Affinity Of Myosin For Actin Research Articles

Abstract 911: New Promoters to Improve the Efficiency of Cardiac Gene Therapies Using 2 Deoxy-ATP

Traditional inotropes work by altering intracellular calcium levels but have many off-target effects. Myosin activators have shown promise for treating the increasing number of heart failure patients today, with one drug in phase three clinical trials. These drugs work by increasing the affinity of myosin for actin. Our lab has demonstrated that the small molecule 2 deoxy-ATP (dATP) can improve both myosin binding to and myosin release from actin. We have also demonstrated the efficacy of a cardiac targeted gene therapy to improve heart function via increasing intracellular dATP levels. This was done by overexpressing ribonucleotide reductase (RNR)—the rate-limiting enzyme in de novo dNTP synthesis. This cardiac therapy, using the cardiac troponin T (cTnT) promoter, was effective in a mouse model but provided variable results in a large animal model due to delivery challenges. Therefore, we are now investigating alternative promoters to deliver dATP to cardiomyocytes and/or other striated muscles. Here we present our investigation of the CK8 promoter for striated muscle specific delivery of RNR. We report that the CK8 promoter was able to produce a greater than fourfold increase in mouse ventricular tissue dATP levels above that seen with the cTnT promoter (2.0 to 8.4 pmol/mg; P<0.01). RNR was also delivered to skeletal muscle with a twofold increase in gastrocnemius dATP levels (0.62 to 1.31 pmol/mg; P<0.01). We have also been investigating cardiac fibroblasts as a route to deliver dATP to cardiomyocytes. We have shown that in vitro fibroblasts can transmit dATP to coupled cardiomyocytes through gap junctions, indicating that overexpressing RNR in cardiac fibroblasts could increase dATP levels in both cell types. Previous research has shown that proliferating fibroblasts have increased levels of dATP, and preliminary results in our lab show that differentiated myofibroblasts have decreased levels of dATP. We therefore hypothesize that increased dATP in cardiac fibroblasts will not only improve contraction in coupled cardiomyocytes but may also inhibit fibroblast transdifferentiation into collagen producing myofibroblasts.

Cardiac myosin activation with 2-deoxy-ATP via increased electrostatic interactions with actin

The naturally occurring nucleotide 2-deoxy-adenosine 5'-triphosphate (dATP) can be used by cardiac muscle as an alternative energy substrate for myosin chemomechanical activity. We and others have previously shown that dATP increases contractile force in normal hearts and models of depressed systolic function, but the structural basis of these effects has remained unresolved. In this work, we combine multiple techniques to provide structural and functional information at the angstrom-nanometer and millisecond time scales, demonstrating the ability to make both structural measurements and quantitative kinetic estimates of weak actin-myosin interactions that underpin sarcomere dynamics. Exploiting dATP as a molecular probe, we assess how small changes in myosin structure translate to electrostatic-based changes in sarcomere function to augment contractility in cardiac muscle. Through Brownian dynamics simulation and computational structural analysis, we found that deoxy-hydrolysis products [2-deoxy-adenosine 5'-diphosphate (dADP) and inorganic phosphate (Pi)] bound to prepowerstroke myosin induce an allosteric restructuring of the actin-binding surface on myosin to increase the rate of cross-bridge formation. We then show experimentally that this predicted effect translates into increased electrostatic interactions between actin and cardiac myosin in vitro. Finally, using small-angle X-ray diffraction analysis of sarcomere structure, we demonstrate that the proposed increased electrostatic affinity of myosin for actin causes a disruption of the resting conformation of myosin motors, resulting in their repositioning toward the thin filament before activation. The dATP-mediated structural alterations in myosin reported here may provide insight into an improved criterion for the design or selection of small molecules to be developed as therapeutic agents to treat systolic dysfunction.

