2P194 Single molecule observation of ATP hydrolysis of Myosin V

Recent experiments, drawing upon single-molecule, solution kinetic and structural techniques, have clarified our mechanistic understanding of class V myosins. The findings of the past two years can be summarized as follows: (1) Myosin V is a highly efficient processive motor, surpassing even conventional kinesin in the distance that individual molecules can traverse. (2) The kinetic scheme underlying ATP turnover resembles those of myosins I and II but with rate constants tuned to favor strong binding to actin. ADP release precedes dissociation from actin and is rate-limiting in the cycle. (3) Myosin V walks in strides averaging ∼36 nm, the long pitch pseudo-repeat of the actin helix, each step coupled to a single ATP hydrolysis. Such a unitary displacement, the largest molecular step size measured to date, is required for a processive myosin motor to follow a linear trajectory along a helical actin track.

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Structural dynamics of P-type ATPase ion pumps

Biochemical Society Transactions ◽

10.1042/bst20190124 ◽

2019 ◽

Vol 47 (5) ◽

pp. 1247-1257 ◽

Cited By ~ 17

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.

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Hydrolysis of nucleoside triphosphates catalyzed by the recA protein of Escherichia coli. Steady state kinetic analysis of ATP hydrolysis.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)68922-2 ◽

1981 ◽

Vol 256 (16) ◽

pp. 8845-8849 ◽

Cited By ~ 8

Author(s):

G.M. Weinstock ◽

K. McEntee ◽

I.R. Lehman

Keyword(s):

Escherichia Coli ◽

Steady State ◽

Kinetic Analysis ◽

Atp Hydrolysis ◽

Reca Protein ◽

Steady State Kinetic ◽

Nucleoside Triphosphates ◽

Hydrolysis Of ◽

State Kinetic

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Single–motor mechanics and models of the myosin motor

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2000.0585 ◽

2000 ◽

Vol 355 (1396) ◽

pp. 441-447 ◽

Cited By ~ 37

Author(s):

T. Yanagida ◽

S. Esaki ◽

A. Hikikoshi Iwane ◽

Y. Inoue ◽

A. Ishijima ◽

...

Keyword(s):

Single Molecule ◽

Atp Hydrolysis ◽

Accurate Determination ◽

Step Size ◽

Single Molecule Level ◽

Detection Techniques ◽

Myosin Motor ◽

Mechanochemical Coupling ◽

Chemical Events

Recent progress in single–molecule detection techniques is remarkable. These techniques have allowed the accurate determination of myosin–head–induced displacements and how mechanical cycles are coupled to ATP hydrolysis, by measuring individual mechanical events and chemical events of actomyosin directly at the single–molecule level. Here we review our recent work in which we have made detailed measurements of myosin step size and mechanochemical coupling, and propose a model of the myosin motor.

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

Journal of the American Chemical Society ◽

10.1021/ja211027m ◽

2012 ◽

Vol 134 (20) ◽

pp. 8447-8454 ◽

Cited By ~ 64

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

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The role of bound potassium ions in the hydrolysis of low concentrations of adenosine triphosphate by preparations of membrane fragments from ox brain cerebral cortex

Biochemical Journal ◽

10.1042/bj1200015 ◽

1970 ◽

Vol 120 (1) ◽

pp. 15-24 ◽

Cited By ~ 18

Author(s):

P. S. G. Goldfarb ◽

R. Rodnight

Keyword(s):

