scholarly journals Electrostatic interaction of loop 1 and backbone of human cardiac myosin regulates the rate of ATP induced actomyosin dissociation

2021 ◽  
Author(s):  
Akhil Gargey Iragavarapu ◽  
Yuri Nesmelov
Keyword(s):  
Electrostatic Interactions ◽  
Myosin Head ◽  
Salt Bridge ◽  
C2c12 Cells ◽  
Atp Binding ◽  
Cardiac Myosin ◽  
Wild Type ◽  
Transient Kinetics ◽  
Double Mutation ◽  
Adp Release

Double mutation D208Q:K450L was introduced in the beta isoform of human cardiac myosin to remove the salt bridge D208:K450 connecting loop 1 and the seven stranded beta sheet within the myosin head. Beta isoform specific salt bridge D208:K450 was previously discovered in the molecular dynamics simulations. It was proposed that loop 1 modulates nucleotide affinity to actomyosin and we hypothesized that the electrostatic interactions between loop 1 and myosin head backbone regulates ATP binding to and ADP dissociation from actomyosin, and therefore, the time of the strong actomyosin binding. Wild type and the mutant of the myosin head construct (843 amino acid residues) were expressed in differentiated C2C12 cells, and the kinetics of ATP induced actomyosin dissociation and ADP release were characterized using transient kinetics spectrophotometry. Both constructs exhibit a fast rate of ATP binding to actomyosin and a slow rate of ADP dissociation, showing that ADP release limits the time of the strongly bound state of actomyosin. We observed a faster rate of ATP induced actomyosin dissociation with the mutant, compared to the wild type actomyosin. The rate of ADP release from actomyosin remains the same for the mutant and the wild type actomyosin. We conclude that the flexibility of loop 1 is a factor affecting the rate of ATP binding to actomyosin and actomyosin dissociation. We observed no effect of loop 1 flexibility on the rate of ADP release from actomyosin.

scholarly journals The time of strong actomyosin binding depends on electrostatic interactions within the force generating region in human cardiac myosin

2020 ◽  
Author(s):  
Akhil Gargey ◽  
Shiril Bhardwaj Iragavarapu ◽  
Alexander V. Grdzelishvili ◽  
Yuri E. Nesmelov
Keyword(s):  
Electrostatic Interactions ◽  
Myosin Head ◽  
Bound State ◽  
Myosin Isoforms ◽  
Salt Bridges ◽  
Cardiac Myosin ◽  
Wild Type ◽  
Transient Kinetics ◽  
Adp Release ◽  
Dynamics Simulations

AbstractTwo single mutations, R694N and E45Q, were introduced in the beta isoform of human cardiac myosin to remove permanent salt bridges E45:R694 and E98:R694 in the force-generating region of myosin head. Beta isoform-specific bridges E45:R694 and E98:R694 were discovered in the molecular dynamics simulations of the alpha and beta myosin isoforms. Alpha and beta isoforms exhibit different kinetics, ADP dissociates slower from actomyosin containing beta myosin isoform, therefore, beta myosin stays strongly bound to actin longer. We hypothesize that the electrostatic interactions in the force-generating region modulate affinity of ADP to actomyosin, and therefore, the time of the strong actomyosin binding. Wild type and the mutants of the myosin head construct (1-843 amino acid residues) were expressed in differentiated C2C12 cells, and duration of the strongly bound state of actomyosin was characterized using transient kinetics spectrophotometry. All myosin constructs exhibited a fast rate of ATP binding to actomyosin and a slow rate of ADP dissociation, showing that ADP release limits the time of the strongly bound state of actomyosin. Mutant R694N showed faster rate of ADP release from actomyosin, compared to the wild type and the E45Q mutant, thus confirming that electrostatic interactions within the force-generating region of human cardiac myosin regulate ADP release and the duration of the strongly bound state of actomyosin.

scholarly journals Electrostatic Interactions within Human Cardiac Myosin Head Modulate Its Kinetics

2020 ◽  
Vol 118 (3) ◽  
pp. 433a
Author(s):  
Akhil Gargey ◽  
Shiril Bharadwaj ◽  
Yaroslav V. Tkachev ◽  
Yuri E. Nesmelov
Keyword(s):  
Electrostatic Interactions ◽  
Myosin Head ◽  
Cardiac Myosin

scholarly journals Point Mutations in Human β Cardiac Myosin Heavy Chain Have Differential Effects on Sarcomeric Structure and Assembly: An ATP Binding Site Change Disrupts Both Thick and Thin Filaments, Whereas Hypertrophic Cardiomyopathy Mutations Display Normal Assembly

