RESUMO
The biochemical SRX (super-relaxed) state of myosin has been defined as a low ATPase activity state. This state can conserve energy when the myosin is not recruited for muscle contraction. The SRX state has been correlated with a structurally defined ordered (versus disordered) state of muscle thick filaments. The two states may be linked via a common interacting head motif (IHM) where the two heads of heavy meromyosin (HMM), or myosin, fold back onto each other and form additional contacts with S2 and the thick filament. Experimental observations of the SRX, IHM, and the ordered form of thick filaments, however, do not always agree, and result in a series of unresolved paradoxes. To address these paradoxes, we have reexamined the biochemical measurements of the SRX state for porcine cardiac HMM. In our hands, the commonly employed mantATP displacement assay was unable to quantify the population of the SRX state with all data fitting very well by a single exponential. We further show that mavacamten inhibits the basal ATPases of both porcine ventricle HMM and S1 (Ki, 0.32 and 1.76 µM respectively) while dATP activates HMM cooperatively without any evidence of an SRX state. A combination of our experimental observations and theories suggests that the displacement of mantATP in purified proteins is not a reliable assay to quantify the SRX population. This means that while the structurally defined IHM and ordered thick filaments clearly exist, great care must be employed when using the mantATP displacement assay.
Assuntos
Trifosfato de Adenosina , Ensaios Enzimáticos , Miosina não Muscular Tipo IIA , Suínos , ortoaminobenzoatos , Animais , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Benzilaminas/farmacologia , Ensaios Enzimáticos/métodos , Ensaios Enzimáticos/normas , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/enzimologia , Ventrículos do Coração/metabolismo , Contração Miocárdica , Subfragmentos de Miosina/química , Subfragmentos de Miosina/metabolismo , Miosina não Muscular Tipo IIA/química , Miosina não Muscular Tipo IIA/metabolismo , ortoaminobenzoatos/metabolismo , Uracila/análogos & derivados , Uracila/farmacologiaRESUMO
BACKGROUND: Associations between actin filaments (AFs) and intermediate filaments (IFs) are frequently observed in living cells. The crosstalk between these cytoskeletal components underpins cellular organization and dynamics; however, the molecular basis of filamentous interactions is not fully understood. Here, we describe the mode of interaction between AFs and desmin IFs (DIFs) in a reconstituted in vitro system. METHODS: AFs (rabbit skeletal muscle) and DIFs (chicken gizzard) were labeled with fluorescent dyes. DIFs were immobilized on a heavy meromyosin (HMM)-coated collodion surface. HMM-driven AFs with ATP hydrolysis was assessed in the presence of DIFs. Images of single filaments were obtained using fluorescence microscopy. Vector changes in the trajectories of single AFs were calculated from microscopy images. RESULTS: AF speed transiently decreased upon contact with DIF. The difference between the incoming and outgoing angles of a moving AF broadened upon contact with a DIF. A smaller incoming angle tended to result in a smaller outgoing angle in a nematic manner. The percentage of moving AFs decreased with an increasing DIF density, but the speed of the moving AFs was similar to that in the no-desmin control. An abundance of DIFs tended to exclude AFs from the HMM-coated surfaces. CONCLUSIONS: DIFs agitate the movement of AFs with the orientation. DIFs can bind to HMMs and weaken actin-myosin interactions. GENERAL SIGNIFICANCE: The study indicates that apart from the binding strength, the accumulation of weak interactions characteristic of filamentous structures may affect the dynamic organization of cell architecture.
