conformational changes of a GPCR 51 and photophysical properties of photosynthetic proteins by lifetime 52,53 , reversible binding and aggregation by diffusion and electromobility analysis 46,54,55 and precise intramolecular distance measurements by smFRET 38,48,56 .…”

“…The ABEL trap for quantitative single nanoparticle spectroscopy in solution was invented by A. E. Cohen and W. E. Moerner [31][32][33][34] . Here, we analyzed the brightness of the NTSR1-SMALPs in our confocal ABEL trap [35][36][37][38] . We found different fluorescence intensity levels in SMALPs derived from HEK293T cells expressing NTSR1-mNeonGreen either before or after stimulation with its ligand neurotensin.…”