Effects of the cardiomyopathy-causing E244D mutation of troponin T on the structures of cardiac thin filaments studied by small-angle X-ray scattering

Small-angle X-ray scattering experiments were carried out to investigate the structural changes of cardiac thin filaments induced by the cardiomyopathy-causing E244D mutation in troponin T (TnT). We examined native thin filaments (NTF) from a bovine heart, reconstituted thin filaments containing human cardiac wild-type Tn (WTF), and filaments containing the E244D mutant of Tn (DTF), in the absence and presence of Ca2+. Analysis by model calculation showed that upon Ca2+-activation, tropomyosin (Tm) and Tn in the WTF and NTF moved together in a direction to expose myosin-binding sites on actin. On the other hand, Tm and Tn of the DTF moved in the opposite directions to each other upon Ca2+-activation. These movements caused Tm to expose more myosin-binding sites on actin than the WTF, suggesting that the affinity of myosin for actin is higher for the DTF. Thus, the mutation-induced structural changes in thin filaments would increase the number of myosin molecules bound to actin compared with the WTF, resulting in the force enhancement observed for the E244D mutation.

2-deoxy-ATP Alters Myosin Structure to Enhance Cross-Bridge Cycling and Improve Cardiac Function

We previously reported that increased cellular 2-deoxy-ATP (dATP) augments contraction, enhancing the rate and magnitude of both contraction and relaxation in intact cardiomyocytes, force production in skinned cardiac muscle, and fractional shortening of the whole heart. To better understand the mechanism by which dATP enhances cardiac function, we used molecular dynamics (MD) simulations to study the pre- and post-powerstroke states of myosin (PDB ID: 1VOM and 1MMA) bound to either Mg2+.(d)ADP.Pi (pre-powerstroke) or Mg2+.(d)ADP (post-powerstroke) for 50ns. In both states, dATP binding to myosin alters the conformation of the nucleotide binding pocket and the actin binding surface. In the pre-powerstroke state, myosin made fewer contacts with dADP than with ADP and this structural change translated to increased exposure of polar residues on the actin-binding surface of myosin. Since the initial acto-myosin interaction is primarily electrostatic, the affinity of myosin for actin may thus be enhanced with dADP.Pi. In post-powerstroke simulations, dADP binding reduced the exposure of actin binding residues on myosin somewhat. This may translate into improved ability for myosin detachment from actin at the end of the powerstroke in the presence of dADP vs. ADP. Furthermore, stopped-flow spectroscopy demonstrated that myosin has 40-80% weaker binding affinity for dADP than ADP, which may also contribute to faster myosin detachment. Together, these data demonstrate that dADP binding to myosin significantly alters the conformation of myosin, which may translate to faster cross-bridge cycling due to both enhanced myosin binding in the pre-powerstroke state and faster myosin detachment from actin in the post-powerstroke state. Ongoing studies examining the effect of enhanced cross-bridge cycling with elevated dATP in infarcted hearts suggest that dATP may reduce the loss of systolic function. Supported by HL111197 (MR), WT085309 (MAG), GM50789 (VD) and T32EB001650 (SGN).

A Troponin-T Mutation Initiates Cardiac and Skeletal Myopathy due to Impaired Inhibition of Contraction in Drosophila Melanogaster

Muscle contraction results from a series of molecular events that involve transient interactions between myosin-containing thick and actin-containing thin filaments. The high affinity of myosin for actin suggests that without regulation, muscle would remain in a continuous state of contraction. Regulation of striated muscle contraction is primarily achieved by Ca2+-dependent modulation of myosin crossbridge cycling on actin by the thin filament (TF) troponin-tropomyosin complex. Alterations in various subunits of the complex trigger contractile dysregulation and myopathy. For example, point mutations located over a span of ten amino acids (130-39) of human cardiac troponin T (cTnT) are associated with distinct cardiomyopathic responses. The Drosophila up101 (E88K) mutation localizes to the end of this well-conserved region of TnT. Here we examined the consequences of the lesion on distinct muscle types from the fly. Cardiac performance and morphology were assessed using direct immersion DIC optics, high-speed video imaging and motion analysis. Relative to controls, up101 hearts displayed a phenotype reminiscent of human restrictive cardiomyopathy. End-diastolic and end-systolic dimensions and percent fractional shortening were significantly reduced. Furthermore systolic intervals were significantly prolonged. This suggests TF dysregulation initiates excessive periods of force production and diastolic dysfunction. To characterize the fundamental cause of cardiac remodeling, we isolated TFs from the indirect flight muscles (IFM). The constitutively expressed up101 mutation results in flightlessness due to severe IFM hypercontraction. Electron microscopy and three-dimensional reconstruction of IFM TFs revealed Ca+2-dependent tropomyosin movement, typical of control filaments, was not a general feature of the TnT mutants. The vast majority of Ca+2-free mutant TFs exhibited tropomyosin associated with successive actin monomers over actin subdomains 3 and 4, distal to known myosin binding sites. Thus, up101 TnT likely promotes TF dysinhibition and ubiquitous myopathic remodeling.

