scholarly journals Simple mechanism whereby the F1-ATPase motor rotates with near-perfect chemomechanical energy conversion

2015 ◽  
Vol 112 (31) ◽  
pp. 9626-9631 ◽  
Author(s):  
Ei-ichiro Saita ◽  
Toshiharu Suzuki ◽  
Kazuhiko Kinosita ◽  
Masasuke Yoshida
Keyword(s):  
Physical Model ◽  
Single Molecule ◽  
Atp Hydrolysis ◽  
Rotation Angle ◽  
Mechanical Work ◽  
F1 Atpase ◽  
The Third ◽  
Γ Subunit ◽  
Simple Physical Model ◽  
Central Shaft

F1-ATPase is a motor enzyme in which a central shaft γ subunit rotates 120° per ATP in the cylinder made of α3β3 subunits. During rotation, the chemical energy of ATP hydrolysis (ΔGATP) is converted almost entirely into mechanical work by an elusive mechanism. We measured the force for rotation (torque) under various ΔGATP conditions as a function of rotation angles of the γ subunit with quasi-static, single-molecule manipulation and estimated mechanical work (torque × traveled angle) from the area of the function. The torque functions show three sawtooth-like repeats of a steep jump and linear descent in one catalytic turnover, indicating a simple physical model in which the motor is driven by three springs aligned along a 120° rotation angle. Although the second spring is unaffected by ΔGATP, activation of the first spring (timing of the torque jump) delays at low [ATP] (or high [ADP]) and activation of the third spring delays at high [Pi]. These shifts decrease the size and area of the sawtooth (magnitude of the work). Thus, F1-ATPase responds to the change of ΔGATP by shifting the torque jump timing and uses ΔGATP for the mechanical work with near-perfect efficiency.

Molecular Mechanism of ATP Hydrolysis in F1-ATPase Revealed by Molecular Simulations and Single-Molecule Observations

2012 ◽  
Vol 134 (20) ◽  
pp. 8447-8454 ◽  
Author(s):  
Shigehiko Hayashi ◽  
Hiroshi Ueno ◽  
Abdul Rajjak Shaikh ◽  
Myco Umemura ◽  
Motoshi Kamiya ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Single Molecule ◽  
Atp Hydrolysis ◽  
Molecular Simulations ◽  
F1 Atpase

scholarly journals Regulation of the thermoalkaliphilic F1-ATPase from Caldalkalibacillus thermarum

2016 ◽  
Vol 113 (39) ◽  
pp. 10860-10865 ◽  
Author(s):  
Scott A. Ferguson ◽  
Gregory M. Cook ◽  
Martin G. Montgomery ◽  
Andrew G. W. Leslie ◽  
John E. Walker
Keyword(s):  
Conformational Changes ◽  
Catalytic Domain ◽  
Atp Hydrolysis ◽  
Hydrolytic Activity ◽  
Magnesium Ion ◽  
Geobacillus Stearothermophilus ◽  
F1 Atpase ◽  
Phosphate Ion ◽  
Γ Subunit ◽  
Open Conformation

The crystal structure has been determined of the F1-catalytic domain of the F-ATPase from Caldalkalibacillus thermarum, which hydrolyzes adenosine triphosphate (ATP) poorly. It is very similar to those of active mitochondrial and bacterial F1-ATPases. In the F-ATPase from Geobacillus stearothermophilus, conformational changes in the ε-subunit are influenced by intracellular ATP concentration and membrane potential. When ATP is plentiful, the ε-subunit assumes a “down” state, with an ATP molecule bound to its two C-terminal α-helices; when ATP is scarce, the α-helices are proposed to inhibit ATP hydrolysis by assuming an “up” state, where the α-helices, devoid of ATP, enter the α3β3-catalytic region. However, in the Escherichia coli enzyme, there is no evidence that such ATP binding to the ε-subunit is mechanistically important for modulating the enzyme’s hydrolytic activity. In the structure of the F1-ATPase from C. thermarum, ATP and a magnesium ion are bound to the α-helices in the down state. In a form with a mutated ε-subunit unable to bind ATP, the enzyme remains inactive and the ε-subunit is down. Therefore, neither the γ-subunit nor the regulatory ATP bound to the ε-subunit is involved in the inhibitory mechanism of this particular enzyme. The structure of the α3β3-catalytic domain is likewise closely similar to those of active F1-ATPases. However, although the βE-catalytic site is in the usual “open” conformation, it is occupied by the unique combination of an ADP molecule with no magnesium ion and a phosphate ion. These bound hydrolytic products are likely to be the basis of inhibition of ATP hydrolysis.