Assuntos
Citoesqueleto de Actina , Filamentos Intermediários , Animais , Coelhos , Filamentos Intermediários/metabolismo , Desmina/análise , Desmina/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Miosinas/metabolismo , Subfragmentos de Miosina/análise , Subfragmentos de Miosina/química , Subfragmentos de Miosina/metabolismoRESUMO
BACKGROUND: The interaction of N-terminal extension of the myosin A1 essential light chain (A1 ELC) with actin is receiving increasing attention as a target in utilizing synthetic A1 ELC N-terminal-derived peptides in cardiac dysfunction therapy. METHODS: To elucidate the mechanism by which these peptides regulate actin-myosin interaction, here we have investigated their effects on the myosin subfragment 1 (S1)-induced polymerization of G-actin. RESULTS: The MLCFpep and MLCSpep peptides spanning the 3-12 of A1 ELC sequences from fast and slow skeletal muscle, respectively, increased the rate of actin polymerization not only by S1(A2) but also the rate of S1(A1)-induced actin polymerization, suggesting that they did not interfere with the direct binding of A1 ELC with actin. The efficiency of actin polymerization in the presence of the N-terminal ELC peptides depended on their sequence. Substitution of aspartic acid for neutral asparagine at position 5 of MLCFpep dramatically enhanced its ability to stimulate S1-induced polymerization and enabled it to initiate polymerization of G-actin in the absence of S1. CONCLUSIONS: These and other results presented in this work suggest that the modulation of myosin motor activity by N-terminal ELC peptides is exerted through a change in actin filament conformation rather than through blocking the A1 ELC-actin interaction. GENERAL SIGNIFICANCE: The results imply the possibility of enhancing therapeutic effects of these peptides by modifications of their sequence.
Assuntos
Actinas , Cadeias Leves de Miosina , Actinas/metabolismo , Músculo Esquelético/metabolismo , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/metabolismo , Subfragmentos de Miosina/química , Subfragmentos de Miosina/metabolismoRESUMO
Time-resolved cryo-electron microscopy (TrEM) allows the study of proteins under non-equilibrium conditions on the millisecond timescale, permitting the analysis of large-scale conformational changes or assembly and disassembly processes. However, the technique is developing and there have been few comparisons with other biochemical kinetic studies. Using current methods, the shortest time delay is on the millisecond timescale (â¼5-10â ms), given by the delay between sample application and vitrification, and generating longer time points requires additional approaches such as using a longer delay line between the mixing element and nozzle, or an incubation step on the grid. To compare approaches, the reaction of ATP with the skeletal actomyosin S1 complex was followed on grids prepared with a 7-700â ms delay between mixing and vitrification. Classification of the cryo-EM data allows kinetic information to be derived which agrees with previous biochemical measurements, showing fast dissociation, low occupancy during steady-state hydrolysis and rebinding once ATP has been hydrolysed. However, this rebinding effect is much less pronounced when on-grid mixing is used and may be influenced by interactions with the air-water interface. Moreover, in-flow mixing results in a broader distribution of reaction times due to the range of velocities in a laminar flow profile (temporal spread), especially for longer time delays. This work shows the potential of TrEM, but also highlights challenges and opportunities for further development.
Assuntos
Microscopia Crioeletrônica/instrumentação , Microscopia Crioeletrônica/métodos , Microfluídica/métodos , Músculo Esquelético/metabolismo , Subfragmentos de Miosina/química , Manejo de Espécimes/métodos , Animais , CoelhosRESUMO
Phosphates are commonly included in meat processing, where oxidation is inevitable, to improve water binding. This present study attempted to reveal the interactive roles of protein oxidation and tetrasodium pyrophosphate (TSPP) on the crosslinking pattern of myosin mediated by transglutaminase (TGase). Mild oxidation at 1 mM H2O2 facilitated the TGase-initiated crosslinking, with the dominate crosslinking site shifted from S1 (in nonoxidized myosin) to Rod. The introduction of TSPP alleviated the oxidation stress on proteins, and was conductive to the crosslinking reaction notably at the LMM domain. The crosslinking sites in untreated myosin were identified as Gln-613 (S1) and Gln-1498 (LMM) by amino-acid sequence analysis, while strongly oxidation resulted in the loss of Gln-1498. Contrastively, four new reactive crosslinking sites were generated by TSPP, one (Gln-558/Gln-567) located on S1 and three (Gln-1362, Gln-1374, and Gln-1423/Gln-1426) on LMM. Yet, Gln-1362 was eliminated under strong oxidation at 50 mM H2O2.
