Rabbit Skeletal Muscle Myosin Research Articles

Synthesis of C-ring-modified blebbistatin derivatives and evaluation of their myosin II ATPase inhibitory potency

(S)-Blebbistatin is a micromolar myosin II ATPase inhibitor that is extensively used in research. In search of analogs with improved potency, we have synthesized for the first time C-ring modified analogs. We introduced hydroxymethyl or allyloxymethyl functionalities in search of additional favorable interactions and a more optimal filling of the binding pocket. Unfortunately, the resulting compounds did not significantly inhibit the ATPase activity of rabbit skeletal-muscle myosin II. This and earlier reports suggest that rational design of potent myosin II inhibitors based on the architecture of the blebbistatin binding pocket is an ineffective strategy.

The structure of the actin-smooth muscle myosin motor domain complex in the rigor state.

Myosin-based motility utilizes catalysis of ATP to drive the relative sliding of F-actin and myosin. The earliest detailed model based on cryo-electron microscopy (cryoEM) and X-ray crystallography postulated that higher actin affinity and lever arm movement were coupled to closure of a feature of the myosin head dubbed the actin-binding cleft. Several studies since then using crystallography of myosin-V and cryoEM structures of F-actin bound myosin-I, -II and -V have provided details of this model. The smooth muscle myosin II interaction with F-actin may differ from those for striated and non-muscle myosin II due in part to different lengths of important surface loops. Here we report a ∼6 Å resolution reconstruction of F-actin decorated with the nucleotide-free recombinant smooth muscle myosin-II motor domain (MD) from images recorded using a direct electron detector. Resolution is highest for F-actin and the actin-myosin interface (3.5-4 Å) and lowest (∼6-7 Å) for those parts of the MD at the highest radius. Atomic models built into the F-actin density are quite comparable to those previously reported for rabbit muscle actin and show density from the bound ADP. The atomic model of the MD, is quite similar to a recently published structure of vertebrate non-muscle myosin II bound to F-actin and a crystal structure of nucleotide free myosin-V. Larger differences are observed when compared to the cryoEM structure of F-actin decorated with rabbit skeletal muscle myosin subfragment 1. The differences suggest less closure of the 50 kDa domain in the actin bound skeletal muscle myosin structure.

Nanothermometry Measure of Muscle Efficiency.

Despite recent advances in thermometry, determination of temperature at the nanometer scale in single molecules to live cells remains a challenge that holds great promise in disease detection among others. In the present study, we use a new approach to nanometer scale thermometry with a spatial and thermal resolution of 80 nm and 1 mK respectively, by directly associating 2 nm cadmium telluride quantum dots (CdTe QDs) to the subject under study. The 2 nm CdTe QDs physically adhered to bovine cardiac and rabbit skeletal muscle myosin, enabling the determination of heat released when ATP is hydrolyzed by both myosin motors. Greater heat loss reflects less work performed by the motor, hence decreased efficiency. Surprisingly, we found rabbit skeletal myosin to be more efficient than bovine cardiac. We have further extended this approach to demonstrate the gain in efficiency of Drosophila melanogaster skeletal muscle overexpressing the PGC-1α homologue spargel, a known mediator of improved exercise performance in humans. Our results establish a novel approach to determine muscle efficiency with promise for early diagnosis and treatment of various metabolic disorders including cancer.

A highly soluble, non-phototoxic, non-fluorescent blebbistatin derivative.

