Faculty Opinions recommendation of Single molecule mechanics resolves the earliest events in force generation by cardiac myosin.

Key steps of cardiac mechanochemistry, including the force-generating working stroke and the release of phosphate (Pi), occur rapidly after myosin-actin attachment. An ultra-high-speed optical trap enabled direct observation of the timing and amplitude of the working stroke, which can occur within <200 μs of actin binding by β-cardiac myosin. The initial actomyosin state can sustain loads of at least 4.5 pN and proceeds directly to the stroke or detaches before releasing ATP hydrolysis products. The rates of these processes depend on the force. The time between binding and stroke is unaffected by 10 mM Pi which, along with other findings, indicates the stroke precedes phosphate release. After Pi release, Pi can rebind enabling reversal of the working stroke. Detecting these rapid events under physiological loads provides definitive indication of the dynamics by which actomyosin converts biochemical energy into mechanical work.

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Single molecule mechanics resolves the earliest events in force generation by cardiac myosin

10.1101/683623 ◽

2019 ◽

Author(s):

Michael S. Woody ◽

Donald A. Winkelmann ◽

Marco Capitanio ◽

E. Michael Ostap ◽

Yale E. Goldman

Keyword(s):

Single Molecule ◽

High Speed ◽

Atp Hydrolysis ◽

Optical Trap ◽

Force Generation ◽

Actin Binding ◽

Cardiac Myosin ◽

Phosphate Release ◽

Hydrolysis Products ◽

Key Steps

AbstractKey steps of cardiac mechanochemistry, including the force-generating working stroke and the release of phosphate (Pi), occur rapidly after myosin-actin attachment. An ultra-high-speed optical trap enabled direct observation of the timing and amplitude of the working stroke, which can occur within <200 μs of actin binding by β-cardiac myosin. The initial actomyosin state can sustain loads of at least 4.5 pN and proceeds directly to the stroke or detaches before releasing ATP hydrolysis products. The rates of these processes depend on the force. The time between binding and stroke is unaffected by 10 mM Pi which, along with other findings, indicates the stroke precedes phosphate release. After Pi release, Pi can rebind enabling reversal of the working stroke. Detecting these rapid events under physiological loads provides definitive indication of the dynamics by which actomyosin converts biochemical energy into mechanical work.

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Force Generation by Membrane-Bound Myo1c, a Single Molecule Study

Biophysical Journal ◽

10.1016/j.bpj.2012.11.3547 ◽

2013 ◽

Vol 104 (2) ◽

pp. 642a

Author(s):

Serapion Pyrpassopoulos ◽

Henry Shuman ◽

E. Michael Ostap

Keyword(s):

Single Molecule ◽

Force Generation ◽

Membrane Bound

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Altered C10 domain in cardiac myosin binding protein-C results in hypertrophic cardiomyopathy

Cardiovascular Research ◽

10.1093/cvr/cvz111 ◽

2019 ◽

Vol 115 (14) ◽

pp. 1986-1997 ◽

Cited By ~ 5

Author(s):

Diederik W D Kuster ◽

Thomas L Lynch ◽

David Y Barefield ◽

Mayandi Sivaguru ◽

Gina Kuffel ◽

...

Keyword(s):

Hypertrophic Cardiomyopathy ◽

Protein C ◽

Binding Protein ◽

Force Generation ◽

Contractile Dysfunction ◽

Cardiac Myosin ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Myosin Binding

Abstract Aims A 25-base pair deletion in the cardiac myosin binding protein-C (cMyBP-C) gene (MYBPC3), proposed to skip exon 33, modifies the C10 domain (cMyBP-CΔC10mut) and is associated with hypertrophic cardiomyopathy (HCM) and heart failure, affecting approximately 100 million South Asians. However, the molecular mechanisms underlying the pathogenicity of cMyBP-CΔC10mutin vivo are unknown. We hypothesized that expression of cMyBP-CΔC10mut exerts a poison polypeptide effect leading to improper assembly of cardiac sarcomeres and the development of HCM. Methods and results To determine whether expression of cMyBP-CΔC10mut is sufficient to cause HCM and contractile dysfunction in vivo, we generated transgenic (TG) mice having cardiac-specific protein expression of cMyBP-CΔC10mut at approximately half the level of endogenous cMyBP-C. At 12 weeks of age, significant hypertrophy was observed in TG mice expressing cMyBP-CΔC10mut (heart weight/body weight ratio: 4.43 ± 0.11 mg/g non-transgenic (NTG) vs. 5.34 ± 0.25 mg/g cMyBP-CΔC10mut, P < 0.05). Furthermore, haematoxylin and eosin, Masson’s trichrome staining, as well as second-harmonic generation imaging revealed the presence of significant fibrosis and a greater relative nuclear area in cMyBP-CΔC10mut hearts compared with NTG controls. M-mode echocardiography analysis revealed hypercontractile hearts (EF: 53.4%±2.9% NTG vs. 66.4% ± 4.7% cMyBP-CΔC10mut; P < 0.05) and early diastolic dysfunction (E/E′: 28.7 ± 3.7 NTG vs. 46.3 ± 8.4 cMyBP-CΔC10mut; P < 0.05), indicating the presence of an HCM phenotype. To assess whether these changes manifested at the myofilament level, contractile function of single skinned cardiomyocytes was measured. Preserved maximum force generation and increased Ca2+-sensitivity of force generation were observed in cardiomyocytes from cMyBP-CΔC10mut mice compared with NTG controls (EC50: 3.6 ± 0.02 µM NTG vs. 2.90 ± 0.01 µM cMyBP-CΔC10mut; P < 0.0001). Conclusion Expression of cMyBP-C protein with a modified C10 domain is sufficient to cause contractile dysfunction and HCM in vivo.

