scholarly journals Stepwise nucleosome translocation by RSC remodeling complexes

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Bryan T Harada ◽  
William L Hwang ◽  
Sebastian Deindl ◽  
Nilanjana Chatterjee ◽  
Blaine Bartholomew ◽  
...  
Keyword(s):  
Free Energy ◽  
Atpase Activity ◽  
Binding Site ◽  
Single Molecule ◽  
Chromatin Structure ◽  
Atp Hydrolysis ◽  
Size Distributions ◽  
Dna Translocation ◽  
Step Size ◽  
Single Molecule Fret

The SWI/SNF-family remodelers regulate chromatin structure by coupling the free energy from ATP hydrolysis to the repositioning and restructuring of nucleosomes, but how the ATPase activity of these enzymes drives the motion of DNA across the nucleosome remains unclear. Here, we used single-molecule FRET to monitor the remodeling of mononucleosomes by the yeast SWI/SNF remodeler, RSC. We observed that RSC primarily translocates DNA around the nucleosome without substantial displacement of the H2A-H2B dimer. At the sites where DNA enters and exits the nucleosome, the DNA moves largely along or near its canonical wrapping path. The translocation of DNA occurs in a stepwise manner, and at both sites where DNA enters and exits the nucleosome, the step size distributions exhibit a peak at approximately 1–2 bp. These results suggest that the movement of DNA across the nucleosome is likely coupled directly to DNA translocation by the ATPase at its binding site inside the nucleosome.

scholarly journals Single–motor mechanics and models of the myosin motor

2000 ◽  
Vol 355 (1396) ◽  
pp. 441-447 ◽  
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.

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

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.

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

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.

scholarly journals Slow domain reconfiguration causes power-law kinetics in a two-state enzyme

2018 ◽  
Vol 115 (3) ◽  
pp. 513-518 ◽  
Author(s):  
Iris Grossman-Haham ◽  
Gabriel Rosenblum ◽  
Trishool Namani ◽  
Hagen Hofmann
Keyword(s):  
Free Energy ◽  
Single Molecule ◽  
Power Law ◽  
Energy Landscape ◽  
Rate Equations ◽  
Free Energy Landscape ◽  
Closed Domain ◽  
Single Molecule Fret ◽  
Power Law Kinetics ◽  
Interdomain Interactions

Protein dynamics are typically captured well by rate equations that predict exponential decays for two-state reactions. Here, we describe a remarkable exception. The electron-transfer enzyme quiescin sulfhydryl oxidase (QSOX), a natural fusion of two functionally distinct domains, switches between open- and closed-domain arrangements with apparent power-law kinetics. Using single-molecule FRET experiments on time scales from nanoseconds to milliseconds, we show that the unusual open-close kinetics results from slow sampling of an ensemble of disordered domain orientations. While substrate accelerates the kinetics, thus suggesting a substrate-induced switch to an alternative free energy landscape of the enzyme, the power-law behavior is also preserved upon electron load. Our results show that the slow sampling of open conformers is caused by a variety of interdomain interactions that imply a rugged free energy landscape, thus providing a generic mechanism for dynamic disorder in multidomain enzymes.

Tropomyosin-Troponin-Induced Changes in the Partitioning of Free Energy Release of Actomyosin-Catalyzed ATP Hydrolysis as Measured by ATP-Phosphate Exchange

1980 ◽  
Vol 35 (5-6) ◽  
pp. 431-438 ◽  
Author(s):  
Peter Dancker
Keyword(s):  
Free Energy ◽  
Atpase Activity ◽  
Energy Release ◽  
Atp Hydrolysis ◽  
Free Energy Change ◽  
Energy Change ◽  
Experimental Conditions ◽  
Mean Values ◽  
Induced Changes ◽  
Phosphate Exchange

Abstract ATPase activity and ATP-Pi exchange of unregulated (without tropomyosin-troponin) and regulated (with tropomyosin-troponin) acto-HMM were measured in media containing 0.2 mg/ml actin, HMM, and (when present) tropomyosin-troponin, 2 mM MgCl2, 10 m M KCl, 2 mM NaN3, 10 mM Pi(pH 7.0), 3 mM ATP. The following mean values for ATPase activity and for the rate of incorporation of P, into ATP (each per mg HMM and per min) were obtained: unregulated acto-HMM 0.33 nmol Pi and 0.33 nmol Pi, regulated acto-HMM 0.54 nmol Pi and 1.06 nmol P*. The ratio of P4 incorporation rate to ATPase activity was 1.01 × 10-3 for unregulated and 2.02 × 10-3 for regulated acto-HMM. From these ratios and from the overall free energy change of ATP hydrolysis it was calculated that under the prevailing experimental conditions in unregulated acto-HMM 62% and in regulated acto-HMM 66% of the free energy change of ATP hydrolysis occurs after the release of phosphate from actomyosin. It is probably this part of the free energy change that is used by the muscle for the performance of work.