2,4-Dinitrophenol reduces the reactivity of Lys553 in the lower 50-kDa region of myosin subfragment 1

2,4-Dinitrophenol (DNP) increases the affinity of myosin for actin and accelerates its Mg 2+ATPase activity, suggesting that it acts on a region of the myosin head that transmits conformational changes to actin- and ATP-binding sites. The binding site/s for DNP are unknown; however similar hydrophobic compounds bind to the 50-kDa subfragment of the myosin head, near the actin-binding interface. In this region, a helix-loop-helix motif contains Lys553, which is specifically labeled with the fluorescent probe 6-[fluorescein-5(and 6)-carboxamido] hexanoic acid succinimidyl ester (FHS). This reaction is sensitive to conformational changes in the helix-loop-helix and the labeling efficiency was reduced when S1 was bound to actin, DNP or nucleotide analogs. The nucleotide analogs had a range of effects (PPi > ADP·AlF 4 − > ADP) irrespective of the open-closed state of switch 2. The greatest reduction in labeling was in the presence of actin or DNP. When we measured the effect of each ligand on the fluorescence of FHS previously attached to S1, only DNP quenched the emission. Together, the results suggest that the helix-loop-helix region is flexible, it is part of the communication pathway between the ATP- and actin-binding sites of myosin and it is proximal to the region of myosin where DNP binds.

Regulation of Myosin Motility by D-Loop of Actin

Subdomain 2 of actin, which contains the DNase I binding loop (D-loop, residues 38-52), slightly changes its conformation during actin polymerization and interacts with the C-terminus of the adjacent subunit in actin filament. This region is suggested to be important for actin-myosin interaction: it was found that binding of myosin II induces conformational changes in subdomain 2 and the proteolytic digestion of the D-loop disturbs the motor function of myosin II. However, although as many as 24 classes of myosin have already been found and their in vivo roles are completely different, the contribution of the D-loop to actin-myosin interaction has so far been studied only for myosin II.In this study, to determine whether the D-loop contributes to the interaction with myosin V and if so, in what way it affects its motor function, we prepared actins modified in the D-loop and analyzed the effects of modifications on the motile properties of myosins II and V. We found that the D-loop modifications, namely, the proteolytic digestion with subtilisin and the M47A point mutation, significantly decreased the gliding velocity on myosin II-HMM in an in vitro motility assay, due to a weaker generated force. On the other hand, single molecules of myosin V walked with the same velocity on both the wild-type and modified actins; however, the run lengths decreased sharply, correlating with a lower affinity of myosin for actin due to the D-loop modifications.These results show that the D-loop strongly modulates the force generation by myosin II and the processivity of myosin V, presumably affecting actin-myosin interaction in the A.M.ADP.Pi state of both myosins. Our findings are important to understand the principles how an actin molecule may regulate diverse in vivo functions of various myosin isoforms.

D-loop of Actin Differently Regulates the Motor Function of Myosins II and V

To gain more information on the manner of actin-myosin interaction, we examined how the motile properties of myosins II and V are affected by the modifications of the DNase I binding loop (D-loop) of actin, performed in two different ways, namely, the proteolytic digestion with subtilisin and the M47A point mutation. In an in vitro motility assay, both modifications significantly decreased the gliding velocity on myosin II-heavy meromyosin due to a weaker generated force but increased it on myosin V. On the other hand, single molecules of myosin V "walked" with the same velocity on both the wild-type and modified actins; however, the run lengths decreased sharply, correlating with a lower affinity of myosin for actin due to the D-loop modifications. The difference between the single-molecule and the ensemble measurements with myosin V indicates that in an in vitro motility assay the non-coordinated multiple myosin V molecules impose internal friction on each other via binding to the same actin filament, which is reduced by the weaker binding to the modified actins. These results show that the D-loop strongly modulates the force generation by myosin II and the processivity of myosin V, presumably affecting actin-myosin interaction in the actomyosin-ADP.P(i) state of both myosins.