Assuntos
Proteínas de Bactérias/metabolismo , Difosfatos/química , Subfragmentos de Miosina/metabolismo , Transglutaminases/metabolismo , Sequência de Aminoácidos , Animais , Peróxido de Hidrogênio/química , Carne/análise , Subfragmentos de Miosina/química , Oxirredução , SuínosRESUMO
A hallmark feature of myosin-II is that it can spontaneously self-assemble into bipolar synthetic thick filaments (STFs) in low-ionic-strength buffers, thereby serving as a reconstituted in vitro model for muscle thick filaments. Although these STFs have been extensively used for structural characterization, their functional evaluation has been limited. In this report, we show that myosins in STFs mirror the more electrostatic and cooperative interactions that underlie the energy-sparing super-relaxed (SRX) state, which are not seen using shorter myosin subfragments, heavy meromyosin (HMM) and myosin subfragment 1 (S1). Using these STFs, we show several pathophysiological insults in hypertrophic cardiomyopathy, including the R403Q myosin mutation, phosphorylation of myosin light chains, and an increased ADP:ATP ratio, destabilize the SRX population. Furthermore, WT myosin containing STFs, but not S1, HMM, or STFs-containing R403Q myosin, recapitulated the ADP-induced destabilization of the SRX state. Studies involving a clinical-stage small-molecule inhibitor, mavacamten, showed that it is more effective in not only increasing myosin SRX population in STFs than in S1 or HMM but also in increasing myosin SRX population equally well in STFs made of healthy and disease-causing R403Q myosin. Importantly, we also found that pathophysiological perturbations such as elevated ADP concentration weakens mavacamten's ability to increase the myosin SRX population, suggesting that mavacamten-bound myosin heads are not permanently protected in the SRX state but can be recruited into action. These findings collectively emphasize that STFs serve as a valuable tool to provide novel insights into the myosin SRX state in healthy, diseased, and therapeutic conditions.
Assuntos
Benzilaminas/química , Benzilaminas/metabolismo , Miosinas/metabolismo , Uracila/análogos & derivados , Trifosfato de Adenosina/metabolismo , Animais , Humanos , Músculo Esquelético/metabolismo , Contração Miocárdica/fisiologia , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/metabolismo , Subfragmentos de Miosina/química , Subfragmentos de Miosina/metabolismo , Miosinas/química , Fosforilação/fisiologia , Uracila/química , Uracila/metabolismoRESUMO
The phosphorylated and unphosphorylated forms of tropomyosin Tpm1.1(α) are prepared from adult rabbit heart and compared biochemically. Electrophoresis confirms the high level of enrichment of the chromatography fractions and is consistent with a single site of phosphorylation. Covalently bound phosphate groups at position 283 of Tpm1.1(α) increase the rate of digestion at Leu-169, suggestive of a conformational rearrangement that extends to the midregion. Such a rearrangement, which is supported by ellipticity measurements between 25 and 42 °C, is consistent with a phosphorylation-mediated tightening of the interaction between various myofilament components. In a nonradioactive, co-sedimentation assay [30 mM KCl, 1 mM Mg(II), and 4 °C], phosphorylated Tpm1.1(α) displays a higher affinity for F-actin compared to that of the unphosphorylated control (Kd, 0.16 µM vs 0.26 µM). Phosphorylation decreases the concentration of thin filaments (pCa 4 plus ATP) required to attain a half-maximal rate of release of product from a pre-power stroke complex [myosin-S1-2-deoxy-3-O-(N-methylanthraniloyl)ADP-Pi], as investigated by double-mixing stopped-flow fluorescence, suggestive of a change in the proportion of active (turned on) and inactive (turned off) conformers, but similar maximum rates of product release are observed with either type of reconstituted thin filament. Phosphorylated thin filaments (pCa 4 and 8) display a higher affinity for myosin-S1(ADP) versus the control scenario without affecting isotherm steepness. Specific activities of ATP and Tpm1.1(α) are determined during an in vitro incubation of rat cardiac tissue [12 day-old, 50% phosphorylated Tpm1.1(α)] with [32P]orthophosphate. The incorporation of an isotope into tropomyosin lags behind that of ATP by a factor of approximately 10, indicating that transfer is a comparatively slow process.