Blebbistatin is a commonly used molecular tool for the specific inhibition of various myosin II isoforms both in vitro and in vivo. Despite its popularity, the use of blebbistatin is hindered by its poor water-solubility (below 10 micromolar in aqueous buffer) and blue-light sensitivity, resulting in the photoconversion of the molecule, causing severe cellular phototoxicity in addition to its cytotoxicity. Furthermore, blebbistatin forms insoluble aggregates in water-based media above 10 micromolar with extremely high fluorescence and also high adherence to different types of surfaces, which biases its experimental usage. Here, we report a highly soluble (440 micromolar in aqueous buffer), non-fluorescent and photostable C15 amino-substituted derivative of blebbistatin, called para-aminoblebbistatin. Importantly, it is neither photo- nor cytotoxic, as demonstrated on HeLa cells and zebrafish embryos. Additionally, para-aminoblebbistatin bears similar myosin II inhibitory properties to blebbistatin or para-nitroblebbistatin (not to be confused with the C7 substituted nitroblebbistatin), tested on rabbit skeletal muscle myosin S1 and on M2 and HeLa cells. Due to its drastically improved solubility and photochemical feature, as well as lack of photo- or cytotoxicity, para-aminoblebbistatin may become a feasible replacement for blebbistatin, especially at applications when high concentrations of the inhibitor or blue light irradiation is required.

Exome Genotyping Links Venous Thrombosis Risk with the Skeletal Muscle Myosin Gene Cluster and Leads to Discovery of New Family of Procoagulant Factors

Venous thromboembolism (VTE) exacts a severe toll, and its toll persists even after the acute thrombotic event is resolved. Although several low frequency, racially-specific, genetic variants have been linked to VTE (e.g., factor V Leiden, prothrombin nt20210A, protein S Tokushima, Protein C R147W and del-Lys150), these polymorphisms fail to explain the majority of VTE risk. These variants are causally linked to increased VTE risk and, based on their racial-specific occurrence, they likely provided evolutionary benefits. Thus, to discover new rare missense functional variants linked to VTE, we used the new Affymetrix Axiom array to determine ~ 300,000 mostly exomic rare variant polymorphisms in the Scripps venous thrombosis registry (N=214 controls and 107 VTE cases) and gained multiple novel insights. When recurrent VTE cases were analyzed, 34 SNPs, including two F5 SNPs (rs6025 and rs6687813), were significant (p<0.05) after FDR correction. Analysis of clustering for these SNPs revealed associations of VTE recurrence risk with skeletal muscle myosin (MYH) rare SNPs (chr 17p13.1) in a highly conserved region with clustered myosin heavy chain genes (MYH1, 2, 4, 8). Among 11 rare MYH SNPs, rs111567318 was significantly linked to VTE (p=8.55x10-6, FDR p<0.05). Notably, 16 of 107 (15. %) VTE subjects had ≥ one SNP in the MYH chr17 cluster compared to only 1 of 212 (0.5 %) controls.Studies were initiated to define procoagulant or anticoagulant properties of skeletal muscle myosins which heretofore had no known function in blood clotting and which are broadly found in the body, even in plasma (e.g., at 20 nM). Remarkably, purified rabbit skeletal muscle myosin enhanced TF-induced thrombin generation in plasma. In neat plasma, anti-myosin antibodies reduced TF-induced thrombin generation, suggesting the contribution of endogenous plasma myosin to plasma thrombin generation. Skeletal muscle myosin also enhanced recalcification-induced thrombin generation in plasma. Studies to tests if prothrombinase activity is promoted by skeletal muscle myosin in purified prothrombinase (IIase) assays (factor Xa, factor Va, and II plus Ca++, with or without phospholipid added) showed that skeletal muscle myosin greatly enhanced IIase activity. When factor (F) Va was absent, myosin did not detectably enhance IIase activity, showing FVa was required. The myosin heavy chain can be split into 1 light meromyosin (LMM) and 1 heavy meromyosin (HMM) domains which can be further split into 2 globular subfragments (S1) and 1 rod-shaped subfragment (S2). HMM enhanced IIase activity whereas the myosin S1 subfragment did not, suggesting the S2 subfragment and LMM were required for the procoagulant activity. When prothrombin cleavage kinetics in the presence of skeletal muscle myosin were studied using PAGE, negligible amounts of meizothrombin were observed while fragment 1.2 and prothrombin 2 (or a-thrombin) bands were observed. This implies that skeletal muscle myosin promotes the prothrombin cleavage pathway similar to that enhanced by platelets, but not similar to that enhanced by phospholipid vesicles. The IIase activity of plasma-derived, purified extra cellular vesicles (ECVs) was markedly inhibited (> 60 %) by anti-skeletal muscle myosin antibodies, suggesting that a major fraction of procoagulant extracellular vesicle procoagulant activity involves skeletal muscle myosin. In purified systems, the apparent Kd of skeletal myosin in the presence of FVa for FXa was 9 nM.In summary, we found that MYH SNPs may be linked to VTE risk which led to the discovery of a new family of procoagulant proteins, skeletal myosins, that bind FXa, stimulate thrombin generation in plasma and in purified systems, thus paving the way for future genetic and functional VTE research. DisclosuresNo relevant conflicts of interest to declare.

