Investigation of the Recovery Stroke and ATP Hydrolysis and Changes Caused Due to the Cardiomyopathic Point Mutations in Human Cardiac β Myosin

2021 ◽  
Author(s):  
Ananya Chakraborti ◽  
Anthony P. Baldo ◽  
Jil C. Tardiff ◽  
Steven D. Schwartz
Keyword(s):  
Atp Hydrolysis ◽  
Point Mutations ◽  
Recovery Stroke

scholarly journals Selective Perturbation of the Myosin Recovery Stroke by Point Mutations at the Base of the Lever Arm Affects ATP Hydrolysis and Phosphate Release

2007 ◽  
Vol 282 (24) ◽  
pp. 17658-17664 ◽  
Author(s):  
András Málnási-Csizmadia ◽  
Judit Tóth ◽  
David S. Pearson ◽  
Csaba Hetényi ◽  
László Nyitray ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Point Mutations ◽  
Phosphate Release ◽  
Lever Arm ◽  
Recovery Stroke

scholarly journals Mechanochemical Function of Myosin II: Investigation into the Recovery Stroke and ATP Hydrolysis

2020 ◽  
Vol 124 (45) ◽  
pp. 10014-10023
Author(s):  
Anthony P. Baldo ◽  
Jil C. Tardiff ◽  
Steven D. Schwartz
Keyword(s):  
Atp Hydrolysis ◽  
Myosin Ii ◽  
Recovery Stroke

scholarly journals Genetic and biochemical analysis of Msh2p-Msh6p: role of ATP hydrolysis and Msh2p-Msh6p subunit interactions in mismatch base pair recognition.

1997 ◽  
Vol 17 (5) ◽  
pp. 2436-2447 ◽  
Author(s):  
E Alani ◽  
T Sokolsky ◽  
B Studamire ◽  
J J Miret ◽  
R S Lahue
Keyword(s):  
Base Pair ◽  
Biochemical Analysis ◽  
Atp Hydrolysis ◽  
Point Mutations ◽  
Binding Domain ◽  
Dominant Negative ◽  
Mutant Protein ◽  
Atp Binding ◽  
Mutant Proteins ◽  
Mismatch Recognition

Recent studies have shown that Saccharomyces cerevisiae Msh2p and Msh6p form a complex that specifically binds to DNA containing base pair mismatches. In this study, we performed a genetic and biochemical analysis of the Msh2p-Msh6p complex by introducing point mutations in the ATP binding and putative helix-turn-helix domains of MSH2. The effects of these mutations were analyzed genetically by measuring mutation frequency and biochemically by measuring the stability, mismatch binding activity, and ATPase activity of msh2p (mutant msh2p)-Msh6p complexes. A mutation in the ATP binding domain of MSH2 did not affect the mismatch binding specificity of the msh2p-Msh6p complex; however, this mutation conferred a dominant negative phenotype when the mutant gene was overexpressed in a wild-type strain, and the mutant protein displayed biochemical defects consistent with defects in mismatch repair downstream of mismatch recognition. Helix-turn-helix domain mutant proteins displayed two different properties. One class of mutant proteins was defective in forming complexes with Msh6p and also failed to recognize base pair mismatches. A second class of mutant proteins displayed properties similar to those observed for the ATP binding domain mutant protein. Taken together, these data suggested that the proposed helix-turn-helix domain of Msh2p was unlikely to be involved in mismatch recognition. We propose that the MSH2 helix-turn-helix domain mediates changes in Msh2p-Msh6p interactions that are induced by ATP hydrolysis; the net result of these changes is a modulation of mismatch recognition.

scholarly journals Oligomerization of EpsE Coordinates Residues from Multiple Subunits to Facilitate ATPase Activity

2011 ◽  
Vol 286 (12) ◽  
pp. 10378-10386 ◽  
Author(s):  
Marcella Patrick ◽  
Konstantin V. Korotkov ◽  
Wim G. J. Hol ◽  
Maria Sandkvist
Keyword(s):  
Atpase Activity ◽  
Conformational Changes ◽  
Active Site ◽  
Structural Changes ◽  
Atp Hydrolysis ◽  
Hydrolytic Enzymes ◽  
Point Mutations ◽  
Nucleotide Binding ◽  
Type Ii ◽  
Arginine Residues