Potassium Chloride ◽

Magnesium Chloride ◽

Adenosine Triphosphatase ◽

Time Course ◽

Atp Hydrolysis ◽

Protein Ratio ◽

Low Concentrations ◽

Hydrolysis Of ◽

Emission Flame

1. The intrinsic Na+, K+, Mg2+ and Ca2+ contents of a preparation of membrane fragments from ox brain were determined by emission flame photometry. 2. Centrifugal washing of the preparation with imidazole-buffered EDTA solutions decreased the bound Na+ from 90±20 to 24±12, the bound K+ from 27±3 to 7±2, the bound Mg2+ from 20±2 to 3±1 and the bound calcium from 8±1 to <1nmol/mg of protein. 3. The activities of the Na++K++Mg2+-stimulated adenosine triphosphatase and the Na+-dependent reaction forming bound phosphate were compared in the unwashed and washed preparations at an ATP concentration of 2.5μm (ATP/protein ratio 12.5pmol/μg). 4. The Na+-dependent hydrolysis of ATP as well as the plateau concentration of bound phosphate and the rate of dephosphorylation were decreased in the washed preparation. The time-course of formation and decline of bound phosphate was fully restored by the addition of 2.5μm-magnesium chloride and 2μm-potassium chloride. Addition of 2.5μm-magnesium chloride alone fully restored the plateau concentration of bound phosphate, but the rate of dephosphorylation was only slightly increased. Na+-dependent ATP hydrolysis was partly restored with 2.5μm-magnesium chloride; addition of K+ in the range 2–10μm-potassium chloride then further restored hydrolysis but not to the control rate. 5. Pretreatment of the washed preparation at 0°C with 0.5nmol of K+/mg of protein so that the final added K+ in the reaction mixture was 0.1μm restored the Na+-dependent hydrolysis of ATP and the time-course of the reaction forming bound phosphate. 6. The binding of [42K]potassium chloride by the washed membrane preparation was examined. Binding in a solution containing 10nmol of K+/mg of protein was linear over a period of 20min and was inhibited by Na+. Half-maximal inhibition of 42K+-binding required a 100-fold excess of sodium chloride. 7. It was concluded (a) that a significant fraction of the apparent Na+-dependent hydrolysis of ATP observed in the unwashed preparation is due to activation by bound K+ and Mg2+ of the Na++K++Mg2+-stimulated adenosine triphosphatase system and (b) that the enzyme system is able to bind K+ from a solution of 0.5μm-potassium chloride.

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Meiosis-specific recombinase Dmc1 is a potent inhibitor of the Srs2 antirecombinase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1810457115 ◽

2018 ◽

Vol 115 (43) ◽

pp. E10041-E10048 ◽

Cited By ~ 14

Author(s):

J. Brooks Crickard ◽

Kyle Kaniecki ◽

Youngho Kwon ◽

Patrick Sung ◽

Eric C. Greene

Keyword(s):

Chromosome Segregation ◽

Single Molecule ◽

Atp Hydrolysis ◽

Potent Inhibitor ◽

Chromosomal Rearrangements ◽

Motor Protein ◽

Product Formation ◽

Gross Chromosomal Rearrangements ◽

Strand Annealing ◽

Hydrolysis Activity

Cross-over recombination products are a hallmark of meiosis because they are necessary for accurate chromosome segregation and they also allow for increased genetic diversity during sexual reproduction. However, cross-overs can also cause gross chromosomal rearrangements and are therefore normally down-regulated during mitotic growth. The mechanisms that enhance cross-over product formation upon entry into meiosis remain poorly understood. In Saccharomyces cerevisiae, the Superfamily 1 (Sf1) helicase Srs2, which is an ATP hydrolysis-dependent motor protein that actively dismantles recombination intermediates, promotes synthesis-dependent strand annealing, the result of which is a reduction in cross-over recombination products. Here, we show that the meiosis-specific recombinase Dmc1 is a potent inhibitor of Srs2. Biochemical and single-molecule assays demonstrate that Dmc1 acts by inhibiting Srs2 ATP hydrolysis activity, which prevents the motor protein from undergoing ATP hydrolysis-dependent translocation on Dmc1-bound recombination intermediates. We propose a model in which Dmc1 helps contribute to cross-over formation during meiosis by antagonizing the antirecombinase activity of Srs2.

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Hierarchical coupling between ATP hydrolysis and Hsp90’s client binding site

10.1101/2020.02.15.950725 ◽

2020 ◽

Author(s):

Steffen Wolf ◽

Benedikt Sohmen ◽

Björn Hellenkamp ◽

Johann Thurn ◽

Gerhard Stock ◽

...