X-Ray Diffraction from Insect Flight Muscle Fibers with Exchanged Contractile Proteins

By recording fast X-ray diffraction movies from live bumblebee, we have shown that specific X-ray signals (the reciprocal intensity changes of the 111 and 201 reflections) arise in the stretch phase of the flight muscle (IFM), and from this observation and model calculations we have suggested that the stretch-induced distortion of actin-bound low-force myosin head is the trigger for stretch activation (SA). However, there are still some unsolved questions: (1) does IFM myosin have special sensitivity to stretch? (2) Is it the very regular spatial arrangement of proteins in IFM sarcomere that causes SA regardless of the source of myosin? (3) Does the IFM-specific troponin keep the myosin from generating force prematurely? To answer these questions we have started experiments to exchange contractile proteins within IFM sarcomere with those from other sources. It is shown that, when IFM fibers from crane fly are incubated in a solution containing fluorescent rabbit skeletal muscle myosin, it is incorporated into IFM sarcomere. This technique utilizes the properties of myosins that have different extractability in KCl solutions. The IFM fibers with rabbit myosin showed a series of strong X-ray meridional reflections with a basic repeat of 14.5 nm. On the other hand, the control IFM fibers incubated in the same KCl solution without rabbit myosin seem to retain their endogenous myosin molecules. These results suggest that, even at KCl concentrations too low to extract endogenous myosin, myosin exchange occurs if exogenous solubilized myosin is present in the surrounding medium. We also plan to extract the thin filaments in IFM and to replace them with actin filaments from other sources.

Sea Lily Muscle Lacks a Troponin-Regulatory System, While it Contains Paramyosin

Troponin, a Ca(2+)-dependent regulator of striated muscle contraction, has been characterized in vertebrates, protochordates (amphioxus and ascidian), and many invertebrate animals that are categorized in protostomes, but it has not been detected in echinoderms, such as sea urchin and sea cucumber, members of subphylum Eleutherozoa. In this study, we examined the muscle of a species of isocrinid sea lilies, a member of subphylum Pelmatozoa, that constitute the most basal group of extant echinoderms to clarify whether troponin is lacking from the early evolution of echinoderms. Native thin filaments were released from the muscle homogenates in a relaxing buffer containing ATP and EGTA, a Ca(2+)-chelator, and were collected by ultra-centrifugation. Actin and tropomyosin, but not a troponin-like protein, were detected in the filament preparation. The filaments increased Mg(2+)-ATPase activity of rabbit skeletal muscle myosin irrespective of the presence or absence of Ca(2+). The results indicate that Ca(2+)-sensitive factor, troponin, is lacking in the thin filaments of sea lily muscle as in those of the other echinoderms, sea urchin and sea cucumber. On the other hand, a paramyosin-like protein that is absent from chordates was detected in sea lily muscle as in the muscles of the other echinoderms and invertebrate animals of protostomes.