scholarly journals Method to extract multiple states in F1-ATPase rotation experiments from jump distributions

2019 ◽  
Vol 116 (51) ◽  
pp. 25456-25461
Author(s):  
Sándor Volkán-Kacsó ◽  
Luan Q. Le ◽  
Kaicheng Zhu ◽  
Haibin Su ◽  
Rudolph A. Marcus
Keyword(s):  
Relaxation Time ◽  
Single Molecule ◽  
Rotation Angle ◽  
Bimodal Distribution ◽  
Atp Binding ◽  
Angular Range ◽  
Β Subunit ◽  
Consistency Test ◽  
F1 Atpase ◽  
Adp Release

A method is proposed for analyzing fast (10 μs) single-molecule rotation trajectories in F1adenosinetriphosphatase (F1-ATPase). This method is based on the distribution of jumps in the rotation angle that occur in the transitions during the steps between subsequent catalytic dwells. The method is complementary to the “stalling” technique devised by H. Noji et al. [Biophys. Rev.9, 103–118, 2017], and can reveal multiple states not directly detectable as steps. A bimodal distribution of jumps is observed at certain angles, due to the system being in either of 2 states at the same rotation angle. In this method, a multistate theory is used that takes into account a viscoelastic fluctuation of the imaging probe. Using an established sequence of 3 specific states, a theoretical profile of angular jumps is predicted, without adjustable parameters, that agrees with experiment for most of the angular range. Agreement can be achieved at all angles by assuming a fourth state with an ∼10 μs lifetime and a dwell angle about 40° after the adenosine 5′-triphosphate (ATP) binding dwell. The latter result suggests that the ATP binding in one β subunit and the adenosine 5′-diphosphate (ADP) release from another β subunit occur via a transient whose lifetime is ∼10 μs and is about 6 orders of magnitude smaller than the lifetime for ADP release from a singly occupiedF1-ATPase. An internal consistency test is given by comparing 2 independent ways of obtaining the relaxation time of the probe. They agree and are ∼15 μs.

scholarly journals Efficient conversion of chemical energy into mechanical work by Hsp70 chaperones

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Salvatore Assenza ◽  
Alberto Stefano Sassi ◽  
Ruth Kellner ◽  
Benjamin Schuler ◽  
Paolo De Los Rios ◽  
...  
Keyword(s):  
Free Energy ◽  
Single Molecule ◽  
Atp Hydrolysis ◽  
Critical Role ◽  
Quantitative Agreement ◽  
Mechanical Work ◽  
Chemical Energy ◽  
Coarse Grained ◽  
Theoretical Approaches ◽  
Non Equilibrium

Hsp70 molecular chaperones are abundant ATP-dependent nanomachines that actively reshape non-native, misfolded proteins and assist a wide variety of essential cellular processes. Here, we combine complementary theoretical approaches to elucidate the structural and thermodynamic details of the chaperone-induced expansion of a substrate protein, with a particular emphasis on the critical role played by ATP hydrolysis. We first determine the conformational free-energy cost of the substrate expansion due to the binding of multiple chaperones using coarse-grained molecular simulations. We then exploit this result to implement a non-equilibrium rate model which estimates the degree of expansion as a function of the free energy provided by ATP hydrolysis. Our results are in quantitative agreement with recent single-molecule FRET experiments and highlight the stark non-equilibrium nature of the process, showing that Hsp70s are optimized to effectively convert chemical energy into mechanical work close to physiological conditions.

scholarly journals A rotary molecular motor that can work at near 100% efficiency

2000 ◽  
Vol 355 (1396) ◽  
pp. 473-489 ◽  
Author(s):  
Kazuhiko Kinosita ◽  
Ryohei Yasuda ◽  
Hiroyuki Noji ◽  
Kengo Adachi
Keyword(s):  
Free Energy ◽  
Actin Filament ◽  
Single Molecule ◽  
High Efficiency ◽  
Atp Hydrolysis ◽  
Molecular Motor ◽  
Constant Torque ◽  
Γ Subunit ◽  
Work Done