1997 ◽  
Vol 137 (1) ◽  
pp. 131-140 ◽  
Author(s):  
K. David Becker ◽  
Kim R. Gottshall ◽  
Reed Hickey ◽  
Jean-Claude Perriard ◽  
Kenneth R. Chien
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Subcellular Localization ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Point Mutations ◽  
Neonatal Rat ◽  
Atp Binding ◽  
Cardiac Myosin ◽  
Wild Type ◽  
Thick Filaments

Hypertrophic cardiomyopathy is a human heart disease characterized by increased ventricular mass, focal areas of fibrosis, myocyte, and myofibrillar disorganization. This genetically dominant disease can be caused by mutations in any one of several contractile proteins, including β cardiac myosin heavy chain (βMHC). To determine whether point mutations in human βMHC have direct effects on interfering with filament assembly and sarcomeric structure, full-length wild-type and mutant human βMHC cDNAs were cloned and expressed in primary cultures of neonatal rat ventricular cardiomyocytes (NRC) under conditions that promote myofibrillogenesis. A lysine to arginine change at amino acid 184 in the consensus ATP binding sequence of human βMHC resulted in abnormal subcellular localization and disrupted both thick and thin filament structure in transfected NRC. Diffuse βMHC K184R protein appeared to colocalize with actin throughout the myocyte, suggesting a tight interaction of these two proteins. Human βMHC with S472V mutation assembled normally into thick filaments and did not affect sarcomeric structure. Two mutant myosins previously described as causing human hypertrophic cardiomyopathy, R249Q and R403Q, were competent to assemble into thick filaments producing myofibrils with well defined I bands, A bands, and H zones. Coexpression and detection of wild-type βMHC and either R249Q or R403Q proteins in the same myocyte showed these proteins are equally able to assemble into the sarcomere and provided no discernible differences in subcellular localization. Thus, human βMHC R249Q and R403Q mutant proteins were readily incorporated into NRC sarcomeres and did not disrupt myofilament formation. This study indicates that the phenotype of myofibrillar disarray seen in HCM patients which harbor either of these two mutations may not be directly due to the failure of the mutant myosin heavy chain protein to assemble and form normal sarcomeres, but may rather be a secondary effect possibly resulting from the chronic stress of decreased βMHC function.

scholarly journals Role of Electrostatic Interactions in the Isoform-Specific Rate of ADP Release from Human Cardiac Myosin

2018 ◽  
Vol 114 (3) ◽  
pp. 140a-141a
Author(s):  
Akhil Gargey ◽  
Jinghua Ge ◽  
Yaroslav Tkachev ◽  
Yuri Nesmelov
Keyword(s):  
Electrostatic Interactions ◽  
Specific Rate ◽  
Cardiac Myosin ◽  
Adp Release

scholarly journals Electrostatic Interactions Within Loop 1 and the Force Generation Region of Human Cardiac Myosin Affect the Rate of Actomyosin Dissociation and ADP Release

2019 ◽  
Vol 116 (3) ◽  
pp. 259a-260a
Author(s):  
Akhil Gargey ◽  
Jinghua Ge ◽  
Alex Grdzelishvili ◽  
Yaroslav Tkachev ◽  
Yuri E. Nesmelov
Keyword(s):  
Electrostatic Interactions ◽  
Force Generation ◽  
Cardiac Myosin ◽  
Adp Release ◽  
Generation Region

scholarly journals Electron transfer by human wild-type and A287P mutant P450 oxidoreductase assessed by transient kinetics: functional basis of P450 oxidoreductase deficiency

2015 ◽  
Vol 468 (1) ◽  
pp. 25-31 ◽  
Author(s):  
Yi Jin ◽  
Mo Chen ◽  
Trevor M. Penning ◽  
Walter L. Miller
Keyword(s):  
Electron Transfer ◽  
European Ancestry ◽  
Wild Type ◽  
Transient Kinetics ◽  
Skeletal Malformation ◽  
P450 Oxidoreductase ◽  
Sex Development ◽  
Cytochrome P450 Oxidoreductase ◽  
Flavin Binding ◽  
Human Wild Type

We show that the cytochrome P450 oxidoreductase (POR) A287P variant, which can lead to skeletal malformation and disordered sex development, found in 40% of patients of European ancestry is characterized by deficient flavin binding and impaired electron transfer from NADPH.

scholarly journals Insights on the mechanisms of Ca2+ regulation of connexin26 hemichannels revealed by human pathogenic mutations (D50N/Y)

