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 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 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 Hexameric helicase G40P unwinds DNA in single base pair steps

2018 ◽  
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

AbstractMost 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. Single ATPγS binding could stall unwinding, demonstrating highly coordinated ATP hydrolysis between the 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.

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.

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 Human HELB is a processive motor protein which catalyses RPA clearance from single-stranded DNA

2021 ◽  
Author(s):  
Silvia Hormeno ◽  
Oliver J Wilkinson ◽  
Clara Aicart-Ramos ◽  
Sahiti Kuppa ◽  
Edwin Antony ◽  
...  
Keyword(s):  
Dna Replication ◽  
Direct Observation ◽  
Single Molecule ◽  
Atp Hydrolysis ◽  
Motor Protein ◽  
Dna Unwinding ◽  
Helicase Activity ◽  
Dna Loops ◽  
Single Stranded Dna ◽  
A Site

Human HELB is a poorly-characterised helicase suggested to play both positive and negative regulatory roles in DNA replication and recombination. In this work, we used bulk and single molecule approaches to characterise the biochemical activities of HELB protein with a particular focus on its interactions with RPA and RPA-ssDNA filaments. HELB is a monomeric protein which binds tightly to ssDNA with a site size of ~20 nucleotides. It couples ATP hydrolysis to translocation along ssDNA in the 5′-to-3′ direction accompanied by the formation of DNA loops and with an efficiency of 1 ATP per base. HELB also displays classical helicase activity but this is very weak in the absence of an assisting force. HELB binds specifically to human RPA which enhances its ATPase and ssDNA translocase activities but inhibits DNA unwinding. Direct observation of HELB on RPA nucleoprotein filaments shows that translocating HELB concomitantly clears RPA from single-stranded DNA.

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 Hierarchical dynamics in allostery following ATP hydrolysis monitored by single molecule FRET measurements and MD simulations

2021 ◽  
Author(s):  
Steffen Wolf ◽  
Benedikt Sohmen ◽  
Björn Hellenkamp ◽  
Johann Thurn ◽  
Gerhard Stock ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Single Molecule ◽  
Atp Hydrolysis ◽  
Md Simulations ◽  
Large Protein ◽  
Single Molecule Fret ◽  
Fluorescence Methods ◽  
Dynamics Simulations

We report on a study that combines advanced fluorescence methods with molecular dynamics simulations to cover timescales from nanoseconds to milliseconds for a large protein, the chaperone Hsp90.

scholarly journals Structural basis for polarized elongation of actin filaments

2020 ◽  
Author(s):  
Vilmos Zsolnay ◽  
Harshwardhan H. Katkar ◽  
Steven Z. Chou ◽  
Thomas D. Pollard ◽  
Gregory A. Voth
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Actin Filaments ◽  
Atp Hydrolysis ◽  
Underlying Mechanism ◽  
Structural Basis ◽  
Dnase 1 ◽  
Dynamics Simulations ◽  
Pointed End ◽  
Kinetics Of

AbstractActin filaments elongate and shorten much faster at their barbed end than their pointed end, but the molecular basis of this difference has not been understood. We use all-atom molecular dynamics simulations to investigate the properties of subunits at both ends of the filament. The terminal subunits tend towards conformations that resemble actin monomers in solution, while contacts with neighboring subunits progressively flatten the conformation of internal subunits. At the barbed end the terminal subunit is loosely tethered by its DNase-1 loop to the third subunit, because its monomer-like conformation precludes stabilizing contacts with the penultimate subunit. The motions of the terminal subunit make the partially flattened penultimate subunit accessible for binding monomers. At the pointed end, unique contacts between the penultimate and terminal subunits are consistent with existing cryo-EM maps, limit binding to incoming monomers, and flatten the terminal subunit, which likely promotes ATP hydrolysis and rapid phosphate release. These structures explain the distinct polymerization kinetics of the two ends.Significance StatementEukaryotic cells utilize actin filaments to move, change shape, divide, and transport cargo. Decades of experiments have established that actin filaments elongate and shorten significantly faster from one end than the other, but the underlying mechanism for this asymmetry has not been explained. We used molecular dynamics simulations to investigate the structures of the actin filament ends in the ATP, ADP plus γ-phosphate, and ADP nucleotide states. We characterize the structures of actin subunits at both ends of the filament, explain the mechanisms leading to these differences, and connect the divergent structural properties of the two ends to their distinct polymerization rate constants.

scholarly journals Structural basis for relief of the sarcoplasmic reticulum Ca2+-ATPase inhibition by phospholamban at saturating Ca2+ conditions

2018 ◽  
Author(s):  
Eli Fernández-de Gortari ◽  
L. Michel Espinoza-Fonseca
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Structural Features ◽  
Structural Basis ◽  
Initial Structure ◽  
Structural Mechanism ◽  
Cytosolic Side ◽  
Dynamics Simulations ◽  
Atpase Inhibition ◽  
Inhibitory Interactions ◽  
Serca Inhibition

AbstractWe have performed extensive atomistic molecular dynamics simulations to probe the structural mechanism for relief of sarcoplasmic reticulum Ca2+-ATPase (SERCA) inhibition by phospholamban (PLB) at saturating Ca2+ conditions. Reversal of SERCA-PLB inhibition by saturating Ca2+ operates as a physiological rheostat to reactivate SERCA function in the absence of PLB phosphorylation. Simulation of the inhibitory complex at super-physiological Ca2+ concentrations ([Ca2+]=10 mM) revealed that calcium ions interact primarily with SERCA and the lipid headgroups, but not with the cytosolic domain of PLB or the cytosolic side of the SERCA-PLB interface. At this [Ca2+], a single Ca2+ ion is translocated from the cytosol to the transmembrane transport sites. We used this Ca2+-bound complex as an initial structure to simulate the effects of saturating Ca2+ at physiological conditions ([Ca2+]total≈400 μM). At these conditions, ~30% of the Ca2+-bound complexes exhibit structural features that correspond to an inhibited state. However, in ~70% of the Ca2+-bound complexes, Ca2+ moves to transport site I, recruits Glu771 and Asp800, and disrupts key inhibitory contacts involving conserved PLB residue Asn34. Structural analysis showed that Ca2+ induces only local changes in interresidue inhibitory interactions, but does not induce dissociation, repositioning or changes in the structural dynamics of PLB. Upon relief of SERCA inhibition, Ca2+ binding produces a productive site I configuration that is sufficient for subsequent SERCA activation. We propose that at saturating [Ca2+] and in the absence of PLB phosphorylation, binding of a single Ca2+ ion in the transport sites rapidly shifts the equilibrium toward a non-inhibited SERCA-PLB complex.

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