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.

PROTEIN MOTOR F1 AS A MODEL SYSTEM FOR FLUCTUATION THEORIES OF NON-EQUILIBRIUM STATISTICAL MECHANICS

2012 ◽  
Vol 11 (03) ◽  
pp. 1241001 ◽  
Author(s):  
KUMIKO HAYASHI ◽  
RYUNOSUKE HAYASHI
Keyword(s):  
Statistical Mechanics ◽  
Single Molecule ◽  
Nonequilibrium Statistical Mechanics ◽  
Model System ◽  
Fluctuation Theorem ◽  
Biological Model ◽  
Equilibrium Statistical Mechanics ◽  
Γ Subunit ◽  
Protein Motor ◽  
Rotary Motor

F1-ATPase (F1) is a rotary motor protein in which the rotor γ subunit rotates in the α3β3 ring hydrolyzing adenosine-5′-triphosphate (ATP). Several fluctuation theories of nonequilibrium statistical mechanics have been applied recently to the single-molecule experiments on F1. For example, the fluctuation theorem, a recent achievement in the field of nonequilibrium statistical mechanics, has been suggested to be useful for measuring the rotary torque of F1. In this paper, we introduce F1 as a good biological model for experimentally testing the theories of nonequilibrium statistical mechanics.

scholarly journals Extreme parsimony in ATP consumption by 20S complexes in the global disassembly of single SNARE complexes

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Changwon Kim ◽  
Min Ju Shon ◽  
Sung Hyun Kim ◽  
Gee Sung Eun ◽  
Je-Kyung Ryu ◽  
...  
Keyword(s):  
Energy Efficiency ◽  
Single Molecule ◽  
Molecular Motors ◽  
Atp Hydrolysis ◽  
Snare Complex ◽  
Atp Binding ◽  
Attachment Protein ◽  
Receptor Complexes ◽  
Protein Receptor ◽  
Sensitive Factor

AbstractFueled by ATP hydrolysis in N-ethylmaleimide sensitive factor (NSF), the 20S complex disassembles rigid SNARE (soluble NSF attachment protein receptor) complexes in single unraveling step. This global disassembly distinguishes NSF from other molecular motors that make incremental and processive motions, but the molecular underpinnings of its remarkable energy efficiency remain largely unknown. Using multiple single-molecule methods, we found remarkable cooperativity in mechanical connection between NSF and the SNARE complex, which prevents dysfunctional 20S complexes that consume ATP without productive disassembly. We also constructed ATP hydrolysis cycle of the 20S complex, in which NSF largely shows randomness in ATP binding but switches to perfect ATP hydrolysis synchronization to induce global SNARE disassembly, minimizing ATP hydrolysis by non-20S complex-forming NSF molecules. These two mechanisms work in concert to concentrate ATP consumption into functional 20S complexes, suggesting evolutionary adaptations by the 20S complex to the energetically expensive mechanical task of SNARE complex disassembly.

scholarly journals Biosynthesis and Characterization of Silver Nanoparticles From Pantoea agglomerans and Some of Their Antibacterial Activities

2020 ◽  
Vol 31 (3) ◽  
pp. 1
Author(s):  
Layla Abdul-Hamid Said
Keyword(s):  
Escherichia Coli ◽  
Silver Nanoparticles ◽  
Room Temperature ◽  
Antibacterial Activities ◽  
Multidrug Resistant ◽  
Pantoea Agglomerans ◽  
Force Microscopy ◽  
Atomic Force ◽  
And Staphylococcus Aureus

Recently, the biosynthesis of nanoparticles from bacteria have attracted attention, this study has been made for biosynthesize and characterizes silver nanoparticles (AgNPs) from local clinical isolate Pantoea agglomerans. The ability of those particles to inhibit the virulence factors biofilm and hemolysin produced by some local clinical multidrug-resistant human pathogenes including Acinetobactor haemolyticus, Escherichia coli, Serratia marcescens and Staphylococcus aureus were investigated by treating all of the test isolates with sub-MIC(16 mg/ml) AgNPs. The AgNPs produced were characterized using Atomic Force Microscopy (AFM). Pantoea agglomerans were found to have the ability to synthesize AgNPs at room temperature within 24hrs and were spherical in shape as depicted by AFM. The AgNPs produced exhibited a potential antibiofilm and hemolysin inhibition activities against tested pathogens.

Collective Dynamics of Kinesin-1 Motor Proteins Transporting a Common Load

2007 ◽  
Author(s):  
Adam G. Hendricks ◽  
Bogdan I. Epureanu ◽  
Edgar Meyho¨fer
Keyword(s):  
Single Molecule ◽  
Collective Behavior ◽  
Intracellular Transport ◽  
Atp Hydrolysis ◽  
Mechanistic Model ◽  
State Behavior ◽  
Directed Movement ◽  
The Difference ◽  
Transient And Steady State

Kinesin-1 is a motor protein essential to intracellular transport that converts the energy from ATP hydrolysis to directed movement along microtubules. Experimental and theoretical characterization of kinesin-1 has focused on single-molecule experiments. These experiments show that one motor is capable of transporting a cargo at speeds of about 1 μm/sec and maintaining contact with the microtubule for about 100 steps. In the cell, it is widely thought that several kinesin-1 motors cooperate to transport a cargo. Through a mechanistic model, we have extended the theoretical analysis of kinesin to describing transient and steady state behavior. A transient description is essential when studying collective behavior, as interaction between motors introduces time-varying loads. Herein, we interpret the kinesin motors as nonlinear, non-smooth oscillators and we employ metrics to characterize their cooperativity and to quantify their synchronization. These metrics are used to investigate the effect of the cargo linker stiffness, the load, and the difference in intrinsic velocity on the synchronization of two mechanically coupled motors.

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.

Permeation of Antibiotics through Escherichia coli OmpF and OmpC Porins

2010 ◽  
Vol 15 (3) ◽  
pp. 302-307 ◽  
Author(s):  
Kozhinjampara R. Mahendran ◽  
Mohamed Kreir ◽  
Helge Weingart ◽  
Niels Fertig ◽  
Mathias Winterhalter
Keyword(s):  
Escherichia Coli ◽  
Single Molecule ◽  
Single Channel ◽  
Patch Clamp Technique ◽  
Single Molecule Level ◽  
Solution Exchange ◽  
Automated Patch Clamp ◽  
Low Capacitance ◽  
Bacterial Porins

A chip-based automated patch-clamp technique provides an attractive biophysical tool to quantify solute permeation through membrane channels. Proteo–giant unilamellar vesicles (proteo-GUVs) were used to form a stable lipid bilayer across a micrometer-sized hole. Because of the small size and hence low capacitance of the bilayer, single-channel recordings were achieved with very low background noise. The latter allowed the characterization of the influx of 2 major classes of antibiotics—cephalosporins and fluoroquinolones—through the major Escherichia coli porins OmpF and OmpC. Analyzing the ion current fluctuations in the presence of antibiotics revealed transport properties that allowed the authors to determine the mode of permeation. The chip-based setup allows rapid solution exchange and efficient quantification of antibiotic permeation through bacterial porins on a single-molecule level.

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