2013 ◽  
Vol 142 (1) ◽  
pp. 23-35 ◽  
Author(s):  
William Lopez ◽  
Jorge Gonzalez ◽  
Yu Liu ◽  
Andrew L. Harris ◽  
Jorge E. Contreras
Keyword(s):  
Essential Role ◽  
Negative Charge ◽  
Salt Bridge ◽  
Cysteine Residue ◽  
Wild Type ◽  
Closed State ◽  
Large Size ◽  
Deactivation Kinetics ◽  
Connexin Hemichannel

Because of the large size and modest selectivity of the connexin hemichannel aqueous pore, hemichannel opening must be highly regulated to maintain cell viability. At normal resting potentials, this regulation is achieved predominantly by the physiological extracellular Ca2+ concentration, which drastically reduces hemichannel activity. Here, we characterize the Ca2+ regulation of channels formed by wild-type human connexin26 (hCx26) and its human mutations, D50N/Y, that cause aberrant hemichannel opening and result in deafness and skin disorders. We found that in hCx26 wild-type channels, deactivation kinetics are accelerated as a function of Ca2+ concentration, indicating that Ca2+ facilitates transition to, and stabilizes, the closed state of the hemichannels. The D50N/Y mutant hemichannels show lower apparent affinities for Ca2+-induced closing than wild-type channels and have more rapid deactivation kinetics, which are Ca2+ insensitive. These results suggest that D50 plays a role in (a) stabilizing the open state in the absence of Ca2+, and (b) facilitating closing and stabilization of the closed state in the presence of Ca2+. To explore the role of a negatively charged residue at position 50 in regulation by Ca2+, this position was substituted with a cysteine residue, which was then modified with a negatively charged methanethiosulfonate reagent, sodium (2-sulfanoethyl) methanethiosulfonate (MTSES)−. D50C mutant hemichannels display properties similar to those of D50N/Y mutants. Recovery of the negative charge with chemical modification by MTSES− restores the wild-type Ca2+ regulation of the channels. These results confirm the essential role of a negative charge at position 50 for Ca2+ regulation. Additionally, charge-swapping mutagenesis studies suggest involvement of a salt bridge interaction between D50 and K61 in the adjacent connexin subunit in stabilizing the open state in low extracellular Ca2+. Mutant cycle analysis supports a Ca2+-sensitive interaction between these two residues in the open state of the channel. We propose that disruption of this interaction by extracellular Ca2+ destabilizes the open state and facilitates hemichannel closing. Our data provide a mechanistic understanding of how mutations at position 50 that cause human diseases are linked to dysfunction of hemichannel gating by external Ca2+.

scholarly journals Time-Resolved FRET Confirms Human Cardiac Myosin Head-Tail Interaction

2020 ◽  
Vol 118 (3) ◽  
pp. 427a
Author(s):  
Alexandra N. Hurst ◽  
Shiril Bhardwaj ◽  
Akhil Gargey ◽  
Yuri Nesmelov
Keyword(s):  
Myosin Head ◽  
Cardiac Myosin ◽  
Time Resolved

scholarly journals Rational design of new NO and redox sensitivity into connexin26 hemichannels

Open Biology ◽  
2015 ◽  
Vol 5 (2) ◽  
pp. 140208 ◽  
Author(s):  
Louise Meigh ◽  
Daniel Cook ◽  
Jie Zhang ◽  
Nicholas Dale
Keyword(s):  
Redox Potential ◽  
Rational Design ◽  
Salt Bridge ◽  
Disulfide Bridge ◽  
Wild Type ◽  
No Donors ◽  
Bridge Formation ◽  
Redox Sensitivity ◽  
Intracellular Redox

CO 2 directly opens hemichannels of connexin26 (Cx26) by carbamylating K125, thereby allowing salt bridge formation with R104 of the neighbouring subunit in the connexin hexamer. The formation of the inter-subunit carbamate bridges within the hexameric hemichannel traps it in the open state. Here, we use insights derived from this model to test whether the range of agonists capable of opening Cx26 can be extended by promoting the formation of analogous inter-subunit bridges via different mechanisms. The mutation K125C gives potential for nitrosylation on Cys125 and formation of an SNO bridge to R104 of the neighbouring subunit. Unlike wild-type Cx26 hemichannels, which are insensitive to NO and NO 2 − , hemichannels comprising Cx26 K125C can be opened by NO 2 − and NO donors. However, NO 2 − was unable to modulate the doubly mutated (K125C, R104A) hemichannels, indicating that an inter-subunit bridge between C125 and R104 is required for the opening action of NO 2 − . In a further test, we introduced two mutations into Cx26, K125C and R104C, to allow disulfide bridge formation across the inter-subunit boundary. These doubly mutated hemichannels open in response to changes in intracellular redox potential.

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