A novel stopped-flow method for measuring the affinity of actin for myosin head fragments using ?g quantities of protein

It has been well established that muscle contraction is driven by structural rearrangements within myosin during its ATPase cycle and interaction with actin. The crystal structures of myosin (Fig. 1) solved in different nucleotide states have provided a framework for examining the structural basis of the ATPase cycle of myosin (1-6). In addition, solution techniques that include monitoring the intrinsic fluorescence of myosin have proven extremely valuable for examining the enzymatic and kinetic properties of the molecule (7-13). The following kinetic scheme of the myosin MgATPase cycle was developed based on observed changes in intrinsic fluorescence corresponding to structural changes within myosin, where M represents myosin and an asterisk represents enhanced protein fluorescence (14).In this reaction myosin first forms a collision complex with ATP followed by an isomerization to the M*⅐ATP complex, which results in the first level of fluorescence enhancement (*). During the rapid reversible process of ATP hydrolysis, a structural change induces an additional fluorescence enhancement (**). Then, following hydrolysis of ATP the fluorescence decreases back to the first level of enhancement (*), which is thought to correspond to the rate-limiting structural change resulting in phosphate release. The phosphate release step then shifts myosin from a weak to strong binding conformation and is the step believed to be associated with force generation during muscle contraction. The release of ADP is a much faster two-step process in which the second step results in a decrease in fluorescence to basal levels. Thus, examining the conformational changes that result in alterations in intrinsic tryptophan fluorescence may lead to important insights about the structural properties of the myosin MgATPase cycle.Dynamic structural information about domain motions within myosin during its MgATPase cycle has been pursued extensively (reviewed in Ref. 15). Indeed, several studies have demonstrated key rearrangements in the light chain-binding region (residues 781-820 highlighted in pink in Fig. 1), also referred to as the lever arm, providing a structural mechanism for force generation (reviewed in Ref. 16). Electron paramagnetic resonance studies, utilizing a probe on the regulatory light chain have shown a 30°rotation of this region (17), suggesting that the lever arm region rotates relative to the catalytic domain during muscle contraction. In addition, fluorescence studies utilizing a fluorescent probe at the regulatory light chain demonstrated rotation of the lever arm in contracting muscle fibers (15). Cryo-electron microscopy studies have revealed that smooth muscle myosin decorated on actin filaments undergoes a large structural change (30 -35 Å) of the lever arm upon ADP release (18). Furthermore, nucleotide analogs, which trap myosin in specific nucleotide states, have been useful in elucidating the structural properties of the normally short-lived stages of the MgATPase cycle. Structural studies suggest that MgADP-BeF ...

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Tryptophan 512 Is Sensitive to Conformational Changes in the Rigid Relay Loop of Smooth Muscle Myosin during the MgATPase Cycle

Yengo

Chrin

Rovner³

et al. 2000

“…Pyrene-labeled Actin Experiments-Actin was labeled with pyrene iodoacetamide and treated with phalloidin (45,46). The actin binding assay was performed in rigor or in the presence of MgADP (MgADP state) as described previously (46 -48).…”

Section: Methodsmentioning

confidence: 99%

The Myosin Cardiac Loop Participates Functionally in the Actomyosin Interaction

Ajtai

Garamszegi

Watanabe

et al. 2004

The motor protein myosin in association with actin transduces chemical free energy in ATP into work in the form of actin translation against an opposing force. Mediating the actomyosin interaction in myosin is an actin binding site distributed among several peptides on the myosin surface including surface loops contributing to affinity and actin regulation of myosin ATPase. A structured surface loop on ␤-cardiac myosin, the cardiac or C-loop, was recently demonstrated to affect myosin ATPase and was indirectly implicated in the actomyosin interaction. The C-loop is a conserved feature of all myosin isoforms with crystal structures, suggesting that it is an essential part of the core energy transduction machinery. It is shown here that proteolytic digestion of the C-loop in ␤-cardiac myosin eliminates actin-activated myosin ATPase and reduces actomyosin affinity in rigor more than 100-fold. Studies of C-loop function in smooth muscle myosin were also undertaken using sitedirected mutagenesis. Mutagenesis of a single charged residue in the C-loop of smooth muscle myosin alters actomyosin affinity and doubles myosin in vitro motility and actin-activated ATPase velocities, thereby involving a charged region of the loop in the actomyosin interaction. It appears likely that the C-loop is an essential electrostatic binding site for actin involved in modulation of actomyosin affinity and regulation of actomyosin ATPase velocity.The motor protein myosin is an ATPase and an actin-binding protein transducing ATP free energy into work. In muscle, myosin, actin, and ATP constitute the unitary work producer. The cyclical interaction of these components, a sequence of states each characterized as a static relation between the proteins and an intermediate in the degradation of ATP, is a contraction cycle. In a contraction cycle, myosin hydrolyzes ATP in its active site and forms a weak association with actin at a separate actin binding site. Release of phosphate from the active site initiates strong binding to actin and a large conformation change in myosin that produces work in the form of actin translation against a load. Several solved crystal structures represent intermediates in ATP hydrolysis and define myosin conformation changes in the absence of actin (1-6). Presently, we lack a detailed understanding of actomyosin structure and in particular how the elements making up the actomyosin interface contribute to mutual affinity, actin regulation of phosphate release, and the ability of myosin-bound nucleotide to modulate actomyosin affinity.Myosin consists of a globular head domain containing the enzymatic portion of the molecule and a tail involved in filament assembly. The globular head separated from its tail portion is called subfragment 1 (S1). 1 S1 interacts with filamentous actin (F-actin) consisting of polymerized actin monomers. An atomic model for F-actin built from monomeric actin crystal structures (7-9) satisfies x-ray diffraction data restraints (10) and is consistent with electron microscopic imagery (11). In...

“…Despite the structural similarities between myosins and kinesins, the kinetic and thermodynamic mechanisms are quite different. For example, upon ATP binding to the actomyosin complex, myosin⅐ATP rapidly detaches from the filament before ATP hydrolysis (10), whereas ATP binding to the microtubule⅐kinesin complex does not typically dissociate the motor from the filament, rather it remains tightly bound and undergoes hydrolysis on the filament before detaching (11). Nevertheless, the rate-limiting reaction in the ATP hydrolysis cycle for both motors is stimulated in the presence of their respective filaments.…”

mentioning

confidence: 99%

Metal Switch-controlled Myosin II from Dictyostelium discoideum Supports Closure of Nucleotide Pocket during ATP Binding Coupled to Detachment from Actin Filaments

Cochran

Thompson

Kull

2013

Background:Myosins convert the energy of ATP hydrolysis into force production. Results: Substitution of the metal-interacting serine in switch-1 with cysteine renders the motor sensitive to manganese. Conclusion: This technology provides a reversible structural linkage between the nucleotide pocket and actin-binding region in the myosin motor domain. Significance: Understanding the ATPase mechanism requires a description of allosteric communication in molecular motors.