Modulation of the Cardiac Sarcomere by a Small Molecule Agent MYK0000461: A Potential Therapeutic for the Treatment of Genetic Hypertrophic Cardiomyopathies

We have identified a small molecule inhibitor, MY461, of the cardiac myosin ATPase. This agent was characterized in steady state and transient kinetic assays to understand its mechanism of action. MY461 decreases the steady-state rate of the ATPase activity of purified bovine β-cardiac myosin subfragment-1 (S1) as well as that of bovine cardiac myofibrils, wild type and the mutant R453C of recombinant human β-cardiac myosin S1. We also find that MY461 inhibits cardiac myosin selectively as compared to systems containing rabbit skeletal or chicken smooth muscle myosins Analysis of the individual steps of the chemo-mechanical cycle of cardiac myosin suggests that MY461 exerts its effect by inhibiting the actin-stimulated release of phosphate, presumably by stabilizing the detached state of cardiac myosin prior to the release of phosphate. We find no evidence to suggest that MYK000461 inhibits cardiac myosin in a strongly bound state and no other steps in the chemo-mechanical cycle are affected by MY461. Thus, the enzymatic step governing the weak to strong transition of S1 binding to actin is inhibited without affecting the release from the strongly bound states. This decrease in the rate of transition from the weak to strongly bound state should decrease force production and may underlie its ability to decrease cardiac contractility in cellular and in vivo models of cardiac function. An agent such as MY461 could potentially be used to treat cardiac disorders that stem from hyper contractility such as the genetic hypertrophic cardiomyopathies (HCM). By decreasing the net force of contraction and restoring it back to normal level could potentially be useful in treating patients that suffer from this disease.

Nonlinear Cross-Bridge Elasticity and Post-Power-Stroke Events in Fast Skeletal Muscle Actomyosin

Generation of force and movement by actomyosin cross-bridges is the molecular basis of muscle contraction, but generally accepted ideas about cross-bridge properties have recently been questioned. Of the utmost significance, evidence for nonlinear cross-bridge elasticity has been presented. We here investigate how this and other newly discovered or postulated phenomena would modify cross-bridge operation, with focus on post-power-stroke events. First, as an experimental basis, we present evidence for a hyperbolic [MgATP]-velocity relationship of heavy-meromyosin-propelled actin filaments in the in vitro motility assay using fast rabbit skeletal muscle myosin (28–29°C). As the hyperbolic [MgATP]-velocity relationship was not consistent with interhead cooperativity, we developed a cross-bridge model with independent myosin heads and strain-dependent interstate transition rates. The model, implemented with inclusion of MgATP-independent detachment from the rigor state, as suggested by previous single-molecule mechanics experiments, accounts well for the [MgATP]-velocity relationship if nonlinear cross-bridge elasticity is assumed, but not if linear cross-bridge elasticity is assumed. In addition, a better fit is obtained with load-independent than with load-dependent MgATP-induced detachment rate. We discuss our results in relation to previous data showing a nonhyperbolic [MgATP]-velocity relationship when actin filaments are propelled by myosin subfragment 1 or full-length myosin. We also consider the implications of our results for characterization of the cross-bridge elasticity in the filament lattice of muscle.

Structural Dynamics of Actin during Active Interaction with Myosin depends on the Isoform of the Essential Light Chain