A single molecule of F 1 –ATPase is by itself a rotary motor in which a central γ–subunit rotates against a surrounding cylinder made of α 3 β 3 –subunits. Driven by the three βs that sequentially hydrolyse ATP, the motor rotates in discrete 120° steps, as demonstrated in video images of the movement of an actin filament bound, as a marker, to the central γ–subunit. Over a broad range of load (hydrodynamic friction against the rotating actin filament) and speed, the F motor produces a constant torque of ca . 40 pN nm. The work done in a 120° step, or the work per ATP molecule, is thus ca . 80 pN nm. In cells, the free energy of ATP hydrolysis is ca . 90 pN nm per ATP molecule, suggesting that the F 1 motor can work at near 100% efficiency. We confirmed in vitro that F 1 indeed does ca . 80 pN nm of work under the condition where the free energy per ATP is 90 pN nm. The high efficiency may be related to the fully reversible nature of the F 1 motor: the ATP synthase, of which F 1 is a part, is considered to synthesize ATP from ADP and phosphate by reverse rotation of the F motor. Possible mechanisms of F 1 rotation are discussed.

scholarly journals High-resolution single-molecule characterization of the enzymatic states in Escherichia coli F 1 -ATPase

2013 ◽  
Vol 368 (1611) ◽  
pp. 20120023 ◽  
Author(s):  
Thomas Bilyard ◽  
Mayumi Nakanishi-Matsui ◽  
Bradley C. Steel ◽  
Teuta Pilizota ◽  
Ashley L. Nord ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Single Molecule ◽  
Room Temperature ◽  
Atp Hydrolysis ◽  
Chemical Processes ◽  
Atp Binding ◽  
Γ Subunit ◽  
Rotary Motor ◽  
Resolution Single

The rotary motor F 1 -ATPase from the thermophilic Bacillus PS3 (TF 1 ) is one of the best-studied of all molecular machines. F 1 -ATPase is the part of the enzyme F 1 F O -ATP synthase that is responsible for generating most of the ATP in living cells. Single-molecule experiments have provided a detailed understanding of how ATP hydrolysis and synthesis are coupled to internal rotation within the motor. In this work, we present evidence that mesophilic F 1 -ATPase from Escherichia coli (EF 1 ) is governed by the same mechanism as TF 1 under laboratory conditions. Using optical microscopy to measure rotation of a variety of marker particles attached to the γ-subunit of single surface-bound EF 1 molecules, we characterized the ATP-binding, catalytic and inhibited states of EF 1 . We also show that the ATP-binding and catalytic states are separated by 35±3°. At room temperature, chemical processes occur faster in EF 1 than in TF 1 , and we present a methodology to compensate for artefacts that occur when the enzymatic rates are comparable to the experimental temporal resolution. Furthermore, we show that the molecule-to-molecule variation observed at high ATP concentration in our single-molecule assays can be accounted for by variation in the orientation of the rotating markers.

scholarly journals F1-ATPase conformational cycle from simultaneous single-molecule FRET and rotation measurements

2016 ◽  
Vol 113 (21) ◽  
pp. E2916-E2924 ◽  
Author(s):  
Mitsuhiro Sugawa ◽  
Kei-ichi Okazaki ◽  
Masaru Kobayashi ◽  
Takashi Matsui ◽  
Gerhard Hummer ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Single Molecule ◽  
Conformational Changes ◽  
Molecular Motor ◽  
Principal Component ◽  
Structural Basis ◽  
F1 Atpase ◽  
Single Molecule Fret ◽  
Mechanochemical Coupling ◽  
Central Shaft

Despite extensive studies, the structural basis for the mechanochemical coupling in the rotary molecular motor F1-ATPase (F1) is still incomplete. We performed single-molecule FRET measurements to monitor conformational changes in the stator ring-α3β3, while simultaneously monitoring rotations of the central shaft-γ. In the ATP waiting dwell, two of three β-subunits simultaneously adopt low FRET nonclosed forms. By contrast, in the catalytic intermediate dwell, two β-subunits are simultaneously in a high FRET closed form. These differences allow us to assign crystal structures directly to both major dwell states, thus resolving a long-standing issue and establishing a firm connection between F1 structure and the rotation angle of the motor. Remarkably, a structure of F1 in an ε-inhibited state is consistent with the unique FRET signature of the ATP waiting dwell, while most crystal structures capture the structure in the catalytic dwell. Principal component analysis of the available crystal structures further clarifies the five-step conformational transitions of the αβ-dimer in the ATPase cycle, highlighting the two dominant modes: the opening/closing motions of β and the loosening/tightening motions at the αβ-interface. These results provide a new view of tripartite coupling among chemical reactions, stator conformations, and rotary angles in F1-ATPase.