EpsE is an ATPase that powers transport of cholera toxin and hydrolytic enzymes through the Type II secretion (T2S) apparatus in the Gram-negative bacterium, Vibrio cholerae. On the basis of structures of homologous Type II/IV secretion ATPases and our biochemical data, we believe that EpsE is active as an oligomer, likely a hexamer, and the binding, hydrolysis, and release of nucleotide cause EpsE to undergo dynamic structural changes, thus converting chemical energy to mechanical work, ultimately resulting in extracellular secretion. The conformational changes that occur as a consequence of nucleotide binding would realign conserved arginines (Arg210, Arg225, Arg320, Arg324, Arg336, and Arg369) from adjoining domains and subunits to complete the active site around the bound nucleotide. Our data suggest that these arginines are essential for ATP hydrolysis, although their roles in shaping the active site of EpsE are varied. Specifically, we have shown that replacements of these arginine residues abrogate the T2S process due to a reduction of ATPase activity yet do not have any measurable effect on nucleotide binding or oligomerization of EpsE. We have further demonstrated that point mutations in the EpsE intersubunit interface also reduce ATPase activity without disrupting oligomerization, strengthening the idea that residues from multiple subunits must precisely interact in order for EpsE to be sufficiently active to support T2S. Our findings suggest that the action of EpsE is similar to that of other Type II/IV secretion ATPase family members, and thus these results may be widely applicable to the family as a whole.

scholarly journals Functional Role of Class III Myosins in Hair Cells

2021 ◽  
Vol 9 ◽  
Author(s):  
Joseph A. Cirilo ◽  
Laura K. Gunther ◽  
Christopher M. Yengo
Keyword(s):  
Hearing Loss ◽  
Hair Cells ◽  
Atp Hydrolysis ◽  
Point Mutations ◽  
Special Focus ◽  
Myosin Isoforms ◽  
Class Iii ◽  
Myosin Motors ◽  
And Function

Cytoskeletal motors produce force and motion using the energy from ATP hydrolysis and function in a variety of mechanical roles in cells including muscle contraction, cargo transport, and cell division. Actin-based myosin motors have been shown to play crucial roles in the development and function of the stereocilia of auditory and vestibular inner ear hair cells. Hair cells can contain hundreds of stereocilia, which rely on myosin motors to elongate, organize, and stabilize their structure. Mutations in many stereocilia-associated myosins have been shown to cause hearing loss in both humans and animal models suggesting that each myosin isoform has a specific function in these unique parallel actin bundle-based protrusions. Here we review what is known about the classes of myosins that function in the stereocilia, with a special focus on class III myosins that harbor point mutations associated with delayed onset hearing loss. Much has been learned about the role of the two class III myosin isoforms, MYO3A and MYO3B, in maintaining the precise stereocilia lengths required for normal hearing. We propose a model for how class III myosins play a key role in regulating stereocilia lengths and demonstrate how their motor and regulatory properties are particularly well suited for this function. We conclude that ongoing studies on class III myosins and other stereocilia-associated myosins are extremely important and may lead to novel therapeutic strategies for the treatment of hearing loss due to stereocilia degeneration.

scholarly journals The Iron-containing Domain Is Essential in Rad3 Helicases for Coupling of ATP Hydrolysis to DNA Translocation and for Targeting the Helicase to the Single-stranded DNA-Double-stranded DNA Junction

2007 ◽  
Vol 283 (3) ◽  
pp. 1732-1743 ◽  
Author(s):  
Robert A. Pugh ◽  
Masayoshi Honda ◽  
Haley Leesley ◽  
Alvin Thomas ◽  
Yuyen Lin ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Point Mutations ◽  
Structural Features ◽  
Fine Tuning ◽  
Dna Interactions ◽  
Double Stranded Dna ◽  
Distinct Disease ◽  
Relevant Point ◽  
Conserved Core ◽  
Xpd Helicase