Assuntos
Tropomiosina/química , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Actinas/química , Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Técnicas In Vitro , Cinética , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Miocárdio/química , Miocárdio/metabolismo , Subfragmentos de Miosina/química , Subfragmentos de Miosina/metabolismo , Fosforilação , Conformação Proteica , Estabilidade Proteica , Proteólise , Coelhos , Ratos , Serina/química , Tropomiosina/metabolismo , Troponina/química , Troponina/metabolismoRESUMO
High-speed atomic force microscopy (HS-AFM) can be used to study dynamic processes with real-time imaging of molecules within 1- to 5-nm spatial resolution. In the current study, we evaluated the 3-state model of activation of cardiac thin filaments (cTFs) isolated as a complex and deposited on a mica-supported lipid bilayer. We studied this complex for dynamic conformational changes 1) at low and high [Ca2+] (pCa 9.0 and 4.5), and 2) upon myosin binding to the cTF in the nucleotide-free state or in the presence of ATP. HS-AFM was used to directly visualize the tropomyosin-troponin complex and Ca2+-induced tropomyosin movements accompanied by structural transitions of actin monomers within cTFs. Our data show that cTFs at relaxing or activating conditions are not ultimately in a blocked or activated state, respectively, but rather the combination of states with a prevalence that is dependent on the [Ca2+] and the presence of weakly or strongly bound myosin. The weakly and strongly bound myosin induce similar changes in the structure of cTFs as confirmed by the local dynamical displacement of individual tropomyosin strands in the center of a regulatory unit of cTF at the relaxed and activation conditions. The displacement of tropomyosin at the relaxed conditions had never been visualized directly and explains the ability of myosin binding to TF at the relaxed conditions. Based on the ratios of nonactivated and activated segments within cTFs, we proposed a mechanism of tropomyosin switching from different states that includes both weakly and strongly bound myosin.
Assuntos
Citoesqueleto de Actina/ultraestrutura , Actinas/ultraestrutura , Subfragmentos de Miosina/ultraestrutura , Tropomiosina/ultraestrutura , Troponina/ultraestrutura , Citoesqueleto de Actina/química , Actinas/química , Animais , Cálcio/metabolismo , Bicamadas Lipídicas/química , Modelos Moleculares , Imagem Molecular , Contração Muscular/genética , Músculo Esquelético/química , Músculo Esquelético/ultraestrutura , Miocárdio/química , Miocárdio/ultraestrutura , Subfragmentos de Miosina/química , Miosinas/química , Ligação Proteica , Coelhos , Sarcômeros/química , Sarcômeros/ultraestrutura , Tropomiosina/química , Troponina/químicaRESUMO
The cross-linking of myosin, an important property in meat processing, has previously been studied in complex myofibril systems to address free radical and microbial transglutaminase (MTG) effects. In the present study, purified myosin from pork Longissimus muscle was modified with H2O2 and 10⯵M Fe to determine the effects of oxidation on the subsequent cross-linking by MTG (E:Sâ¯=â¯1:20) at 4⯰C. When subjected to MTG, the degree of cross-linking in mildly oxidized myosin sample (1â¯mM H2O2) reached 87.6%, compared to 64.7% in MTG-treated nonoxidized myosin and 33.8% in excessively oxidized myosin (200â¯mM H2O2), based on the ε(γ-glutamyl)lysine bond, SDS-PAGE, and solubility analyses. Although S1 in myosin was always the principal cross-linking site, the S2 subfragment became a significant new region of cross-linking when stressed by 1â¯mM H2O2. These findings may guide the MTG application in meat processing where oxidation is not inhibited.
Assuntos
Subfragmentos de Miosina/química , Transglutaminases/química , Reagentes de Ligações Cruzadas/química , Dipeptídeos/química , Eletroforese em Gel de Poliacrilamida , Peróxido de Hidrogênio/química , Oxirredução , Carne Vermelha , Solubilidade , Transglutaminases/metabolismoRESUMO
Due to their high aspect ratio and increased surface-to-foot-print area, arrays of vertical semiconductor nanowires are used in numerous biological applications, such as cell transfection and biosensing. Here we focus on two specific valuable biosensing approaches that, so far, have received relatively limited attention in terms of their potential capabilities: cellular mechanosensing and lightguiding-induced enhanced fluorescence detection. Although proposed a decade ago, these two applications for using vertical nanowire arrays have only very recently achieved significant breakthroughs, both in terms of understanding their fundamental phenomena, and in the ease of their implementation. We review the status of the field in these areas and describe significant findings and potential future directions.
