The regulatory switch of F₁-ATPase studied by single-molecule FRET in the ABEL trap

Abstract

F1-ATPase is the soluble portion of the membrane-embedded enzyme FoF1-ATP synthase that catalyzes the production of adenosine triphosphate in eukaryotic and eubacterial cells. In reverse, the F1 part can also hydrolyze ATP quickly at three catalytic binding sites. Therefore, catalysis of ‘non-productive’ ATP hydrolysis by F1 (or FoF1) must be minimized in the cell. In bacteria, the ε subunit is thought to control and block ATP hydrolysis by mechanically inserting its C-terminus into the rotary motor region of F1. We investigate this proposed mechanism by labeling F1 specifically with two fluorophores to monitor the C-terminus of the ε subunit by Förster resonance energy transfer. Single F1 molecules are trapped in solution by an Anti-Brownian electrokinetic trap which keeps the FRET-labeled F1 in place for extended observation times of several hundreds of milliseconds, limited by photobleaching. FRET changes in single F1 and FRET histograms for different biochemical conditions are compared to evaluate the proposed regulatory mechanism.

Aerobic Growth of Escherichia coli Is Reduced, and ATP Synthesis Is Selectively Inhibited when Five C-terminal Residues Are Deleted from the ϵ Subunit of ATP Synthase

“…Initial studies with isolated F 1 (59,60) showed bimodal distribution of FRET efficiencies that correlate with the ⑀ C and ⑀ X states, and nucleotides shifted the balance between the two FRET states in agreement The specific activity of F 1 (-␦⑀) alone was 60.9 mol/min/mg. For each data set, the curve shown is from a nonlinear regression fit to a quadratic equation described in Ref.…”

Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics

“…It is difficult to characterize such spatially and temporally inhomogeneous dynamics in bulk solution by an ensemble-averaged measurement, especially in proteins that undergo multiple-conformation transformations. In such cases, single-molecule spectroscopy is a powerful approach to analyze protein conformational states and dynamics under physiological conditions, and can provide a molecular-level perspective on how a protein's structural dynamics link to its functional mechanisms (12)(13)(14)(15)(16)(17)(18)(19)(20)(21).…”

Regulatory conformational changes of the Ɛ subunit in single FRET-labeled F0F1-ATP synthase

“…Separately we purified a cysteine mutant of the ε subunit, ε99C, for labeling with Atto647N. F 1 -γ108-atto488 and ε99-atto647N were combined to yield F 1 -γ108-atto488/ε99-atto647N as described previously [47] (see also S. D. Bockenhauer et al [69] , preprint available at http://arxiv.org/abs/1402.1845). To complete F 1 with all 9 subunits, we added an excess of additional δ that was overexpressed [44] and purified separately.…”