Muscle contraction results from cyclic interaction of myosin and actin, driven by ATP hydrolysis, where the actomyosin complex undergoes transitions between weakly and strongly bound structural states. Rabbit skeletal muscle myosin has two isoforms of the essential light chain (ELC), designated A1 and A2. These isoforms have significant effects on myosin's interaction with actin, resulting in higher catalytic efficiency for A1 but higher in vitro motility and muscle shortening velocity for A2. In order to understand the structural basis of these differences, we have used time-resolved phosphorescence anisotropy (TPA) to investigate the effects of ELC isoforms on the microsecond dynamics of actin during interaction with skeletal muscle myosin in the absence and presence of saturating ATP. Actin was labeled with a phosphorescent probe at C374, and the myosin head (S1) was separated into isoenzymes containing A1 (S1A1) and A2 (S1A2) by ion-exchange chromatography. As previously reported, S1A1 exhibited substantially lower ATPase activity at saturating actin but substantially higher apparent actin affinity, resulting in higher catalytic efficiency. In the absence of ATP, each isoenzyme increased actin's final anisotropy similarly, indicating similar restriction of the amplitude of intrafilament rotational motions in the strong-binding (rigor) state of actomyosin. In contrast, in the presence of saturating ATP, the final anisotropy of actin was substantially higher during interaction with S1A1 than with S1A2. Thus, S1A1 was more effective in restricting actin dynamics during the active interaction of actin and myosin, suggesting greater population of the strong-binding state of actomyosin. We conclude that ELC of skeletal muscle myosin modulates strong-to-weak structural transitions during the actomyosin ATPase cycle in an isoform-dependent manner, and thus modulates the contractile function of actomyosin.

Structural Transitions within the Actomyosin Complex in the Presence of Saturating ATP, Detected by Time-Resolved FRET

Myosin generates force by cycling between actin-attached (strong binding) and actin-detached (weak binding) states during the actomyosin ATPase cycle. However, it remains unclear what specific conformational changes in myosin take place upon binding to actin, and how these structural changes lead to product release and force. To characterize these changes, we have used site-directed FRET to determine distance between specific sites in actin and myosin subfragment 1 (S1) in the presence and absence of ATP. Rabbit skeletal muscle myosin has two essential light chain (ELC) isoforms, A1 and A2.The ELC isoform has significant effects on the interaction of S1 with actin, resulting in higher catalytic efficiency for the S1A1-isoenzyme but higher in vitro motility and muscle shortening velocity for S1A2.We have focused on S1A1, since it binds to actin in the presence of ATP with higher affinity. Actin was labeled at C374 with a donor, AlexaFluor 568. A1 light chain, was expressed,purified and labeled at C177 with an acceptor, AlexaFluor 647. The labeled A1 was exchanged into myosin S1, and S1A1 was isolated using a Talon affinity resin. The efficiency of energy transfer between C374 of actin and C177 of A1 was measured by time-resolved fluorescence, allowing us to detect directly the FRET within the actin-S1A1 complex, even in the presence of saturating ATP. Addition of ATP to the strongly bound complex resulted in a significant decrease in FRET within the weakly bound complex, corresponding to a significant increase in the donor-acceptor distance. We conclude that the transition between strongly and weakly bound states of acto-S1 is associated with a structural transition in the acto-S1 complex that affects the distance from actin to the light-chain domain of myosin.

Nuclear Calcium Signaling in Spinal Neurons Drives a Genomic Program Required for Persistent Inflammatory Pain

Persistent pain induced by noxious stimuli is characterized by the transition from normosensitivity to hypersensitivity. Underlying mechanisms are not well understood, although gene expression is considered important. Here, we show that persistent nociceptive-like activity triggers calcium transients in neuronal nuclei within the superficial spinal dorsal horn, and that nuclear calcium is necessary for the development of long-term inflammatory hypersensitivity. Using a nucleus-specific calcium signal perturbation strategy in vivo complemented by gene profiling, bioinformatics, and functional analyses, we discovered a pain-associated, nuclear calcium-regulated gene program in spinal excitatory neurons. This includes C1q, a modulator of synaptic spine morphogenesis, which we found to contribute to activity-dependent spine remodelling on spinal neurons in a manner functionally associated with inflammatory hypersensitivity. Thus, nuclear calcium integrates synapse-to-nucleus communication following noxious stimulation and controls a spinal genomic response that mediates the transition between acute and long-term nociceptive sensitization by modulating functional and structural plasticity.