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Single Molecule Studies of Recombinant Human α- and β-Cardiac Myosin to Elucidate Molecular Mechanism of Familial Hypertrophic and Dilated Cardiomyopathies

Biophysical Journal ◽

10.1016/j.bpj.2011.11.3345 ◽

2012 ◽

Vol 102 (3) ◽

pp. 613a-614a

Author(s):

Jongmin Sung ◽

Elizabeth Choe ◽

Mary Elting ◽

Suman Nag ◽

Shirley Sutton ◽

...

Keyword(s):

Molecular Mechanism ◽

Single Molecule ◽

Cardiac Myosin ◽

Single Molecule Studies

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Mutations in the catalytic domain of human β-cardiac myosin that cause early onset hypertrophic cardiomyopathy significantly increase the fundamental parameters that determine ensemble force and velocity

10.1101/067066 ◽

2016 ◽

Cited By ~ 1

Author(s):

Arjun S. Adhikari ◽

Kristina B. Kooiker ◽

Chao Liu ◽

Saswata S. Sarkar ◽

Daniel Bernstein ◽

...

Keyword(s):

Hypertrophic Cardiomyopathy ◽

Single Molecule ◽

Early Onset ◽

Catalytic Domain ◽

Cardiovascular Disorder ◽

Cardiac Myosin ◽

Binding Studies ◽

Fundamental Parameters ◽

Biomechanical Parameters ◽

Myosin Motors

AbstractHypertrophic cardiomyopathy (HCM) is a heritable cardiovascular disorder that affects 1 in 500 people. In infants it can be particularly severe and it is the leading cause of sudden cardiac death in pediatric populations. A high percentage of HCM is attributed to mutations in β-cardiac myosin, the motor protein that powers ventricular contraction. This study reports how two mutations that cause early-onset HCM, D239N and H251N, affect the mechanical output of human β-cardiac myosin at the molecular level. We observe extremely large increases (25% – 95%) in the actin gliding velocity, single molecule intrinsic force, and ATPase activity of the two mutant myosin motors compared to wild type myosin. In contrast to previous studies of HCM-causing mutations in human β-cardiac myosin, these mutations were striking in that they caused changes in biomechanical parameters that were both greater in magnitude and more uniformly consistent with a hyper-contractile phenotype. In addition, S1-S2 binding studies revealed a significant decrease in affinity of the H251N motor for S2, suggesting that this mutation may further increase hyper-contractility by releasing active motors from a sequestered state. This report shows, for the first time, a clear and significant gain in function for all tested molecular biomechanical parameters due to HCM mutations in human β-cardiac myosin.

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Mechanism of Force Generation in Kinesin Motility

ASME 2007 Summer Bioengineering Conference ◽

10.1115/sbc2007-175543 ◽

2007 ◽

Author(s):

Wonmuk Hwang ◽

Matthew J. Lang

Keyword(s):

Single Molecule ◽

Forward Bias ◽

Tight Binding ◽

Basic Mechanism ◽

Binding Pocket ◽

Force Generation ◽

Power Stroke ◽

Additional Element ◽

Dynamics Simulations ◽

Motor Head

Conventional kinesin is a dimeric motor protein that uses adenosine triphosphate (ATP) to walk processively along the microtubule. Although its nucleotide dependent conformational switching and binding of the neck linker (NL) on the motor head are known to be key events in kinesin motility, the basic mechanism by which it amplifies a small conformational change upon ATP binding to generate the force of the walking stroke has not been known. We combined structural analysis with a set of molecular dynamics simulations to identify the 9-residue long N-terminal region, which we named the ‘cover strand’ (CS), as an additional element essential for kinesin’s power stroke. It operates by differentially forming a β-sheet with NL when ATP binds, whereby the ‘cover-neck bundle’ (CNB) has an inherent conformational bias that drives NL into its binding pocket on the motor head. After the initial stroke, the later half of NL, starting with the ‘asparagine latch’ in the middle, forms specific bonds with the motor head to ensure tight binding. We constructed the force map generated by CNB, which showed a forward bias in agreement with single molecule motility measurements. Our result is consistent with other experimental observations, including the estimated stall force and the transverse anisotropy. The novel mechanism of force generation by the dynamic folding of CNB appears to hold in various kinesin families, and elucidates the economy in the design principle of the smallest known processive motor.

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