Assuntos
Técnicas Biossensoriais , Mecanotransdução Celular/fisiologia , Nanofios/química , Semicondutores , Biomarcadores/urina , Linhagem Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Interleucina-8/urina , Luz , Células MCF-7 , Subfragmentos de Miosina/química , Subfragmentos de Miosina/metabolismo , Nanofios/ultraestrutura , Espectrometria de Fluorescência/métodos , Fator de Necrose Tumoral alfa/urina , Xylella/citologia , Xylella/fisiologia , Óxido de Zinco/químicaRESUMO
BACKGROUND: The mechanical work and the actin-activated ATP kinetics in skeletal muscles are closely associated with two surface loops that are present in the myosin molecule: loop 1 and loop 2. They are located close to the ATP-loop (loop 1), and the actin binding domain (loop 2). In this study we investigated the roles of loops 1 and 2 in the regulation of the load-dependent velocity of actin sliding and ATPase activity. METHODS: Heavy meromyosin (HMM) from rabbit skeletal muscle was subjected to limited tryptic proteolysis to obtain fragments containing different amounts of loops 1 and 2. The amino-acid sequences of these fragments were confirmed with quantitative mass-spectrometry. The velocity of actin motility propelled by the HMM fragments was measured using in-vitro motility assays, with varying loads induced by the addition of different concentrations of α-actinin. RESULTS: The load-dependent velocity of the myosin-propelled actin motility, and the fraction of actin filaments motility, were decreased in close association with the depletion of loop 1 in the HMM. The ATPase activity was decreased in close association with depletion of loops 1 and 2. CONCLUSIONS: Loop 1 is responsible for regulating the load-dependent velocity of actin motility. GENERAL SIGNIFICANCE: Myosin-actin interaction is closely regulated by two flexible loops in the structure of myosin. The results of this study are important for the understanding of the molecular mechanisms of contraction, and therefore the most basic functions of life, such as locomotion, heart beating, and breathing.
Assuntos
Músculo Esquelético/metabolismo , Subfragmentos de Miosina/metabolismo , Proteólise , Actinas/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Modelos Moleculares , Movimento , Músculo Esquelético/fisiologia , Subfragmentos de Miosina/química , Conformação Proteica , Coelhos , Tripsina/metabolismo , Suporte de CargaRESUMO
Hydrolysis of the triphosphate moiety of ATP, catalyzed by myosin, induces alterations in the affinity of the myosin heads for actin filaments via conformational changes, thereby causing motility of the actomyosin complexes. To elucidate the contribution of the triphosphate group attached to adenosine, we examined the enzymatic activity of heavy meromyosin (HMM) with actin filaments for inorganic tripolyphosphate (3PP) using a Malachite green method and evaluated using fluorescence microscopy the effects of 3PP on actin filament motility on HMM-coated glass slides. In the presence of MgCl2, HMM hydrolyzed 3PP at a maximum rate of 0.016â¯s-1 HMM-1, which was four times lower than the hydrolysis rate of ATP. Tetrapolyphosphate (4PP) was hydrolyzed at a rate similar to that of 3PP hydrolysis. The hydrolysis rates of 3PP and 4PP were enhanced by roughly 10-fold in the presence of actin filaments. In motility assays, the presence of polyphosphates did not lead to the sliding movement of actin filaments. Moreover, in the presence of ATP at low concentrations, the sliding velocity of actin filaments decreased as the concentration of added polyphosphate increased, indicating a competitive binding of polyphosphate to myosin heads with ATP. These results suggested that the energy produced by standalone triphosphate hydrolysis did not induce the unidirectional motion of actomyosin and that the link between triphosphate and adenosine was crucial for motility.
