Conformational selection during weak binding at the actin and myosin interface

The molecular mechanism of the powerstroke in muscle is examined by resonance energy transfer techniques. Recent models suggesting a pre-cocking of the myosin head involving an enormous rotation between the lever arm and the catalytic domain were tested by measuring separation distances among myosin subfragment-2, the nucleotide site, and the regulatory light chain in the presence of nucleotide transition state analogs. Only small changes (<0.5 nm) were detected that are consistent with internal conformational changes of the myosin molecule, but not with extreme differences in the average lever arm position suggested by some atomic models. These results were confirmed by stopped-flow resonance energy transfer measurements during single ATP turnovers on myosin. To examine the participation of actin in the powerstroke process, resonance energy transfer between the regulatory light chain on myosin subfragment-1 and the C-terminus of actin was measured in the presence of nucleotide transition state analogs. The efficiency of energy transfer was much greater in the presence of ADP-AlF(4), ADP-BeF(x), and ADP-vanadate than in the presence of ADP or no nucleotide. These data detect profound differences in the conformations of the weakly and strongly attached cross-bridges that appear to result from a conformational selection that occurs during the weak binding of the myosin head to actin.     

Actin-induced closure of the actin-binding cleft of smooth muscle myosin

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

Switch I closure simultaneously promotes strong binding to actin and ADP in smooth muscle myosin

The motor protein myosin uses energy derived from ATP hydrolysis to produce force and motion. Important conserved components (P-loop, switch I, and switch II) help propagate small conformational changes at the active site into large scale conformational changes in distal regions of the protein. Structural and biochemical studies have indicated that switch I may be directly responsible for the reciprocal opening and closing of the actin and nucleotide-binding pockets during the ATPase cycle, thereby aiding in the coordination of these important substrate-binding sites. Smooth muscle myosin has displayed the ability to simultaneously bind tightly to both actin and ADP, although it is unclear how both substrate-binding clefts could be closed if they are rigidly coupled to switch I. Here we use single tryptophan mutants of smooth muscle myosin to determine how conformational changes in switch I are correlated with structural changes in the nucleotide and actin-binding clefts in the presence of actin and ADP. Our results suggest that a closed switch I conformation in the strongly bound actomyosin-ADP complex is responsible for maintaining tight nucleotide binding despite an open nucleotide-binding pocket. This unique state is likely to be crucial for prolonged tension maintenance in smooth muscle.     

Transient interaction between the N-terminal extension of the essential light chain-1 and motor domain of the myosin head during the ATPase cycle

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-BeF_x complex (stable analog of S1¹-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-BeF_x 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.     

A structural model for actin-induced nucleotide release in myosin

Myosins are molecular motor proteins that harness the chemical energy stored in ATP to produce directed force along actin filaments. Complex communication pathways link the catalytic nucleotide-binding region, the structures responsible for force amplification and the actin-binding domain of myosin. We have crystallized the nucleotide-free motor domain of myosin II in a new conformation in which switch I and switch II, conserved loop structures involved in nucleotide binding, have moved away from the nucleotide-binding pocket. These movements are linked to rearrangements of the actin-binding region, which illuminate a previously unobserved communication pathway between the nucleotide-binding pocket and the actin-binding region, explain the reciprocal relationship between actin and nucleotide affinity and suggest a new mechanism for product release in myosin family motors.     

The calponin regulatory region is intrinsically unstructured: novel insight into actin-calponin and calmodulin-calponin interfaces using NMR spectroscopy

Calponin is an actin- and calmodulin-binding protein believed to regulate the function of actin. Low-resolution studies based on proteolysis established that the recombinant calponin fragment 131–228 contained actin and calmodulin recognition sites but failed to precisely identify the actin-binding determinants. In this study, we used NMR spectroscopy to investigate the structure of this functionally important region of calponin and map its interaction with actin and calmodulin at amino-acid resolution. Our data indicates that the free calponin peptide is largely unstructured in solution, although four short amino-acid stretches corresponding to residues 140–146, 159–165, 189–195, and 199–205 display the propensity to form α-helices. The presence of four sequential transient helices probably provides the conformational malleability needed for the promiscuous nature of this region of calponin. We identified all amino acids involved in actin binding and demonstrated for the first time, to our knowledge, that the N-terminal flanking region of Lys¹³⁷-Tyr¹⁴⁴ is an integral part of the actin-binding site. We have also delineated the second actin-binding site to amino acids Thr¹⁸⁰-Asp¹⁹⁰. Ca²⁺-calmodulin binding extends beyond the previously identified minimal sequence of 153–163 and includes most amino acids within the stretch 143–165. In addition, we found that calmodulin induces chemical shift perturbations of amino acids 188–190 demonstrating for the first time, to our knowledge, an effect of Ca²⁺-calmodulin on this region. The spatial relationship of the actin and calmodulin contacts as well as the transient α-helical structures within the regulatory region of calponin provides a structural framework for understanding the Ca²⁺-dependent regulation of the actin-calponin interaction by calmodulin.     