scholarly journals A Generalized Kinetic Model for Coupling between Stepping and ATP Hydrolysis of Kinesin Molecular Motors

2019 ◽  
Vol 20 (19) ◽  
pp. 4911 ◽  
Author(s):  
Xie ◽  
Guo ◽  
Chen
Keyword(s):  
Kinetic Model ◽  
Atpase Activity ◽  
Single Molecule ◽  
Rate Constants ◽  
Molecular Motors ◽  
Atp Hydrolysis ◽  
Power Production ◽  
Wild Type ◽  
Chemomechanical Coupling ◽  
Generalized Kinetic Model

A general kinetic model is presented for the chemomechanical coupling of dimeric kinesin molecular motors with and without extension of their neck linkers (NLs). A peculiar feature of the model is that the rate constants of ATPase activity of a kinesin head are independent of the strain on its NL, implying that the heads of the wild-type kinesin dimer and the mutant with extension of its NLs have the same force-independent rate constants of the ATPase activity. Based on the model, an analytical theory is presented on the force dependence of the dynamics of kinesin dimers with and without extension of their NLs at saturating ATP. With only a few adjustable parameters, diverse available single molecule data on the dynamics of various kinesin dimers, such as wild-type kinesin-1, kinesin-1 with mutated residues in the NLs, kinesin-1 with extension of the NLs and wild-type kinesin-2, under varying force and ATP concentration, can be reproduced very well. Additionally, we compare the power production among different kinesin dimers, showing that the mutation in the NLs reduces the power production and the extension of the NLs further reduces the power production.

scholarly journals A new view of protein synthesis: Mapping the free energy landscape of the ribosome using single-molecule FRET

Biopolymers ◽  
2008 ◽  
Vol 89 (7) ◽  
pp. 565-577 ◽  
Author(s):  
James B. Munro ◽  
Andrea Vaiana ◽  
Kevin Y. Sanbonmatsu ◽  
Scott C. Blanchard
Keyword(s):  
Free Energy ◽  
Protein Synthesis ◽  
Single Molecule ◽  
Energy Landscape ◽  
Free Energy Landscape ◽  
Single Molecule Fret ◽  
New View

scholarly journals Hexameric helicase G40P unwinds DNA in single base pair steps

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Michael Schlierf ◽  
Ganggang Wang ◽  
Xiaojiang S Chen ◽  
Taekjip Ha
Keyword(s):  
Single Molecule ◽  
Base Pair ◽  
Atp Hydrolysis ◽  
Thermal Fluctuation ◽  
Dna Unwinding ◽  
Single Molecule Fluorescence ◽  
Step Size ◽  
Structural Investigations ◽  
Single Base ◽  
Single Base Pair

Most replicative helicases are hexameric, ring-shaped motor proteins that translocate on and unwind DNA. Despite extensive biochemical and structural investigations, how their translocation activity is utilized chemo-mechanically in DNA unwinding is poorly understood. We examined DNA unwinding by G40P, a DnaB-family helicase, using a single-molecule fluorescence assay with a single base pair resolution. The high-resolution assay revealed that G40P by itself is a very weak helicase that stalls at barriers as small as a single GC base pair and unwinds DNA with the step size of a single base pair. Binding of a single ATPγS could stall unwinding, demonstrating highly coordinated ATP hydrolysis between six identical subunits. We observed frequent slippage of the helicase, which is fully suppressed by the primase DnaG. We anticipate that these findings allow a better understanding on the fine balance of thermal fluctuation activation and energy derived from hydrolysis.

Single-Molecule FRET Studies of Counterion Effects on the Free Energy Landscape of Human Mitochondrial Lysine tRNA

Biochemistry ◽  
2011 ◽  
Vol 50 (15) ◽  
pp. 3107-3115 ◽  
Author(s):  
Kirsten Dammertz ◽  
Martin Hengesbach ◽  
Mark Helm ◽  
G. Ulrich Nienhaus ◽  
Andrei Yu. Kobitski
Keyword(s):  
Free Energy ◽  
Single Molecule ◽  
Energy Landscape ◽  
Free Energy Landscape ◽  
Single Molecule Fret ◽  
Lysine Trna

Myosin learns to walk

2001 ◽  
Vol 114 (11) ◽  
pp. 1981-1998
Author(s):  
Amit Mehta
Keyword(s):  
Single Molecule ◽  
Atp Hydrolysis ◽  
Kinetic Scheme ◽  
Myosin V ◽  
Step Size ◽  
Class V ◽  
Adp Release ◽  
Solution Kinetic ◽  
Conventional Kinesin ◽  
Rate Limiting

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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