Instability of the S1/S2 Hinge is not Affected by the Myosin Filament

The S1/S2 hinge of myosin is thought to be unstable in some species of myosin such as myosin VI and certain scallop muscle myosin. To examine the stability of the S1/S2 hinge of rabbit skeletal muscle myosin, gravitational force spectrometry was used to measure the separation of myosin heads bound in rigor to actin immobilized on glass microspheres. The molecular lengths of this separation were measured at different levels of applied force up to the limit that the rigor bond would sustain of about 10 pN in these experiments. The molecular lengths were found to increase linearly with the applied force up to lengths exceeding 100 nm which requires a partial unraveling of the S2 coiled coil in the single myosin molecule. Free-fall force spectrometry on such single myosin molecules confirmed that the coiled coil could reversibly unravel and refold at forces less than 13 pN. In order to test whether thick filament formation would affect this instability under force, synthetic cofilaments of proteolytically isolated myosin rod and intact myosin were prepared at different ratios of myosin to rod up to ratios where it is expected that only one intact myosin would be present per myosin rod cofilament. The force-distance relationships for myosin in the cofilament were not substantially different from those of single myosin molecules. These data indicate the possibility that vertebrate skeletal muscle myosin may also utilize S1/S2 hinge unraveling to provide flexibility during crossbridge formation so that a productive binding orientation is achieved. The reversibility of the unraveling of the coiled coil might contribute to the mechanism of motility under some circumstances as has been proposed for non-muscle myosin VI.

The molecular basis of frictional loads in the in vitro motility assay with applications to the study of the loaded mechanochemistry of molecular motors.

Molecular motors convert chemical energy into mechanical movement, generating forces necessary to accomplish an array of cellular functions. Since molecular motors generate force, they typically work under loaded conditions where the motor mechanochemistry is altered by the presence of a load. Several biophysical techniques have been developed to study the loaded behavior and force generating capabilities of molecular motors yet most of these techniques require specialized equipment. The frictional loading assay is a modification to the in vitro motility assay that can be performed on a standard epifluorescence microscope, permitting the high-throughput measurement of the loaded mechanochemistry of molecular motors. Here, we describe a model for the molecular basis of the frictional loading assay by modeling the load as a series of either elastic or viscoelastic elements. The model, which calculates the frictional loads imposed by different binding proteins, permits the measurement of isotonic kinetics, force-velocity relationships, and power curves in the motility assay. We show computationally and experimentally that the frictional load imposed by alpha-actinin, the most widely employed actin binding protein in frictional loading experiments, behaves as a viscoelastic rather than purely elastic load. As a test of the model, we examined the frictional loading behavior of rabbit skeletal muscle myosin under normal and fatigue-like conditions using alpha-actinin as a load. We found that, consistent with fiber studies, fatigue-like conditions cause reductions in myosin isometric force, unloaded sliding velocity, maximal power output, and shift the load at which peak power output occurs.

Direct Observation of the Uncapping of Capping Protein-capped Actin Filaments by CARMIL Homology Domain 3

Bulk solution assays have shown that the isolated CARMIL homology 3 (CAH3) domain from mouse and Acanthamoeba CARMIL rapidly and potently restores actin polymerization when added to actin filaments previously capped with capping protein (CP). To demonstrate this putative uncapping activity directly, we used total internal reflection microscopy to observe single, CP-capped actin filaments before and after the addition of the CAH3 domain from mouse CARMIL-1 (mCAH3). The addition of mCAH3 rapidly restored the polymerization of individual capped filaments, consistent with uncapping. To verify uncapping, filaments were capped with recombinant mouse CP tagged with monomeric green fluorescent protein (mGFP-CP). Restoration of polymerization upon the addition of mCAH3 was immediately preceded by the complete dissociation of mGFP-CP from the filament end, confirming the CAH3-driven uncapping mechanism. Quantitative analyses showed that the percentage of capped filaments that uncapped increased as the concentration of mCAH3 was increased, reaching a maximum of approximately 90% at approximately 250 nm mCAH3. Moreover, the time interval between mCAH3 addition and uncapping decreased as the concentration of mCAH3 increased, with the half-time of CP at the barbed end decreasing from approximately 30 min without mCAH3 to approximately 10 s with a saturating amount of mCAH3. Finally, using mCAH3 tagged with mGFP, we obtained direct evidence that the complex of CP and mCAH3 has a small but measurable affinity for the barbed end, as inferred from previous studies and kinetic modeling. We conclude that the isolated CAH3 domain of CARMIL (and presumably the intact molecule as well) possesses the ability to uncap CP-capped actin filaments.