Keyword(s):

Binding Site ◽

Single Molecule ◽

Information Transfer ◽

Atp Hydrolysis ◽

Structural Information ◽

Substrate Binding ◽

Signal Propagation ◽

Nonequilibrium Molecular Dynamics ◽

Time Resolved ◽

Substrate Binding Site

I.ABSTRACTSeveral indicators for a signal propagation from a binding site to a distant functional site have been found in the Hsp90 dimer. Here we determined a time-resolved pathway from ATP hydrolysis to changes in a distant folding substrate binding site. This was possible by combining single-molecule fluorescence-based methods with extensive atomistic nonequilibrium molecular dynamics simulations. We find that hydrolysis of one ATP effects a structural asymmetry in the full Hsp90 dimer that leads to the collapse of a central folding substrate binding site. Arg380 is the major mediator in transferring structural information from the nucleotide to the substrate binding site. This allosteric process occurs via hierarchical dynamics that involve timescales from picoto milliseconds and length scales from Ångstroms to several nanometers. We presume that similar hierarchical mechanisms are fundamental for information transfer through many other proteins.

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Targeting Mycobacterial F-ATP Synthase C-Terminal α Subunit Interaction Motif on Rotary Subunit γ

Antibiotics ◽

10.3390/antibiotics10121456 ◽

2021 ◽

Vol 10 (12) ◽

pp. 1456

Author(s):

Amaravadhi Harikishore ◽

Chui-Fann Wong ◽

Priya Ragunathan ◽

Dennis Litty ◽

Volker Müller ◽

...

Keyword(s):

Atp Synthase ◽

Atp Hydrolysis ◽

Atp Synthesis ◽

Membrane Vesicles ◽

Α Subunit ◽

Rotational States ◽

C Terminus ◽

Inverted Membrane Vesicles ◽

Hydrolysis Of ◽

Micromolar Range

Mycobacteria regulate their energy (ATP) levels to sustain their survival even in stringent living conditions. Recent studies have shown that mycobacteria not only slow down their respiratory rate but also block ATP hydrolysis of the F-ATP synthase (α3:β3:γ:δ:ε:a:b:b’:c9) to maintain ATP homeostasis in situations not amenable for growth. The mycobacteria-specific α C-terminus (α533-545) has unraveled to be the major regulative of latent ATP hydrolysis. Its deletion stimulates ATPase activity while reducing ATP synthesis. In one of the six rotational states of F-ATP synthase, α533-545 has been visualized to dock deep into subunit γ, thereby blocking rotation of γ within the engine. The functional role(s) of this C-terminus in the other rotational states are not clarified yet and are being still pursued in structural studies. Based on the interaction pattern of the docked α533-545 region with subunit γ, we attempted to study the druggability of the α533-545 motif. In this direction, our computational work has led to the development of an eight-featured α533-545 peptide pharmacophore, followed by database screening, molecular docking, and pose selection, resulting in eleven hit molecules. ATP synthesis inhibition assays using recombinant ATP synthase as well as mycobacterial inverted membrane vesicles show that one of the hits, AlMF1, inhibited the mycobacterial F-ATP synthase in a micromolar range. The successful targeting of the α533-545-γ interaction motif demonstrates the potential to develop inhibitors targeting the α site to interrupt rotary coupling with ATP synthesis.

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Kinetic and structural mechanism for DNA unwinding by a non-hexameric helicase

Nature Communications ◽

10.1038/s41467-021-27304-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Sean P. Carney ◽

Wen Ma ◽

Kevin D. Whitley ◽

Haifeng Jia ◽

Timothy M. Lohman ◽

...

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Atp Hydrolysis ◽

Variable Number ◽

Structural Basis ◽

Variable Step Size ◽

Dna Unwinding ◽

Step Size ◽

Structural Mechanism ◽

Dynamics Simulations

AbstractUvrD, a model for non-hexameric Superfamily 1 helicases, utilizes ATP hydrolysis to translocate stepwise along single-stranded DNA and unwind the duplex. Previous estimates of its step size have been indirect, and a consensus on its stepping mechanism is lacking. To dissect the mechanism underlying DNA unwinding, we use optical tweezers to measure directly the stepping behavior of UvrD as it processes a DNA hairpin and show that UvrD exhibits a variable step size averaging ~3 base pairs. Analyzing stepping kinetics across ATP reveals the type and number of catalytic events that occur with different step sizes. These single-molecule data reveal a mechanism in which UvrD moves one base pair at a time but sequesters the nascent single strands, releasing them non-uniformly after a variable number of catalytic cycles. Molecular dynamics simulations point to a structural basis for this behavior, identifying the protein-DNA interactions responsible for strand sequestration. Based on structural and sequence alignment data, we propose that this stepping mechanism may be conserved among other non-hexameric helicases.

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