Assuntos
Citoesqueleto de Actina/metabolismo , Subfragmentos de Miosina/metabolismo , Polifosfatos/metabolismo , Citoesqueleto de Actina/química , Biocatálise , Hidrólise , Cloreto de Magnésio/química , Microscopia de Fluorescência , Subfragmentos de Miosina/química , Polifosfatos/química , Ligação Proteica , Corantes de Rosanilina/químicaRESUMO
We used transient biochemical and structural kinetics to elucidate the molecular mechanism of mavacamten, an allosteric cardiac myosin inhibitor and a prospective treatment for hypertrophic cardiomyopathy. We find that mavacamten stabilizes an autoinhibited state of two-headed cardiac myosin not found in the single-headed S1 myosin motor fragment. We determined this by measuring cardiac myosin actin-activated and actin-independent ATPase and single-ATP turnover kinetics. A two-headed myosin fragment exhibits distinct autoinhibited ATP turnover kinetics compared with a single-headed fragment. Mavacamten enhanced this autoinhibition. It also enhanced autoinhibition of ADP release. Furthermore, actin changes the structure of the autoinhibited state by forcing myosin lever-arm rotation. Mavacamten slows this rotation in two-headed myosin but does not prevent it. We conclude that cardiac myosin is regulated in solution by an interaction between its two heads and propose that mavacamten stabilizes this state.
Assuntos
Actinas/metabolismo , Benzilaminas/farmacologia , Miosinas Cardíacas/metabolismo , Cardiomiopatia Hipertrófica Familiar/tratamento farmacológico , Subfragmentos de Miosina/metabolismo , Uracila/análogos & derivados , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Regulação Alostérica/efeitos dos fármacos , Benzilaminas/uso terapêutico , Miosinas Cardíacas/química , Cardiomiopatia Hipertrófica Familiar/etiologia , Humanos , Cinética , Subfragmentos de Miosina/química , Estabilidade Proteica/efeitos dos fármacos , Uracila/farmacologia , Uracila/uso terapêuticoRESUMO
The mechanical stability of the myosin subfragment-2 (S2) was tested with simulated force spectroscopy (SFS) and gravitational force spectroscopy (GFS). Experiments examined unzipping S2, since it required less force than stretching parallel to the coiled coil. Both GFS and SFS demonstrated that the force required to destabilize the light meromyosin (LMM) was greater than the force required to destabilize the coiled coil at each of three different locations along S2. GFS data also conveyed that the mechanical stability of the S2 region is independent from its association with the myosin thick filament using cofilaments of myosin tail and a single intact myosin. The C-terminal end of myosin binding protein C (MyBPC) binds to LMM and the N-terminal end can bind either S2 or actin. The force required to destabilize the myosin coiled coil molecule was 3 times greater in the presence of MyBPC than in its absence. Furthermore, the in vitro motility assay with full length slow skeletal MyBPC slowed down the actin filament sliding over myosin thick filaments. This study demonstrates that skeletal MyBPC both enhanced the mechanical stability of the S2 coiled coil and reduced the sliding velocity of actin filaments over polymerized myosin filaments.
Assuntos
Proteínas de Transporte/química , Subfragmentos de Miosina/química , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Animais , Proteínas de Transporte/metabolismo , Subfragmentos de Miosina/metabolismo , Domínios Proteicos , Estabilidade Proteica , Coelhos , Análise EspectralRESUMO
The molecular mechanism of muscle contraction is based on the ATP-dependent cyclic interaction of myosin heads with actin filaments. Myosin head (myosin subfragment-1, S1) consists of two major domains, the motor domain responsible for ATP hydrolysis and actin binding, and the regulatory domain stabilized by light chains. Essential light chain-1 (LC1) is of particular interest since it comprises a unique N-terminal extension (NTE) which can bind to actin thus forming an additional actin-binding site on the myosin head and modulating its motor activity. However, it remains unknown what happens to the NTE of LC1 when the head binds ATP during ATPase cycle and dissociates from actin. We assume that in this state of the head, when it undergoes global ATP-induced conformational changes, the NTE of LC1 can interact with the motor domain. To test this hypothesis, we applied fluorescence resonance energy transfer (FRET) to measure the distances from various sites on the NTE of LC1 to S1 active site in the motor domain and changes in these distances upon formation of S1-ADP-BeFx complex (stable analog of S1∗-AТP state). For this, we produced recombinant LC1 cysteine mutants, which were first fluorescently labeled with 1,5-IAEDANS (donor) at different positions in their NTE and then introduced into S1; the ADP analog (TNP-ADP) bound to the S1 active site was used as an acceptor. The results show that formation of S1-ADP-BeFx complex significantly decreases the distances from Cys residues in the NTE of LC1 to TNP-ADP in the S1 active site; this effect was the most pronounced for Cys residues located near the LC1 N-terminus. These results support the concept of the ATP-induced transient interaction of the LC1 N-terminus with the S1 motor domain.