Novel Mode of Cooperative Binding between Myosin and Mg2+-actin Filaments in the Presence of Low Concentrations of ATP

Cooperative interaction between myosin and actin filaments has been detected by a number of different methods, and has been suggested to have some role in force generation by the actomyosin motor. In this study, we observed the binding of myosin to actin filaments directly using fluorescence microscopy to analyze the mechanism of the cooperative interaction in more detail. For this purpose, we prepared fluorescently labeled heavy meromyosin (HMM) of rabbit skeletal muscle myosin and Dictyostelium myosin II. Both types of HMMs formed fluorescent clusters along actin filaments when added at substoichiometric amounts. Quantitative analysis of the fluorescence intensity of the HMM clusters revealed that there are two distinct types of cooperative binding. The stronger form was observed along Ca2+-actin filaments with substoichiometric amounts of bound phalloidin, in which the density of HMM molecules in the clusters was comparable to full decoration. The novel, weaker form was observed along Mg2+-actin filaments with and without stoichiometric amounts of phalloidin. HMM density in the clusters of the weaker form was several-fold lower than full decoration. The weak cooperative binding required sub-micromolar ATP, and did not occur in the absence of nucleotides or in the presence of ADP and ADP-Vi. The G680V mutant of Dictyostelium HMM, which over-occupies the ADP-Pi bound state in the presence of actin filaments and ATP, also formed clusters along Mg2+-actin filaments, suggesting that the weak cooperative binding of HMM to actin filaments occurs or initiates at an intermediate state of the actomyosin-ADP-Pi complex other than that attained by adding ADP-Vi.

Cross-Linked Long-Pitch Actin Dimer Forms Stoichiometric Complexes with Gelsolin Segment 1 and/or Deoxyribonuclease I That Nonproductively Interact with Myosin Subfragment 1

Actin dimer cross-linked along the long pitch of the F-actin helix by N-(4-azido)-2-nitrophenyl (ANP) was purified by gel filtration. Purified dimers were found to polymerize on increasing the ionic strength, although at reduced rate and extent in comparison with native actin. Purified actin dimer interacts with the actin-binding proteins (ABPs) deoxyribonuclease I (DNase I) and gelsolin segment-1 (G1) as analyzed by gel filtration and native gel electrophoresis. Complex formation of the actin dimer with these ABPs inhibits its ability to polymerize. The interaction with rabbit skeletal muscle myosin subfragment 1 (S1) was analyzed for polymerized actin dimer and dimer complexed with gelsolin segment 1 or DNase I by measurement of the actin-stimulated myosin S1-ATPase and gel filtration. The data obtained indicate binding of subfragment 1 to actin dimer, albeit with considerably lower affinity than to F-actin. Polymerized actin dimer was able to stimulate the S1-ATPase activity to about 50% of the level of native F-actin. In contrast, the actin dimer complexed to DNase I or gelsolin segment 1 or to both proteins was unable to significantly stimulate the S1-ATPase. Similarly, G1:dimer complex at 20 microM stimulated the rate of release of subfragment 1 bound nucleotide (mant-ADP) only 1.6-fold in comparison to about 9-fold by native F-actin at a concentration of 0.5 microM. Using rapid kinetic techniques, a dissociation constant of 2.4 x 10 (-6) M for subfragment 1 binding to G1:dimer was determined in comparison to 3 x 10 (-8) M for native F-actin under identical conditions. Since the rate of association of subfragment 1 to G1:dimer was considerably lower than to native F-actin, we suspect that the ATP-hydrolysis by S1 was catalyzed before its association to the dimer. These data suggest an altered, nonproductive mode for the interaction of subfragment 1 with the isolated long-pitch actin dimer.