Myosin is reversibly inhibited by S-nitrosylation

Nitric oxide (NO*) is synthesized in skeletal muscle and its production increases during contractile activity. Although myosin is the most abundant protein in muscle, it is not known whether myosin is a target of NO* or NO* derivatives. In the present study, we have shown that exercise increases protein S-nitrosylation in muscle, and, among contractile proteins, myosin is the principal target of exogenous SNOs (S-nitrosothiols) in both skinned skeletal muscle fibres and differentiated myotubes. The reaction of isolated myosin with S-nitrosoglutathione results in S-nitrosylation at multiple cysteine thiols and produces two populations of protein-bound SNOs with different stabilities. The less-stable population inhibits the physiological ATPase activity, without affecting the affinity of myosin for actin. However, myosin is neither inhibited nor S-nitrosylated by the NO* donor diethylamine NONOate, indicating a requirement for transnitrosylation between low-mass SNO and myosin cysteine thiols rather than a direct reaction of myosin with NO* or its auto-oxidation products. Interestingly, alkylation of the most reactive thiols of myosin by N-ethylmaleimide does not inhibit formation of a stable population of protein-SNOs, suggesting that these sites are located in less accessible regions of the protein than those that affect activity. The present study reveals a new link between exercise and S-nitrosylation of skeletal muscle contractile proteins that may be important under (patho)physiological conditions.

Actomyosin Interaction: Mechanical and Energetic Properties in Different Nucleotide Binding States

The mechanics of the actomyosin interaction is central in muscle contraction and intracellular trafficking. A better understanding of the events occurring in the actomyosin complex requires the examination of all nucleotide-dependent states and of the energetic features associated with the dynamics of the cross-bridge cycle. The aim of the present study is to estimate the interaction strength between myosin in nucleotide-free, ATP, ADP·Pi and ADP states and actin monomer. The molecular models of the complexes were constructed based on cryo-electron microscopy maps and the interaction properties were estimated by means of a molecular dynamics approach, which simulate the unbinding of the complex applying a virtual spring to the core of myosin protein. Our results suggest that during an ATP hydrolysis cycle the affinity of myosin for actin is modulated by the presence and nature of the nucleotide in the active site of the myosin motor domain. When performing unbinding simulations with a pulling rate of 0.001 nm/ps, the maximum pulling force applied to the myosin during the experiment is about 1nN. Under these conditions the interaction force between myosin and actin monomer decreases from 0.83 nN in the nucleotide-free state to 0.27 nN in the ATP state, and increases to 0.60 nN after ATP hydrolysis and Pi release from the complex (ADP state).

Myosin essential light chain in health and disease

The essential light chain of myosin (ELC) is known to be important for structural stability of the alpha-helical lever arm domain of the myosin head, but its function in striated muscle contraction is poorly understood. Two ELC isoforms are expressed in fast skeletal muscle, a long isoform and its NH(2)-terminal approximately 40 amino acid shorter counterpart, whereas only the long ELC is observed in the heart. Biochemical and structural studies revealed that the NH(2)-terminus of the long ELC can make direct contacts with actin, but the effects of the ELC on the affinity of myosin for actin, ATPase, force, and the kinetics of force generating myosin cross-bridges are inconclusive. Myosin containing the long ELC has been shown to have slower cross-bridge kinetics than myosin with the short isoform. A difference was also reported among myosins with long isoforms. Increased shortening velocity was observed in atrial compared with ventricular muscle fibers. The common findings suggest that ELC provides the fine tuning of the myosin motor function, which is regulated in an isoform and tissue-dependent manner. The functional importance of the ELC is further implicated by the discovery of ELC mutations associated with Familial Hypertrophic Cardiomyopathy. The pathological phenotypes vary in severity, but more notably, almost all ELC mutations result in sudden cardiac death at a young age. This review summarizes the functional roles of striated muscle ELC in normal healthy muscle and in disease. Transgenic animal models and phenotypic characterization of ELC-mediated remodeling of the heart are also discussed.