Myosins Are Motor Proteins of the Actomyosin Motility System: Association with Membranes and with the Signal Pathways

Structural and functional characteristics of the motor proteins of the actomyosin motility system, myosins, which can be grouped into 15 classes, are presented in brief. The structure of the myosin molecule is considered: a conservative motor domain of the head with ATP- and actin-binding sites, a head segment associated with light chains, and a tail, which is variable in various myosins performing different functions. We address the progress in the studies of myosin functioning as a motor in the in vitroassay systems. Not only animal and prokaryotic organisms but also Characean algae and plant pollen tubes contributed considerably to these studies as sources of actin and myosin. Higher-plant myosins are characterized. The data are presented concerning the interaction between some myosin forms and other actin-binding proteins and, on the other hand, the phosphoinositol signal transduction pathway, the integral plasmalemmal proteins, and the proteins of the extracellular matrix. The most important idea formulated in the review is that a dynamic reorganization of the actin cytoskeleton is a structural basis for physiological processes in plants.     

Conformational transitions within the head and at the head-rod junction in smooth muscle myosin studied with a limited proteolysis method

It was previously shown that tryptic digestion of subfragment 1 (S1) of skeletal muscle myosins at 0 degree C results in cleavage of the heavy chain at a specific site located 5 kDa from the NH2-terminus. This cleavage is enhanced by nucleotides and suppressed by actin and does not occur at 25 degrees C, except in the presence of nucleotide. Here we show a similar temperature sensitivity and protection by actin of an analogous chymotryptic cleavage site in the heavy chain of gizzard S1. The results support the view that the myosin head, in general, can exist in two different conformational states even in the absence of nucleotides and actin, and indicate that the heavy chain region 5 kDa from the NH2-terminus is involved in the communication between the sites of nucleotide and actin binding. We also show here for the first time that the S1-S2 junction in gizzard myosin can be cleaved by chymotrypsin and that this cleavage (observed in papain-produced S1 devoid of the regulatory light chain) is also temperature-dependent but insensitive to nucleotides and actin. It is suggested that the temperature-dependent alteration in the flexibility of the head-rod junction, which is apparent from these and similar observations on skeletal muscle myosin [Miller, L. & Reisler, E. (1985) J. Mol. Biol. 182, 271-279; Redowicz, M.J. & Strzelecka-Go?aszewska, H. (1988) Eur. J. Biochem. 177, 615-624], may contribute to the temperature dependence of some steps in the cross-bridge cycle.     

Three-dimensional reconstruction of thin filaments decorated with a Ca2+-regulated myosin

Three-dimensional reconstructions of “barbed” and “blunted” arrowheads (Craig et al., 1980) show that these two forms arise from arrangement of scallop myosin subfragments (S1) that appear about 40 Å longer in the presence of the regulatory light chain than in its absence. A similar difference in apparent length is indicated by images of single myosin subfragments in partially decorated filaments. The extra mass is located at the end of the subfragment furthest from actin, and probably comprises part of the regulatory light chain as well as a segment of the myosin heavy chain. The fact that barbed arrowheads are also formed by myosin subfragments from vertebrate striated and smooth muscles implies that the homologous light chains in these myosins have locations similar to that of the scallop light chain.The scallop light chain probably does not extend into the actin-binding site on the myosin head, and is therefore unlikely to interfere physically with binding. Rather, regulation of actin-myosin interaction by light chains may involve Ca²⁺-dependent changes in the structure of a region near the head-tail junction of myosin.The reconstructions suggest locations for actin and tropomyosin relative to myosin that are similar to those proposed by Taylor & Amos (1981) and are consistent with a revised steric blocking model for regulation by tropomyosin. The identification of actin from these reconstructions is supported by images of partially decorated filaments that display the polarity of the actin helix relative to that of bound myosin subfragments.     