Helicases often achieve functional specificity through utilization of unique structural features incorporated into an otherwise conserved core. The archaeal Rad3 (xeroderma pigmentosum group D protein (XPD)) helicase is a prototypical member of the Rad3 family, distinct from other related (superfamily II) SF2 enzymes because of a unique insertion containing an iron-sulfur (FeS) cluster. This insertion may represent an auxiliary domain responsible for modifying helicase activity or for conferring specificity for selected DNA repair intermediates. The importance of the FeS cluster for the fine-tuning of Rad3-DNA interactions is illustrated by several clinically relevant point mutations in the FeS domain of human Bach1 (FancJ) and XPD helicases that result in distinct disease phenotypes. Here we analyzed the substrate specificity of the Rad3 (XPD) helicase from Ferroplasma acidarmanus (FacRad3) and probed the importance of the FeS cluster for Rad3-DNA interactions. We found that the FeS cluster stabilizes secondary structure of the auxiliary domain important for coupling of single-stranded (ss) DNA-dependent ATP hydrolysis to ssDNA translocation. Additionally, we observed specific quenching of the Cy5 fluorescent dye when the FeS cluster of a bound helicase is positioned in close proximity to a Cy5 fluorophore incorporated into the DNA molecule. Taking advantage of this Cy5 quenching, we developed an equilibrium assay for analysis of the Rad3 interactions with various DNA substrates. We determined that the FeS cluster-containing domain recognizes the ssDNA-double-stranded DNA junction and positions the helicase in an orientation consistent with duplex unwinding. Although it interacts specifically with the junction, the enzyme binds tightly to ssDNA, and the single-stranded regions of the substrate are the major contributors to the energetics of FacRad3-substrate interactions.

scholarly journals Gradual opening of Smc arms in prokaryotic condensin

2021 ◽  
Author(s):  
Roberto Vazquez Nunez ◽  
Yevhen Polyhach ◽  
Young-Min Soh ◽  
Gunnar Jeschke ◽  
Stephan Gruber
Keyword(s):  
Atp Hydrolysis ◽  
Point Mutations ◽  
Coiled Coil ◽  
Resonance Spectroscopy ◽  
Dna Translocation ◽  
Dimerization Domain ◽  
Dna Loop ◽  
Shaped Particle ◽  
Spin Resonance ◽  
Extrusion Reaction

SummaryMulti-subunit SMC ATPases control chromosome superstructure apparently by catalyzing a DNA-loop-extrusion reaction. SMC proteins harbor an ABC-type ATPase ‘head’ and a ‘hinge’ dimerization domain connected by a coiled coil ‘arm’. Two arms in a SMC dimer can co-align, thereby forming a rod-shaped particle. Upon ATP binding, SMC heads engage, and arms are thought to separate. Here, we studied the shape of B. subtilis Smc-ScpAB by electron-spin resonance spectroscopy. Arm separation was readily detected proximal to the heads in the absence of ligands, while separation near the hinge largely depended on ATP and DNA. Artificial blockage of arm opening eliminated DNA stimulation of ATP hydrolysis, but did not prevent basal ATPase activity. We identified an arm-to-arm contact as being important for controlling the molecular transformations. Point mutations at this arm interface eliminate Smc function. We propose that partially open, intermediary conformations provide directionality to SMC DNA translocation by binding suitable DNA substrates.

CRYO-Electron Microscopy of the Acto-Myosin-ATP Complex

1990 ◽  
Vol 48 (1) ◽  
pp. 248-249
Author(s):  
John Trinickt ◽  
Howard White
Keyword(s):  
Electron Microscopy ◽  
Atp Hydrolysis ◽  
Myosin Head ◽  
Bound State ◽  
Cross Linking ◽  
Weakly Bound ◽  
Cryo Electron Microscopy ◽  
Myosin Subfragment 1 ◽  
Attached State ◽  
High Fraction

The primary force of muscle contraction is thought to involve a change in the myosin head whilst attached to actin, the energy coming from ATP hydrolysis. This change in attached state could either be a conformational change in the head or an alteration in the binding angle made with actin. A considerable amount is known about one bound state, the so-called strongly attached state, which occurs in the presence of ADP or in the absence of nucleotide. In this state, which probably corresponds to the last attached state of the force-producing cycle, the angle between the long axis myosin head and the actin filament is roughly 45°. Details of other attached states before and during power production have been difficult to obtain because, even at very high protein concentration, the complex is almost completely dissociated by ATP. Electron micrographs of the complex in the presence of ATP have therefore been obtained only after chemically cross-linking myosin subfragment-1 (S1) to actin filaments to prevent dissociation. But it is unclear then whether the variability in attachment angle observed is due merely to the cross-link acting as a hinge.We have recently found low ionic-strength conditions under which, without resorting to cross-linking, a high fraction of S1 is bound to actin during steady state ATP hydrolysis. The structure of this complex is being studied by cryo-electron microscopy of hydrated specimens. Most advantages of frozen specimens over ambient temperature methods such as negative staining have already been documented. These include improved preservation and fixation rates and the ability to observe protein directly rather than a surrounding stain envelope. In the present experiments, hydrated specimens have the additional benefit that it is feasible to use protein concentrations roughly two orders of magnitude higher than in conventional specimens, thereby reducing dissociation of weakly bound complexes.

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.

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