We determined the effect of Omecamtiv Mecarbil, a novel allosteric effector cardiac muscle myosin, on kinetic and "in vitro" motility properties porcine ventricular heavy meromyosin (PV-HMM). Mecarbil increases equilibrium constant hydrolysis step (M-ATP ⇄ M-ADP-Pi) from 2.4 to 6 as by quench flow, but maximal rates both tryptophan fluorescence increase are unchanged drug. OM also amplitude fast phase phosphate dissociation (AM-ADP-Pi → AM-ADP + Pi) that is associated with force production...

Significance Heartbeats rely on cyclical interactions between myosin thick and actin thin filaments orchestrated by rising falling Ca 2+ . During systole, binds to the filament allows its interaction with produce force required for contraction. The structure of at physiological levels is unknown, which limits our understanding regulation Here, we directly observe structural states along individual systolic show that activated stochastically short-range cooperativity evident only one strand...

We have measured the kinetics of inorganic phosphate (Pi) release during a single turnover actomyosin nucleoside triphosphate (NTP) hydrolysis using double-mixing stopped-flow spectrofluorometer, at very low ionic strength to increase affinity myosin−ATP and myosin−ADP−Pi actin. Myosin subfragment 1 series triphosphates were mixed incubated for ∼1−10 s allow NTP bind myosin generate steady state mixture myosin−NTP myosin−NDP−Pi. The intermediates then with Pi fluorescent probe Pi, based on...

Significance Muscle contraction is required for critical physiological functions. It relies on the interaction of myosin motors with thin filament (TF), which regulated through a translocation tropomyosin surface F-actin by troponin complex in response to Ca 2+ . The lack high-resolution structure TF under relaxing (low-Ca ) and activating (high-Ca conditions impairs our understanding mechanism cardiac muscle regulation. Here we report structures native conditions. Our data lead model...

Mutations in genes encoding myosin, the molecular motor that powers cardiac muscle contraction, and its accessory protein, myosin binding protein C (cMyBP-C), are two most common causes of hypertrophic cardiomyopathy (HCM). Recent studies established N-terminal domains (NTDs) cMyBP-C (e.g., C0, C1, M, C2) can bind to activate or inhibit thin filament (TF). However, mechanism(s) by which NTDs modulate interaction with TF remains unknown contribution each individual NTD activation/inhibition...

Cardiac contraction depends on molecular interactions among sarcomeric proteins coordinated by the rising and falling intracellular Ca

We have measured the steady state kinetics of hydrolysis and presteady binding nucleoside triphosphates GTP, CTP, aza-ATP (1-wetheno-2-aza-ATP), ATP by rabbit skeletal actomyosin-SI.The maximum rates at 10 "C low ionic strength are: 1.9

We have determined the kinetic mechanism and motile properties of switch 1 mutant S217A myosin Va. Phosphate dissociation from V-ADP-Pi (inorganic phosphate) actomyosin rate hydrolysis step (myosin V-ATP → V-ADP-Pi) were all ∼10-fold slower in than wild type (WT) V, resulting a steady-state basal filamentous actin (actin)-activated ATP hydrolysis. Substrate binding ADP kinetics similar to or slightly faster WT V mechanochemical gating rates between trail lead heads is maintained. The...

We have used enzyme kinetics to investigate the molecular mechanism by which N-terminal domains of human and mouse cardiac MyBP-C (C0C1, C1C2, C0C2) affect activation myosin ATP hydrolysis F-actin native porcine thin filaments. N-Terminal cMyBP-C inhibit myosin-S1 ATPase F-actin. However, C1C2 C0C2 produce biphasic activating inhibitory effects on Low ratios filaments activate hydrolysis, but higher as is observed with alone. These data suggest that low concentrations a similar rigor...

The regulation by calcium and rigor-bound myosin-S1 of the rate acceleration 2'-deoxy-3'-O-(N-methylanthraniloyl)ADP (mdADP) release from myosin-mdADP-P(i) skeletal muscle thin filaments (reconstituted actin-tropomyosin-troponin) was measured using double mixing stopped-flow fluorescence with nucleotide substrate 2'-deoxy-3'-O-(N-methylanthraniloyl). predominant mechanism is product dissociation a factor approximately 200 in fully activated conformation (bound rigor S1) relative to inhibited...

We have studied the mechanism of activation native cardiac thin filaments by calcium and rigor myosin. The acceleration rate 2'-deoxy-3'-O-(N-methylanthraniloyl)ADP (mdADP) dissociation from myosin-S1-mdADP-P(i) myosin-S1-mdADP muscle was measured using double mixing stopped-flow fluorescence. Relative to inhibited (no bound or S1), fully activated (with both rigor-S1 bound) increase product physiologically important pre-power stroke myosin-mdADP-P(i) a factor ∼75. This can be compared with...

Regulation by calcium and myosin-S1 of the acceleration rate phosphate release from myosin-ADP-inorganic (M-ADP-Pi) thin filament actin-tropomyosin (Tm)-troponin (Tn), was measured directly using double mixing stopped-flow experiments with fluorescent binding protein. At low without rigor myosin-S1, saturating concentrations filaments accelerate dissociation M-ADP-Pi 8-fold, 0.08 to 0.64 s −1 . If either or is bound filaments, a biphasic process in which fast phase Pi actoTmTnM-ADP-Pi slow...

The missense mutation of Cys442 to Tyr myosin VI causes progressive postlingual sensorineural deafness. Here we report the affects C442Y on kinetics actomyosin ATP hydrolysis mechanism and motor function VI. largest changes in kinetic produced by are about 10-fold increases rate ADP dissociation from both rates acto-C442Y VI-ADP 20–40 times more rapid than steady state cannot be rate-limiting steps presence or absence actin. 2-fold increase actin gliding velocity compared with wild type (WT)...

Mutations in the <i>MYO7A</i> gene, encoding motor protein myosin VIIa, can cause Usher 1B, a deafness/blindness syndrome humans, and shaker-1 phenotype, characterized by deafness, head tossing, circling behavior, mice. Myosin VIIa is responsible for tension bearing transduction mechanism stereocilia melanosome transport retina, line with phenotypic outcomes observed However, effect of mutation, R502P amino acid substitution, on function unclear. To explore this question, we determined...

Cardiac myosin binding protein C (cMyBP-C) modulates cardiac contraction via direct interactions with thick (myosin) and thin (actin) filaments (cTFs). While its C-terminal domains (e.g. C8-C10) anchor cMyBP-C to the backbone of filament, N-terminal (NTDs) C0, C1, M, C2) bind both actin accomplish dual roles inhibiting activating cTFs. positions C1 C2 on cTF have been reported, site M-domain surface is unknown. Here, we used cryo-EM reveal that interacts helix 3 ordered tri-helix bundle...