Structure of the γ–ε complex of cyanobacterial F1-ATPase reveals a suppression mechanism of the γ subunit on ATP hydrolysis in phototrophs

2018 ◽  
Vol 475 (18) ◽  
pp. 2925-2939 ◽  
Author(s):  
Satoshi Murakami ◽  
Kumiko Kondo ◽  
Shinya Katayama ◽  
Satoshi Hara ◽  
Ei-ichiro Sunamura ◽  
...  
Keyword(s):  
Electrostatic Interactions ◽  
Atp Hydrolysis ◽  
Critical Role ◽  
Structural Data ◽  
Hairpin Structure ◽  
Critical Function ◽  
F1 Atpase ◽  
Γ Subunit ◽  
Suppression Mechanism ◽  
Biochemical Analyses

F1-ATPase forms the membrane-associated segment of F0F1-ATP synthase — the fundamental enzyme complex in cellular bioenergetics for ATP hydrolysis and synthesis. Here, we report a crystal structure of the central F1 subcomplex, consisting of the rotary shaft γ subunit and the inhibitory ε subunit, from the photosynthetic cyanobacterium Thermosynechococcus elongatus BP-1, at 1.98 Å resolution. In contrast with their homologous bacterial and mitochondrial counterparts, the γ subunits of photosynthetic organisms harbour a unique insertion of 35–40 amino acids. Our structural data reveal that this region forms a β-hairpin structure along the central stalk. We identified numerous critical hydrogen bonds and electrostatic interactions between residues in the hairpin and the rest of the γ subunit. To elaborate the critical function of this β-hairpin in inhibiting ATP hydrolysis, the corresponding domain was deleted in the cyanobacterial F1 subcomplex. Biochemical analyses of the corresponding α3β3γ complex confirm that the clinch of the hairpin structure plays a critical role and accounts for a significant interaction in the α3β3 complex to induce ADP inhibition during ATP hydrolysis. In addition, we found that truncating the β-hairpin insertion structure resulted in a marked impairment of the interaction with the ε subunit, which binds to the opposite side of the γ subunit from the β-hairpin structure. Combined with structural analyses, our work provides experimental evidence supporting the molecular principle of how the insertion region of the γ subunit suppresses F1 rotation during ATP hydrolysis.

Structural dynamics of P-type ATPase ion pumps

2019 ◽  
Vol 47 (5) ◽  
pp. 1247-1257 ◽  
Author(s):  
Mateusz Dyla ◽  
Sara Basse Hansen ◽  
Poul Nissen ◽  
Magnus Kjaergaard
Keyword(s):  
Structural Dynamics ◽  
Single Molecule ◽  
New Technologies ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved ◽  
Dynamics Simulations ◽  
Types Of Information ◽  
P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

GABAA receptors: Various stoichiometrics of subunit arrangement in α1β3 and α1β3ε receptors

2018 ◽  
Vol 24 (17) ◽  
pp. 1839-1844 ◽  
Author(s):  
Ahmad Tarmizi Che Has ◽  
Mary Chebib
Keyword(s):  
Central Nervous System ◽  
Gabaa Receptors ◽  
Binding Sites ◽  
New Drugs ◽  
Pharmacological Properties ◽  
Receptor Complex ◽  
The Third ◽  
Γ Subunit ◽  
Subunit Stoichiometry ◽  
The Central Nervous System

GABAA receptors are members of the Cys-loop family of ligand-gated ion channels which mediate most inhibitory neurotransmission in the central nervous system. These receptors are pentameric assemblies of individual subunits, including α1-6, β1-3, γ1-3, δ, ε, π, θ and ρ1-3. The majority of receptors are comprised of α, β and γ or δ subunits. Depending on the subunit composition, the receptors are located in either the synapses or extrasynaptic regions. The most abundant receptors are α1βγ2 receptors, which are activated and modulated by a variety of pharmacologically and clinically unrelated agents such as benzodiazepines, barbiturates, anaesthetics and neurosteroids, all of which bind at distinct binding sites located within the receptor complex. However, compared to αβγ, the binary αβ receptors lack a benzodiazepine α-γ2 interface. In pentameric αβ receptors, the third subunit is replaced with either an α1 or a β3 subunit leading to two distinct receptors that differ in subunit stoichiometry, 2α:3β or 3α:2β. The consequence of this is that 3α:2β receptors contain an α-α interface whereas 2α:3β receptors contain a β-β interface. Apart from the replacement of γ by α1 or β3 in binary receptors, the incorporation of ε subunit into GABAA receptors might be more complicated. As the ε subunit is not only capable of substituting the γ subunit, but also replacing the α/β subunits, receptors with altered stoichiometry and different pharmacological properties are produced. The different subunit arrangement of the receptors potentially constructs novel binding sites which may become new targets of the current or new drugs.

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