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

Abstract

F o F 1 -ATP synthases are ubiquitous membrane-bound, rotary motor enzymes that can catalyze ATP synthesis and hydrolysis. Their enzyme kinetics are controlled by internal subunit rotation, by substrate and product concentrations, and by mechanical inhibitory mechanisms but also by the electrochemical potential of protons across the membrane. Single-molecule Forster resonance energy transfer (smFRET) has been used to detect subunit rotation within F o F 1 -ATP synthases embedded in freely diffusing liposomes. We now report that kinetic monitoring of functional rotation can be prolonged from milliseconds to seconds by utilizing an anti-Brownian electrokinetic trap (ABEL trap). These extended observation times allowed us to observe fluctuating rates of functional rotation for individual F o F 1 -liposomes in solution. Broad distributions of ATP-dependent catalytic rates were revealed. The buildup of an electrochemical potential of protons was confirmed to limit the maximum rate of ATP hydrolysis. In the presence of ionophores or uncouplers, the fastest subunit rotation speeds measured in single reconstituted F o F 1 -ATP synthases were 180 full rounds per second. This was much faster than measured by biochemical ensemble averaging, but not as fast as the maximum rotational speed reported previously for isolated single F 1 complexes uncoupled from the membraneembedded F o complex. Further application of ABEL trap measurements should help resolve the mechanistic causes of such fluctuating rates of subunit rotation.

Single-molecule FRET combined with electrokinetic trapping reveals real-time enzyme kinetics of individual F-ATP synthases

“…In contrast, the authors of a comparable study have argued that the high FRET level is a representative of two states that are indistinguishable due to the geometry of the fluorescent labels during rotation. 46 In this case, even if the two states cannot be distinguished by FRET levels, one would expect the enzyme to rest in the high level twice as long as in the low level. Instead, the authors observed two subpopulations with comparable occurrence and duration.…”

“…The simple assignment of FRET levels to molecular states based on structural data and expected fluoropore distances can lead to inconsistancies within the dataset. 46 Detection techniques with immobilised samples and attached labels such as actin filaments, 44,47–50 gold nanorods 51,52 or beads 12,40 seem to have an advantage over single fluorophore detection in resolving rotational steps but risk obstruction by nearby surface interactions that can result in artificially increased dwell times. 37,40,53…”

“…14 Even though individual F O F 1 -ATP synthases display a wide distribution in rotational rates at any given ATP concentration, lower ATP concentrations resulted in slower catalytic rates on average. Another recent study 46 showed an even more in-depth analysis of molecular heterogeneity for the F-ATP synthase at ATP concentrations from 1 mM to 5 μM: the catalytic rates varied substantially by a factor of 20 to 8, depending on the respective ATP concentration. The authors argued that one cause of this static disorder is the buildup of a pmf counteracting the ATP hydrolysis reaction.…”

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

“…Our implementation of a fast FPGA-based ABEL trap was described earlier 6, 35 - 38, 44, 45 and was slightly modified here. Briefly, a linearly polarized continuous-wave laser emitting at 491 nm (Calypso, 50 mW, Cobolt) was attenuated to 40 μW.…”

“…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.…”