Processivity of Chimeric Class V Myosins

Unconventional myosin V takes many 36-nm steps along an actin filament before it dissociates, thus ensuring its ability to move cargo intracellularly over long distances. In the present study we assessed the structural features that affect processive run length by analyzing the properties of chimeras of mouse myosin V and a non-processive class V myosin from yeast (Myo4p) (Reck-Peterson, S. L., Tyska, M. J., Novick, P. J., and Mooseker, M. S. (2001) J. Cell Biol. 153, 1121-1126). Surprisingly a chimera containing the yeast motor domain on the neck and rod of mouse myosin V (Y-MD) showed longer run lengths than mouse wild type at low salt. Run lengths of mouse myosin V showed little salt dependence, whereas those of Y-MD decreased steeply with ionic strength, similar to a chimera containing yeast loop 2 in the mouse myosin V backbone. Loop 2 binds to acidic patches on actin in the weak binding states of the cycle (Volkmann, N., Liu, H., Hazelwood, L., Krementsova, E. B., Lowey, S., Trybus, K. M., and Hanein, D. (2005) Mol. Cell 19, 595-605). Constructs containing yeast loop 2, which has no net charge compared with +6 for wild type, showed a higher K(m) for actin in steady-state ATPase assays. The results imply that a positively charged loop 2 and a high affinity for actin are important to maintain processivity near physiologic ionic strength.

Modeling the structure of the tightly bound actin-myosin complex by molecular mechanics

The structure of the tightly bound complex of the globular myosin head with F-actin is the key to understanding important details of the mechanism of how the actin-myosin motor functions. The current notion on this complex is based on the docking of known atomic structures of constituent proteins into low-resolution electron-density maps. The atomic structure of the complex was refined by the molecular mechanics method, which consists in minimizing the energy of molecular interaction and which makes it possible to optimize not only the relative position of protein backbones as rigid bodies, but also the position of side chains on the protein interface. The structure calculated using ICM-Pro software, on the one hand, is close to the model obtained using electron microscopy; on the other hand, it ensures the best calculated interaction energy and accounts for the results of mutagenesis experiments. On the basis of the structure obtained, we can suggest the molecular mechanisms underlying the actin-activated release of ATP hydrolysis products from myosin and the decrease in the affinity of myosin for actin upon binding of nucleotides.

Loop 1 of Transducer Region in Mammalian Class I Myosin, Myo1b, Modulates Actin Affinity, ATPase Activity, and Nucleotide Access

Loop 1, a flexible surface loop in the myosin motor domain, comprises in part the transducer region that lies near the nucleotide-binding site and is proposed from structural studies to be responsible for the kinetic tuning of product release following ATP hydrolysis (1). Biochemical studies have shown that loop 1 affects the affinity of actin-myosin-II for ADP, motility and the V(max) of the actin-activated Mg2+-ATPase activity, possibly through P(i) release (2-8). To test the influence of loop 1 on the mammalian class I myosin, Myo1b, chimeric molecules in which (i) loop 1 of a truncated form of Myo1b, Myo1b1IQ, was replaced with either loop 1 from other myosins; (ii) loop 1 was replaced with glycine; or (iii) some amino acids in the loop were substituted with alanine and were expressed in baculovirus, and their interactions with actin and nucleotide were evaluated. The steady-state actin-activated ATPase activity; rate of ATP-induced dissociation of actin from Myo1b1IQ; rate of ADP release from actin-Myo1b1IQ; and the affinity of actin for Myo1b1IQ and Myo1b1IQ.ADP differed in the chimeras versus wild type, indicating that loop 1 has a much wider range of effects on the coupling between actin and nucleotide binding events than previously thought. In particular, the biphasic ATP-induced dissociation of actin from actin-Myo1b1IQ was significantly altered in the chimeras. This provided evidence that loop 1 contributes to the accessibility of the nucleotide pocket and is involved in the integration of information from the actin-, nucleotide-, gamma-P(i)-, and calmodulin-binding sites and predicts that loop 1 modulates the load dependence of the motor.

