Kinetics of acto-S1 interaction as a guide to a model for the crossbridge cycle

Geeves, Michael A.; Goody, Roger S.; Gutfreund, H.

doi:10.1007/bf00818255

Cited by 163 publications

(149 citation statements)

References 23 publications

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“…With the sinusoidal analysis method, we characterized the cross-bridge scheme in rabbit psoas fibres (Kawai and Halvorson 1991), soleus slow twitch fibres (Wang and Kawai 1997), ferret cardiac fibres , porcine cardiac fibres , bovine cardiac fibres , and other fast twitch skeletal muscle fibres from the rabbit (Galler et al 2005). The above Scheme 1 is consistent to those deduced from solution studies (Taylor 1979;Geeves et al 1984) of isolated and reconstituted contractile proteins, except that in solution studies the strongly bound AM*DP state has not been identified, the Pi release step (step 5) is practically irreversible (Taylor 1979), and the ATP isomerization step (step 2) is irreversible (k −2 = 0) in the interaction of myosin subfragment 1 and MgATP (Bagshaw and Trentham 1974). The irreversibility (extremely large K) means that the free energy reduction is large because of the relationship: ΔG° = −RT ln K, which means that perhaps half of the ATP hydrolysis energy is dissipated at step 2 and another half at step 5 in the case of solution studies.…”

Section: Methods Of Studying Cross-bridge Kineticssupporting

confidence: 67%

Use of thin filament reconstituted muscle fibres to probe the mechanism of force generation

Kawai

Ishiwata

2006

J Muscle Res Cell Motil

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The technique of selective removal of the thin filament by gelsolin in bovine cardiac muscle fibres, and reconstitution of the thin filament from isolated proteins is reviewed, and papers that used reconstituted preparations are discussed. By comparing the results obtained in the absence/presence of regulatory proteins tropomyosin (Tm) and troponin (Tn), it is concluded that the role of Tm and Tn in force generation is not only to expose the binding site of actin to myosin, but also to modify actin for better stereospecific and hydrophobic interaction with myosin. This conclusion is further supported by experiments that used a truncated Tm mutant and the temperature study of reconstituted fibres. The conclusion is consistent with the hypothesis that there are three states in the thin filament: blocked state, closed state, and open state. Tm is the major player to produce these effects, with Tn playing the role of Ca 2+ sensing and signal transmission mechanism. Experiments that changed the number of negative charges at the N-terminal finger of actin demonstrates that this part of actin is essential to promote the strong interaction between actin and myosin molecules, in addition to the well-known weak interaction that positions the myosin head at the active site of actin prior to force generation. KeywordsActin; Regulatory proteins; Tropomyosin; Troponin; Myosin; Gelsolin; Cross-bridge kinetics; Sinusoidal analysis; BDM Thin filament reconstituted muscle fibre systemFor the purpose of understanding the cross-bridge interaction with actin and the role of regulatory proteins in this interaction, we reconstituted the thin filament in cardiac muscle fibres. This method is schematically represented in Fig. 1. The thin filament is first removed by gelsolin (Fig. 1A to 1B), followed by sequential reconstitution with G-actin (Fig. 1C), and regulatory proteins, tropomyosin (Tm) and troponin (Tn) (Fig. 1D). Gelsolin is an actin and thin filament severing protein, hence its action is specific and it does not disrupt other sarcomeric structure. However, overtreatment with gelsolin would disrupt the Z-line structure, because actin is one of the main constituents in the Z-line. The thin filament reconstitution method was initially developed at Ishiwata's laboratory and the key properties of the Correspondence to: Masataka Kawai, masataka-kawai@uiowa.edu. reconstituted fibres, such as the recovery of isometric tension, the tension-pCa relationship, and the function of spontaneous oscillatory contraction, were examined on skeletal fibres (Funatsu et al. 1994), followed by cardiac fibres (Fujita et al. 1996; Ishiwata 1998, 1999; for brief review see Ishiwata et al. 1998). In these works, it was demonstrated that bovine cardiac fibres were successful as the reconstituted system, in which actin, Tm and Tn can be replaced with those prepared from other muscles, for example, rabbit skeletal muscle proteins (Fujita et al. 1996;Fujita and Ishiwata 1999). The reconstitution method in cardiac fibres was successfully applied at ...

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Section: Methods Of Studying Cross-bridge Kineticssupporting

confidence: 67%

Use of thin filament reconstituted muscle fibres to probe the mechanism of force generation

Kawai

Ishiwata

2006

J Muscle Res Cell Motil

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“…In each cycle, myosin hydrolyzes a single ATP molecule and couples this chemical reaction to the physical state transition between the weak and strong actin-binding states (11 and 12). The weakto-strong actin-binding transition has been thought to play a key role in the force-generation (12). However, when and how the force is generated remains controversial (7 and 13).…”

mentioning

confidence: 99%

Unidirectional Brownian motion observed in an in silico single molecule experiment of an actomyosin motor

Takano

Terada

Sasai

2010

Proc. Natl. Acad. Sci. U.S.A.