Assuntos
Adenosina Trifosfatases/metabolismo , Cadeias Leves de Miosina/metabolismo , Subfragmentos de Miosina/metabolismo , Trifosfato de Adenosina/metabolismo , Domínio Catalítico , Transferência Ressonante de Energia de Fluorescência , Humanos , Modelos Moleculares , Cadeias Leves de Miosina/química , Subfragmentos de Miosina/química , Miosinas/metabolismo , Conformação Proteica , Domínios ProteicosRESUMO
A protein molecule is a dielectric substance, so the binding of a ligand is expected to induce dielectric response in the protein molecule, considering that ligands are charged or polar in general. We previously reported that binding of adenosine triphosphate (ATP) to molecular motor myosin actually induces such a dielectric response in myosin due to the net negative charge of ATP. By this dielectric response, referred to as "dielectric allostery," spatially separated two regions in myosin, the ATP-binding region and the actin-binding region, are allosterically coupled. In this study, from the statistically stringent analyses of the extensive molecular dynamics simulation data obtained in the ATP-free and the ATP-bound states, we show that there exists the dielectric allostery that transmits the signal of ATP binding toward the distant lever-arm region. The ATP-binding-induced electrostatic potential change observed on the surface of the main domain induced a movement of the converter subdomain from which the lever arm extends. The dielectric response was found to be caused by an underlying large-scale concerted rearrangement of the electrostatic bond network, in which highly conserved charged/polar residues are involved. Our study suggests the importance of the dielectric property for molecular machines in exerting their function.
Assuntos
Trifosfato de Adenosina/metabolismo , Subfragmentos de Miosina/metabolismo , Miosina Tipo II/metabolismo , Pectinidae/metabolismo , Animais , Simulação de Dinâmica Molecular , Subfragmentos de Miosina/química , Miosina Tipo II/química , Pectinidae/química , Ligação Proteica , Domínios Proteicos , Eletricidade EstáticaRESUMO
The bioconjugation of a polyoxometalate (POMs), i.e., dodecavanadate (V12O32), to DNA strands produces a functional labeled DNA primer, V12O32-DNA. The grafting of DNA primer onto streptavidin-coated magnetic nanoparticles (SVM) produces a novel composite, V12O32-DNA@SVM. The high binding-affinity of V12O32 with the ATP binding site in myosin subfragment-1 (S1) facilitates favorable adsorption of myosin, with an efficiency of 99.4% when processing 0.1 mL myosin solution (100 µg mL-1) using 0.1 mg composite. Myosin adsorption fits the Langmuir model, corresponding to a theoretical adsorption capacity of 613.5 mg g-1. The retained myosin is readily recovered by 1% SDS (m/m), giving rise to a recovery of 58.7%. No conformational change is observed for myosin after eliminating SDS by ultrafiltration. For practical use, high-purity myosin S1 is obtained by separation of myosin from the rough protein extract from porcine left ventricle, followed by digestion with α-chymotryptic and further isolation of S1 subfragment. The purified myosin S1 is identified with matrix-assisted laser desorption/ionization time-of-flight/mass spectrometry, giving rise to a sequence coverage of 38%.
