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.

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 2P194 Single molecule observation of ATP hydrolysis of Myosin V

2005 ◽  
Vol 45 (supplement) ◽  
pp. S168
Author(s):  
T. Komori ◽  
S. Nishikawa ◽  
T. Ariga ◽  
A.H. Iwane ◽  
H. Yamakawa ◽  
...  
Keyword(s):  
Single Molecule ◽  
Atp Hydrolysis ◽  
Myosin V ◽  
Hydrolysis Of

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 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.

scholarly journals Reexamining the origin of the directionality of myosin V

2017 ◽  
Vol 114 (39) ◽  
pp. 10426-10431 ◽  
Author(s):  
Raphael Alhadeff ◽  
Arieh Warshel
Keyword(s):  
Kinetic Equations ◽  
Experimental Information ◽  
Power Stroke ◽  
Myosin V ◽  
Time Resolved ◽  
Directional Motion ◽  
Long Time ◽  
Adp Release ◽  
Rate Limiting ◽  
Binding Energy Calculations

The nature of the conversion of chemical energy to directional motion in myosin V is examined by careful simulations that include two complementary methods: direct Langevin Dynamics (LD) simulations with a scaled-down potential that provided a detailed time-resolved mechanism, and kinetic equations solution for the ensemble long-time propagation (based on information collected for segments of the landscape using LD simulations and experimental information). It is found that the directionality is due to the rate-limiting ADP release step rather than the potential energy of the lever arm angle. We show that the energy of the power stroke and the barriers involved in it are of minor consequence to the selectivity of forward over backward steps and instead suggest that the selective release of ADP from a postrigor myosin motor head promotes highly selective and processive myosin V. Our model is supported by different computational methods—LD simulations, Monte Carlo simulations, and kinetic equations solution—as well as by structure-based binding energy calculations.

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

2021 ◽  
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. 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 Kinetic analysis methods applied to single motor protein trajectories

2018 ◽  
Author(s):  
A L Nord ◽  
A F Pols ◽  
M Depken ◽  
F Pedaci
Keyword(s):  
Single Molecule ◽  
Molecular Motors ◽  
Thermal Noise ◽  
Molecular Motor ◽  
Electrical Energy ◽  
Flagellar Motor ◽  
Step Size ◽  
Analysis Methods ◽  
Chemical States ◽  
Rate Limiting

Molecular motors convert chemical or electrical energy into mechanical displacement, either linear or rotary. Under ideal circumstances, single-molecule measurements can spatially and temporally resolve individual steps of the motor, revealing important properties of the underlying mechanochemical process. Unfortunately, steps are often hard to resolve, as they are masked by thermal noise. In such cases, details of the mechanochemistry can nonetheless be recovered by analyzing the fluctuations in the recorded traces. Here, we expand upon existing statistical analysis methods, providing two new avenues to extract the motor step size, the effective number of rate-limiting chemical states per translocation step, and the compliance of the link between the motor position and the probe particle. We first demonstrate the power and limitations of these methods using simulated molecular motor trajectories, and we then apply these methods to experimental data of kinesin, the bacterial flagellar motor, and F1-ATPase.

scholarly journals Effect of low pH on single skeletal muscle myosin mechanics and kinetics

2008 ◽  
Vol 295 (1) ◽  
pp. C173-C179 ◽  
Author(s):  
E. P. Debold ◽  
S. E. Beck ◽  
D. M. Warshaw
Keyword(s):  
Skeletal Muscle ◽  
Single Molecule ◽  
Low Ph ◽  
Muscular Fatigue ◽  
Step Size ◽  
Skeletal Muscle Myosin ◽  
In Vitro Motility ◽  
Adp Release ◽  
Muscle Myosin

Acidosis (low pH) is the oldest putative agent of muscular fatigue, but the molecular mechanism underlying its depressive effect on muscular performance remains unresolved. Therefore, the effect of low pH on the molecular mechanics and kinetics of chicken skeletal muscle myosin was studied using in vitro motility (IVM) and single molecule laser trap assays. Decreasing pH from 7.4 to 6.4 at saturating ATP slowed actin filament velocity ( Vactin) in the IVM by 36%. Single molecule experiments, at 1 μM ATP, decreased the average unitary step size of myosin ( d) from 10 ± 2 nm (pH 7.4) to 2 ± 1 nm (pH 6.4). Individual binding events at low pH were consistent with the presence of a population of both productive (average d = 10 nm) and nonproductive (average d = 0 nm) actomyosin interactions. Raising the ATP concentration from 1 μM to 1 mM at pH 6.4 restored d (9 ± 3 nm), suggesting that the lifetime of the nonproductive interactions is solely dependent on the [ATP]. Vactin, however, was not restored by raising the [ATP] (1–10 mM) in the IVM assay, suggesting that low pH also prolongs actin strong binding ( ton). Measurement of ton as a function of the [ATP] in the single molecule assay suggested that acidosis prolongs ton by slowing the rate of ADP release. Thus, in a detachment limited model of motility (i.e., Vactin ∼ d/ ton), a slowed rate of ADP release and the presence of nonproductive actomyosin interactions could account for the acidosis-induced decrease in Vactin, suggesting a molecular explanation for this component of muscular fatigue.

scholarly journals Characterization of the kinetic cycle of an ABC transporter by single-molecule and cryo-EM analyses

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Ling Wang ◽  
Zachary Lee Johnson ◽  
Michael R Wasserman ◽  
Jesper Levring ◽  
Jue Chen ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Chemotherapeutic Agents ◽  
Multidrug Resistance Protein 1 ◽  
Rate Limiting Step ◽  
Single Molecule Fluorescence Spectroscopy ◽  
Resistance Protein ◽  
Rate Limiting

ATP-binding cassette (ABC) transporters are molecular pumps ubiquitous across all kingdoms of life. While their structures have been widely reported, the kinetics governing their transport cycles remain largely unexplored. Multidrug resistance protein 1 (MRP1) is an ABC exporter that extrudes a variety of chemotherapeutic agents and native substrates. Previously, the structures of MRP1 were determined in an inward-facing (IF) or outward-facing (OF) conformation. Here, we used single-molecule fluorescence spectroscopy to track the conformational changes of bovine MRP1 (bMRP1) in real time. We also determined the structure of bMRP1 under active turnover conditions. Our results show that substrate stimulates ATP hydrolysis by accelerating the IF-to-OF transition. The rate-limiting step of the transport cycle is the dissociation of the nucleotide-binding-domain dimer, while ATP hydrolysis per se does not reset MRP1 to the resting state. The combination of structural and kinetic data illustrates how different conformations of MRP1 are temporally linked and how substrate and ATP alter protein dynamics to achieve active transport.

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