120

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The actomyosin molecular motor, the motor composed of myosin II and actin filament, is responsible for muscle contraction, converting chemical energy into mechanical work. Although recent single molecule and structural studies have shed new light on the energy-converting mechanism, the physical basis of the molecular-level mechanism remains unclear because of the experimental limitations. To provide a clue to resolve the controversy between the lever-arm mechanism and the Brownian ratchet-like mechanism, we here report an in silico single molecule experiment of an actomyosin motor. When we placed myosin on an actin filament and allowed myosin to move along the filament, we found that myosin exhibits a unidirectional Brownian motion along the filament. This unidirectionality was found to arise from the combination of a nonequilibrium condition realized by coupling to the ATP hydrolysis and a ratchet-like energy landscape inherent in the actin-myosin interaction along the filament, indicating that a Brownian ratchet-like mechanism contributes substantially to the energy conversion of this molecular motor. M olecular machines in living organisms are nanometer-sized small systems working robustly and efficiently in the presence of the severely disturbing thermal noises. Furthermore, in the case of the molecular motors, the energy supplied by the hydrolysis of adenosine triphosphate (ATP), which is to be converted to the mechanical work, is only an order of magnitude larger than the thermal energy at room temperature (∼20k B T), and hence it seems likely that the molecular machines harness the thermal noise, instead of suppressing it as the human-made machines do (1). This distinct feature of the molecular machines should arise from the atomic structures of the constituent proteins, which is reflected in the current focus of interest in the structural aspect of the molecular machines. However, it should be remembered that the structural aspect is not everything but is inseparably linked to the dynamical and energetical aspects. Therefore, a unified description of structure, dynamics, and energetics is the step necessary to understand the molecular-level physical principle of how the molecular machines work.The actomyosin molecular motor, the system composed of myosin II and actin molecules, is responsible for muscle contraction, and is the most extensively studied molecular motor. It has been shown by single molecule experiments (SME) that a single myosin molecule and an actin filament (polymerized actin) constitute the minimal force-generating unit of this motor (2-4) (see Fig. 1A). Although SME has been a powerful approach to study the dynamical aspect of the force-generating unit (2-4), the limitation of the spatiotemporal resolution of SME has inhibited us from developing the full atomistic picture of the force-generating process. On the other hand, since the currently available high-resolution structures of myosin (5-7) are those obtained in the crystalline environment and in the absence of actin, it remains unc...

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“…1). Although skeletal muscle actomyosin II binds ADP weakly (K d Ͼ 100 M) in a rapid equilibrium (4), smooth muscle myosin (5) and some nonmuscle actomyosin isoforms (6 -10) bind ADP with high (K d Ͻ 10 M) affinities. Several actomyosins with high ADP binding affinities display an ADP-induced rotation of the light chain binding domain (11,12) and are likely to populate multiple actomyosin-ADP states (5)(6)(7).…”

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confidence: 99%

Mechanism of Nucleotide Binding to Actomyosin VI

Robblee¹,

Olivares²,

Cruz

2004

Journal of Biological Chemistry

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We have examined the kinetics of nucleotide binding to actomyosin VI by monitoring the fluorescence of pyrene-labeled actin filaments. ATP binds singleheaded myosin VI following a two-step reaction mechanism with formation of a low affinity collision complex (1/K 1 ‫؍‬ 5.6 mM) followed by isomerization (k ؉2 ‫؍‬ 176 s ؊1 ) to a state with weak actin affinity. The rates and affinity for ADP binding were measured by kinetic competition with ATP. This approach allows a broader range of ADP concentrations to be examined than with fluorescent nucleotide analogs, permitting the identification and characterization of transiently populated intermediates in the pathway. ADP binding to actomyosin VI, as with ATP binding, occurs via a two-step mechanism. The association rate constant for ADP binding is ϳfive times greater than for ATP binding because of a higher affinity in the collision complex (1/K 5b ‫؍‬ 2.2 mM) and faster isomerization rate constant (k ؉5a ‫؍‬ 366 s ؊1 ). By equilibrium titration, both heads of a myosin VI dimer bind actin strongly in rigor and with bound ADP. In the presence of ATP, conditions that favor processive stepping, myosin VI does not dwell with both heads strongly bound to actin, indicating that the second head inhibits strong binding of the lead head to actin. With both heads bound strongly, ATP binding is accelerated 2.5-fold, and ADP binding is accelerated >10-fold without affecting the rate of ADP release. We conclude that the heads of myosin VI communicate allosterically and accelerate nucleotide binding, but not dissociation, when both are bound strongly to actin.The myosin family of molecular motors constitutes a large gene family of proteins that couple the energy from ATP binding, hydrolysis, and product release to force generation along actin filaments (1). At least 18 classes (Classes I-XVIII) make up the family (2). All possess a highly conserved catalytic motor domain (referred to as "head") that binds actin, hydrolyzes ATP, and performs the mechanical work. In nonmuscle cells, cytoplasmic myosins are required for various cellular processes including membrane and RNA trafficking, cell migration, and cytokinesis (3).The nucleotide bound to myosin dictates the affinity for actin filaments (1). In the absence of nucleotide or with bound ADP, myosin binds actin filaments with high (Ͻ1 M) affinity; these states are referred to as "strongly bound states." With ATP or the hydrolysis products, ADP-P i , bound, myosin binds actin with low affinity and rapidly attaches and detaches from the actin filament on a submicrosecond timescale; these nucleotide states are called "weakly bound states." The strong binding states are force-bearing intermediates, but the weak binding states are not. The fraction of the total ATPase cycle time spent strongly bound to actin is called the duty ratio. Because under physiological nucleotide concentrations ATP binding is largely favored over ADP binding, ADP release limits the lifetime of the strongly bound actomyosin states and dictates the duty ratio.Ther...

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Kinetics of acto-S1 interaction as a guide to a model for the crossbridge cycle

Cited by 163 publications

References 23 publications

Use of thin filament reconstituted muscle fibres to probe the mechanism of force generation

Use of thin filament reconstituted muscle fibres to probe the mechanism of force generation

Unidirectional Brownian motion observed in an in silico single molecule experiment of an actomyosin motor

Mechanism of Nucleotide Binding to Actomyosin VI

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