Regulatory conformational changes of the ε subunit in single FRET-labeled FoF1-ATP synthase.

Subunit ε is an intrinsic regulator of the bacterial FoF1-ATP synthase, the ubiquitous membrane-embedded enzyme that utilizes a proton motive force in most organisms to synthesize adenosine triphosphate (ATP). The C-terminal domain of ε can extend into the central cavity formed by the α and ß subunits, as revealed by the recent X-ray structure of the F1 portion of the Escherichia coli enzyme. This insertion blocks the rotation of the central γ subunit and, thereby, prevents wasteful ATP hydrolysis. Here we aim to develop an experimental system that can reveal conditions under which ε inhibits the holoenzyme FoF1-ATP synthase in vitro. Labeling the C-terminal domain of ε and the γ subunit specifically with two different fluorophores for single-molecule Förster resonance energy transfer (smFRET) allowed monitoring of the conformation of ε in the reconstituted enzyme in real time. New mutants were made for future three-color smFRET experiments to unravel the details of regulatory conformational changes in ε.

Elastic deformations of the rotary double motor of single F(o)F(1)-ATP synthases detected in real time by Förster resonance energy transfer.

Elastic conformational changes of the protein backbone are essential for catalytic activities of enzymes. To follow relative movements within the protein, Förster-type resonance energy transfer (FRET) between two specifically attached fluorophores can be applied. FRET provides a precise ruler between 3 and 8nm with subnanometer resolution. Corresponding submillisecond time resolution is sufficient to identify conformational changes in FRET time trajectories. Analyzing single enzymes circumvents the need for synchronization of various conformations. F(O)F(1)-ATP synthase is a rotary double motor which catalyzes the synthesis of adenosine triphosphate (ATP). A proton-driven 10-stepped rotary F(O) motor in the Escherichia coli enzyme is connected to a 3-stepped F(1) motor, where ATP is synthesized. To operate the double motor with a mismatch of step sizes smoothly, elastic deformations within the rotor parts have been proposed by W. Junge and coworkers. Here we extend a single-molecule FRET approach to observe both rotary motors simultaneously in individual F(O)F(1)-ATP synthases at work. We labeled this enzyme with two fluorophores specifically, that is, on the ε- and c-subunits of the two rotors. Alternating laser excitation was used to select the FRET-labeled enzymes. FRET changes indicated associated transient twisting within the rotors of single enzyme molecules during ATP hydrolysis and ATP synthesis. Supported by Monte Carlo simulations of the FRET experiments, these studies reveal that the rotor twisting is greater than 36° and is largely suppressed in the presence of the rotation inhibitor DCCD. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

Three-color Förster resonance energy transfer within single F0F1-ATP synthases: monitoring elastic deformations of the rotary double motor in real time.

Catalytic activities of enzymes are associated with elastic conformational changes of the protein backbone. Förster-type resonance energy transfer, commonly referred to as FRET, is required in order to observe the dynamics of relative movements within the protein. Förster-type resonance energy transfer between two specifically attached fluorophores provides a ruler with subnanometer resolution between 3 and 8 nm, submillisecond time resolution for time trajectories of conformational changes, and single-molecule sensitivity to overcome the need for synchronization of various conformations. F(O)F(1)-ATP synthase is a rotary molecular machine which catalyzes the formation of adenosine triphosphate (ATP). The Escherichia coli enzyme comprises a proton driven 10 stepped rotary F(O) motor connected to a 3-stepped F(1) motor, where ATP is synthesized. This mismatch of step sizes will result in elastic deformations within the rotor parts. We present a new single-molecule FRET approach to observe both rotary motors simultaneously in a single F(O)F(1)-ATP synthase at work. We labeled this enzyme with three fluorophores, specifically at the stator part and at the two rotors. Duty cycle-optimized with alternating laser excitation, referred to as DCO-ALEX, allowed to control enzyme activity and to unravel associated transient twisting within the rotors of a single enzyme during ATP hydrolysis and ATP synthesis. Monte Carlo simulations revealed that the rotor twisting is larger than 36 deg.

36 degrees step size of proton-driven c-ring rotation in FoF1-ATP synthase.

Synthesis of adenosine triphosphate ATP, the 'biological energy currency', is accomplished by F(o)F(1)-ATP synthase. In the plasma membrane of Escherichia coli, proton-driven rotation of a ring of 10 c subunits in the F(o) motor powers catalysis in the F(1) motor. Although F(1) uses 120 degrees stepping during ATP synthesis, models of F(o) predict either an incremental rotation of c subunits in 36 degrees steps or larger step sizes comprising several fast substeps. Using single-molecule fluorescence resonance energy transfer, we provide the first experimental determination of a 36 degrees sequential stepping mode of the c-ring during ATP synthesis.

The proton-translocating a subunit of F0F1-ATP synthase is allocated asymmetrically to the peripheral stalk.

The position of the a subunit of the membrane-integral F0 sector of Escherichia coli ATP synthase was investigated by single molecule fluorescence resonance energy transfer studies utilizing a fusion of enhanced green fluorescent protein to the C terminus of the a subunit and fluorescent labels attached to specific positions of the epsilon or gamma subunits. Three fluorescence resonance energy transfer levels were observed during rotation driven by ATP hydrolysis corresponding to the three resting positions of the rotor subunits, gamma or epsilon, relative to the a subunit of the stator. Comparison of these positions of the rotor sites with those previously determined relative to the b subunit dimer indicates the position of a as adjacent to the b dimer on its counterclockwise side when the enzyme is viewed from the cytoplasm. This relationship provides stability to the membrane interface between a and b2, allowing it to withstand the torque imparted by the rotor during ATP synthesis as well as ATP hydrolysis.

Crystal structure of the archaeal A1Ao ATP synthase subunit B from Methanosarcina mazei Gö1: Implications of nucleotide-binding differences in the major A1Ao subunits A and B.

The A1Ao ATP synthase from archaea represents a class of chimeric ATPases/synthases, whose function and general structural design share characteristics both with vacuolar V1Vo ATPases and with F1Fo ATP synthases. The primary sequences of the two large polypeptides A and B, from the catalytic part, are closely related to the eukaryotic V1Vo ATPases. The chimeric nature of the A1Ao ATP synthase from the archaeon Methanosarcina mazei Gö1 was investigated in terms of nucleotide interaction. Here, we demonstrate the ability of the overexpressed A and B subunits to bind ADP and ATP by photoaffinity labeling. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to map the peptide of subunit B involved in nucleotide interaction. Nucleotide affinities in both subunits were determined by fluorescence correlation spectroscopy, indicating a weaker binding of nucleotide analogues to subunit B than to A. In addition, the nucleotide-free crystal structure of subunit B is presented at 1.5 A resolution, providing the first view of the so-called non-catalytic subunit of the A1Ao ATP synthase. Superposition of the A-ATP synthase non-catalytic B subunit and the F-ATP synthase non-catalytic alpha subunit provides new insights into the similarities and differences of these nucleotide-binding ATPase subunits in particular, and into nucleotide binding in general. The arrangement of subunit B within the intact A1Ao ATP synthase is presented.