Assuntos
DNA/química , Subfragmentos de Miosina/química , Subfragmentos de Miosina/isolamento & purificação , Compostos Organometálicos/química , Vanádio/química , Adsorção , Animais , Miocárdio/química , Nanopartículas/química , Estreptavidina/química , SuínosRESUMO
Heavy meromyosin (HMM) forms clusters along actin filaments under low ATP concentrations. Here, we observed the growth of HMM clusters under low concentrations of ATP in real time using fluorescence microscopy. When actin filaments were loosely immobilized on positively charged lipid bilayers, clusters of HMM-GFP were readily formed. Time-lapse observation revealed that the clusters grew unidirectionally. When we used a mixture of actin filaments and copolymers of actin and acto-S1dC, a chimeric protein of actin and the myosin motor domain, HMM-GFP preferentially formed clusters along the copolymers. We thus suggest that binding of myosin motors carrying ADP and Pi induces unidirectional conformational changes in actin filaments and allosterically recruits more myosin binding. In contrast, when actin filaments and copolymers were anchored to glass substrate via stable biotin-avidin linkage, higher concentrations of HMM-GFP were required to form clusters than on the lipid bilayer. Moreover, actin filaments and copolymers were not discriminated regarding preferential cluster formation. This is presumably because the myosin-induced cooperative conformational changes in actin filaments involve changes in the helical twist. Consistent with this, cofilin clusters, which supertwist the helix, were readily formed along loosely immobilized actin filaments, but not along those anchored via biotin-avidin linkage.
Assuntos
Citoesqueleto de Actina/química , Dictyostelium/química , Subfragmentos de Miosina/química , Proteínas de Protozoários/química , Microscopia de FluorescênciaRESUMO
Mavacamten, formerly known as MYK-461 is a recently discovered novel small-molecule modulator of cardiac myosin that targets the underlying sarcomere hypercontractility of hypertrophic cardiomyopathy, one of the most prevalent heritable cardiovascular disorders. Studies on isolated cells and muscle fibers as well as intact animals have shown that mavacamten inhibits sarcomere force production, thereby reducing cardiac contractility. Initial mechanistic studies have suggested that mavacamten primarily reduces the steady-state ATPase activity by inhibiting the rate of phosphate release of ß-cardiac myosin-S1, but the molecular mechanism of action of mavacamten has not been described. Here we used steady-state and presteady-state kinetic analyses to investigate the mechanism of action of mavacamten. Transient kinetic analyses revealed that mavacamten modulates multiple steps of the myosin chemomechanical cycle. In addition to decreasing the rate-limiting step of the cycle (phosphate release), mavacamten reduced the number of myosin-S1 heads that can interact with the actin thin filament during transition from the weakly to the strongly bound state without affecting the intrinsic rate. Mavacamten also decreased the rate of myosin binding to actin in the ADP-bound state and the ADP-release rate from myosin-S1 alone. We, therefore, conclude that mavacamten acts on multiple stages of the myosin chemomechanical cycle. Although the primary mechanism of mavacamten-mediated inhibition of cardiac myosin is the decrease of phosphate release from ß-cardiac myosin-S1, a secondary mechanism decreases the number of actin-binding heads transitioning from the weakly to the strongly bound state, which occurs before phosphate release and may provide an additional method to modulate myosin function.
Assuntos
Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Benzilaminas/química , Miosinas Cardíacas/química , Subfragmentos de Miosina/química , Sarcômeros/química , Uracila/análogos & derivados , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Miosinas Cardíacas/metabolismo , Cardiomegalia/metabolismo , Bovinos , Subfragmentos de Miosina/metabolismo , Sarcômeros/metabolismo , Uracila/químicaRESUMO
We examined the movement of an actin filament sliding on a mixture of normal and genetically modified myosin molecules that were attached to a glass surface. For this purpose, we used a Dictyostelium G680V mutant myosin II whose release rates of Pi and ADP were highly suppressed relative to normal myosin, leading to a significantly extended life-time of the strongly bound state with actin and virtually no motility. When the mixing ratio of G680V mutant myosin II to skeletal muscle HMM (heavy myosin) was 0.01%, the actin filaments moved intermittently. When they moved, their sliding velocities were about two-fold faster than the velocity of skeletal HMM alone. Furthermore, sliding movements were also faster when the actin filaments were allowed to slide on skeletal muscle HMM-coated glass surfaces in the motility buffer solution containing G680V HMM. In this case no intermittent movement was observed. When the actin filaments used were copolymerized with a fusion protein consisting of Dictyostelium actin and Dictyostelium G680V myosin II motor domain, similar faster sliding movements were observed on skeletal muscle HMM-coated surfaces. The filament sliding velocities were about two-fold greater than the velocities of normal actin filaments. We found that the velocity of actin filaments sliding on skeletal muscle myosin molecules increased in the presence of a non-motile G680V mutant myosin motor.