Mechanism of regulation of Acanthamoeba myosin-IC by heavy-chain phosphorylation.

The ATPase activity of myosin-Is from lower eukaryotes is activated by phosphorylation by the p21-activated kinase family at the TEDS site on an actin-binding surface-loop. This actin-binding loop is the site of a cardiac myosin-II mutation responsible for some forms of familial hypertrophic cardiomyopathy. To determine the mechanism of myosin-I regulation by heavy-chain phosphorylation (HCP) and to better understand the importance of this loop in the function of all myosin isoforms, we performed a kinetic investigation of the regulatory mechanism of the Acanthamoeba myosin-IC motor domain (MIC(IQ)). Phosphorylated and dephosphorylated MIC(IQ) show actin-activated ATPase activity; however, HCP increases the ATPase activity >20-fold. HCP does not greatly affect the rate of phosphate release from MIC in the absence of actin, as determined by single turnover experiments. Additionally, HCP does not significantly affect the affinity of myosin for actin in the absence or presence of ATP, the rate of ATP-induced dissociation of actoMIC(IQ), the affinity of ADP, or the rate of ADP release. Sequential-mix single-turnover experiments show that HCP regulates the rate of phosphate release from actin-bound MIC(IQ). We propose that the TEDS-containing actin-binding loop plays a direct role in regulating phosphate release and the force-generating (A-to-R) transition of myosin-IC.

Actin-induced Closure of the Actin-binding Cleft of Smooth Muscle Myosin

The putative actin-binding interface of myosin is separated by a large cleft that extends into the base of the nucleotide binding pocket, suggesting that it may be important for mediating the nucleotide-dependent changes in the affinity for myosin on actin. We have genetically engineered a truncated version of smooth muscle myosin containing the motor domain and the essential light chain-binding region (MDE), with a single tryptophan residue at position 425 (F425W-MDE) in the actin-binding cleft. Steady-state fluorescence of F425W-MDE demonstrates that Trp-425 is in a more solvent-exposed conformation in the presence of MgATP than in the presence of MgADP or absence of nucleotide, consistent with closure of the actin-binding cleft in the strongly bound states of MgATPase cycle for myosin. Transient kinetic experiments demonstrate a direct correlation between the rates of strong actin binding and the conformation of Trp-425 in the actin-binding cleft, and suggest the existence of a novel conformation of myosin not previously seen in solution or by x-ray crystallography. Thus, these results directly demonstrate that: 1) the conformation of the actin-binding cleft mediates the affinity of myosin for actin in a nucleotide-dependent manner, and 2) actin induces conformational changes in myosin required to generate force and motion during muscle contraction.

X-ray Structures of the Apo and MgATP-bound States ofDictyostelium discoideum Myosin Motor Domain

Myosin is the most comprehensively studied molecular motor that converts energy from the hydrolysis of MgATP into directed movement. Its motile cycle consists of a sequential series of interactions between myosin, actin, MgATP, and the products of hydrolysis, where the affinity of myosin for actin is modulated by the nature of the nucleotide bound in the active site. The first step in the contractile cycle occurs when ATP binds to actomyosin and releases myosin from the complex. We report here the structure of the motor domain of Dictyostelium discoideum myosin II both in its nucleotide-free state and complexed with MgATP. The structure with MgATP was obtained by soaking the crystals in substrate. These structures reveal that both the apo form and the MgATP complex are very similar to those previously seen with MgATPgammaS and MgAMP-PNP. Moreover, these structures are similar to that of chicken skeletal myosin subfragment-1. The crystallized protein is enzymatically active in solution, indicating that the conformation of myosin observed in chicken skeletal myosin subfragment-1 is unable to hydrolyze ATP and most likely represents the pre-hydrolysis structure for the myosin head that occurs after release from actin.

Altered crossbridge kinetics in the alphaMHC403/+ mouse model of familial hypertrophic cardiomyopathy.