The Mechanism of the Reverse Recovery Step, Phosphate Release, and Actin Activation of Dictyostelium Myosin II

The rate-limiting step of the myosin basal ATPase (i.e. in absence of actin) is assumed to be a post-hydrolysis swinging of the lever arm (reverse recovery step), that limits the subsequent rapid product release steps. However, direct experimental evidence for this assignment is lacking. To investigate the binding and the release of ADP and phosphate independently from the lever arm motion, two single tryptophan-containing motor domains of Dictyostelium myosin II were used. The single tryptophans of the W129+ and W501+ constructs are located at the entrance of the nucleotide binding pocket and near the lever arm, respectively. Kinetic experiments show that the rate-limiting step in the basal ATPase cycle is indeed the reverse recovery step, which is a slow equilibrium step (k(forward) = 0.05 s(-1), k(reverse) = 0.15 s(-1)) that precedes the phosphate release step. Actin directly activates the reverse recovery step, which becomes practically irreversible in the actin-bound form, triggering the power stroke. Even at low actin concentrations the power stroke occurs in the actin-attached states despite the low actin affinity of myosin in the pre-power stroke conformation.

Mechanism, Regulation, and Functional Properties of Dictyostelium Myosin-1B

Myosin-1B is one of three long tailed class-1 myosins containing an ATP-insensitive actin-binding site in the tail region that are produced in Dictyostelium discoideum. Myosin-1B localizes to actin-rich structures at the leading edge of migrating cells where it has been implicated in the formation and retraction of membrane projections, the recycling of plasma membrane components, and intracellular particle transport. Here, we have used a combination of molecular engineering approaches to describe the kinetic and motile properties of the myosin-1B motor and its regulation by TEDS site phosphorylation. Our results show that myosin-1B is a low duty ratio motor and displays the fastest nucleotide binding kinetics of any of the Dictyostelium class-1 myosins studied so far. Different from Dictyostelium myosin-1D and myosin-1E, dephosphorylated myosin-1B is not inactivated but moves actin filaments efficiently, albeit at an up to 8-fold slower velocity in the in vitro motility assay. A further difference is that myosin-1B lacks the ability to switch between rapid movement and bearing tension upon physiological changes of free Mg2+ ions. In this respect, its motor properties appear to be more closely related to Dictyostelium myosin-2 and rabbit skeletal muscle myosin.

Effect of nucleotide on interaction of the 567–578 segment of myosin heavy chain with actin

To probe the effect of nucleotide on the formation of ionic contacts between actin and the 567–578 residue loop of the heavy chain of rabbit skeletal muscle myosin subfragment 1 (S1), the complexes between F-actin and proteolytic derivatives of S1 were submitted to chemical cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. We have shown that in the absence of nucleotide both 45 kDa and 5 kDa tryptic derivatives of the central 50 kDa heavy chain fragment of S1 can be cross-linked to actin, whereas in the presence of MgADP.AlF 4, only the 5 kDa fragment is involved in cross-linking reaction. By the identification of the N-terminal sequence of the 5-kDa fragment, we have found that trypsin splits the 50 kDa heavy chain fragment between Lys-572 and Gly-573, the residues located within the 567–578 loop. Using S1 preparations cleaved with elastase, we could show that the residue of 567–578 loop that can be cross-linked to actin in the presence of MgADP.AlF 4 is Lys-574. The observed nucleotide-dependent changes of the actin-subfragment 1 interface indicate that the 567–578 residue loop of skeletal muscle myosin participates in the communication between the nucleotide and actin binding sites.