A mutation in the cardiac beta-myosin heavy chain, Arg403Gln (R403Q), causes a severe form of familial hypertrophic cardiomyopathy (FHC) in humans. We used small-amplitude (0.25%) length-perturbation analysis to examine the mechanical properties of skinned left ventricular papillary muscle strips from mouse hearts bearing the R403Q mutation in the alpha-myosin heavy chain (alphaMHC403/+). Myofibrillar disarray with variable penetrance occurred in the left ventricular free wall of the alphaMHC403/+ hearts. In resting strips (pCa 8), dynamic stiffness was approximately 40% greater than in wild-type strips, consistent with elevated diastolic stiffness reported for murine hearts with FHC. At pCa 6 (submaximal activation), strip isometric tension was approximately 3 times higher than for wild-type strips, whereas at pCa 5 (maximal activation), tension was marginally lower. At submaximal calcium activation the characteristic frequencies of the work-producing (b) and work-absorbing (c) steps of the crossbridge were less in alphaMHC403/+ strips than in wild-type strips (b=11+/-1 versus 15+/-1 Hz; c= 58+/-3 versus 66+/-3 Hz; 27 degrees C). At maximal calcium activation, strip oscillatory power was reduced (0. 53+/-0.25 versus 1.03+/-0.18 mW/mm3; 27 degrees C), which is partly attributable to the reduced frequency b, at which crossbridge work is maximum. The results are consistent with the hypothesis that the R403Q mutation reduces the strong binding affinity of myosin for actin. Myosin heads may accumulate in a preforce state that promotes cooperative activation of the thin filament at submaximal calcium but blunts maximal tension and oscillatory power output at maximal calcium. The calcium-dependent effect of the mutation (whether facilitating or debilitating), together with a variable degree of fibrosis and myofibrillar disorder, may contribute to the diversity of clinical symptoms observed in murine FHC.

The sequence of the myosin 50-20K loop affects Myosin's affinity for actin throughout the actin-myosin ATPase cycle and its maximum ATPase activity.

We are interested in the role that solvent-exposed, proteolytically sensitive surface loops play in myosin function. The 25-50K loop, or loop 1, is near the ATP binding site, while the 50-20K loop (loop 2) is in the actin binding site. Through chimeric studies, we have found that loop 1 affects ADP release [Murphy, C. T., and Spudich, J. A. (1998) Biochemistry 37, 6738-44], while loop 2 affects the actin-activated ATPase activity [Uyeda, T. Q.-P., et al. (1994) Nature 368, 567-9]. In the study described here, we have found that the kcat of the actin-activated ATPase activity is changed by the loop 2 substitutions in a manner that reflects the relative actin-activated ATPase activities of the donor myosins. Additionally, changes in loop 2 affect the affinity of myosin for actin both in the presence and in the absence of nucleotides. Pre-steady-state studies together with the ATPase and affinity data suggest that while loop 2 does not affect interactions between myosin and nucleotide, it plays a role in determining the affinity of myosin for actin in various nucleotide states and in the rate-limiting transition allowing phosphate release.

Xestoquinone activates skeletal muscle actomyosin ATPase by modification of the specific sulfhydryl group in the myosin head probably distinct from sulfhydryl groups SH1 and SH2.

Xestoquinone isolated from a sea sponge Xestospongia sapra inhibited both Ca2+ and K(+)-(EDTA) ATPase of skeletal muscle myosin. The inhibition was abolished in the presence of dithiothreitol. Xestoquinone reacted with 2-mercaptoethanol, a sulfhydryl (SH) compound. Unlike N-ethylmaleimide, a well-known SH reagent, modification of 2 mol of SH groups per myosin by xestoquinone caused a marked increase in the actomyosin ATPase activity. Kinetical analysis of stimulatory effects of xestoquinone indicates a decrease in the actin concentrations which gives half of the maximum velocity (Vmax) of actomyosin ATPase reaction without affecting the Vmax, suggesting an increase in the affinity of myosin for actin. N-Ethylmaleimide can still modify both the SH1 and SH2 groups after modification of 2 mol of SH groups by xestoquinone. Xestoquinone modified myosin SH groups which caused changes in the tryptophan fluorescence intensity and circular dichroism. These results suggest that xestoquinone modifies the specific SH groups in myosin distinct from SH1 and SH2, resulting in activation of actomyosin ATPase. It is also suggested that xestoquinone strengthens the interaction between actin and myosin through conformational change in the myosin molecule.