Combining EPR with Fluorescence Spectroscopy to Monitor Conformational Changes at the Myosin Nucleotide Pocket

We used spin-labeled nucleotide analogs and fluorescence spectroscopy to monitor conformational changes at the nucleotide-binding site of wild-type Dictyostelium discoideum (WT) myosin and a construct containing a single tryptophan at position F239 near the switch 1 loop. Electron paramagnetic resonance (EPR) spectroscopy and tryptophan fluorescence have been used previously to investigate changes at the myosin nucleotide site. A limitation of fluorescence spectroscopy is that it must be done on mutated myosins containing only a single tryptophan. A limitation of EPR spectroscopy is that one infers protein conformational changes from alterations in the mobility of an attached probe. These limitations have led to controversies regarding conclusions reached by the two approaches. For the first time, the data presented here allow direct correlations to be made between the results from the two spectroscopic approaches on the same proteins and extend our previous EPR studies to a nonmuscle myosin. EPR probe mobility indicates that the conformation of the nucleotide pocket of the WT⋅SLADP (spin-labeled ADP) complex is similar to that of skeletal myosin. The pocket is closed in the absence of actin for both diphosphate and triphosphate nucleotide states. In the actin⋅myosin⋅diphosphate state, the pocket is in equilibrium between closed and open conformations, with the open conformation slightly more favorable than that seen for fast skeletal actomyosin. The EPR spectra for the mutant show similar conformations to skeletal myosin, with one exception: in the absence of actin, the nucleotide pocket of the mutant displays an open component that was approximately 4-5 kJ/mol more favorable than in skeletal or WT myosin. These observations resolve the controversies between the two techniques. The data from both techniques confirm that binding of myosin to actin alters the conformation of the myosin nucleotide pocket with similar but not identical energetics in both muscle and nonmuscle myosins.     

Myosin binding to actin. Structural analysis using myosin fragments

The actin-binding property of the myosin head 20 K (K = 10(3) Mr) fragment has been examined by a structural assay. A new fragment is produced by digestion of scallop myosin synthetic filaments with a lysine-specific protease. This fragment consists of the rod together with two "nubs" corresponding to the 20 K fragment, which retain both the regulatory and essential light chains. Myosin filaments, digested for different lengths of time, were mixed with F-actin and visualized by electron microscopy after negative staining. When the head is cleaved, but the head fragments remain associated, the filaments bind actin in an ATP-sensitive manner. Filaments made primarily of the nub-containing fragments, however, bind actin very poorly. In addition, electron microscopic characterization of actin-binding by the isolated tryptic 20 K fragment from chicken myosin indicates that binding of this fragment to actin is probably non-specific. These results suggest that interactions between the 20 K region and the other peptides in the head are essential for actin-binding.     

MgATP binding changes neither the shape nor the flexibility of the heads of single myosin molecules.

The structural mechanism by which myosin heads exert force is unknown. One possibility is that the tight binding of the heads to actin drives them into a force-generating configuration. Another possibility is that the force-generating conformational change is inherent to the myosin heads. In this case the heads would make force by changing their shape according to the species of nucleotide in their active sites, the tight attachment to actin serving only to provide traction. To test this latter possibility, we used negative stain electron microscopy to search for a MgATP-induced shape change in the heads of single myosin molecules. We compared the heads of 10S smooth muscle myosin monomers (wherein MgATP is trapped at the active site) with the MgATP-free heads of 6S monomers. We found that to a resolution of about 2 nm, MgATP binding to the unrestrained myosin head does not drive it to change its shape or its flexibility. This result suggests that the head makes force by virtue of an induced fit to actin.     

Actin and nucleotide induced conformational changes in the vicinity of Lys553 in myosin subfragment 1.

Bertrand et al. [Bertrand, R., Derancourt, J. & Kassab, R. (1995) Biochemistry 34, 9500-9507] reported that 6-[fluoresceine-5(and 6)-carboxamido] hexanoic acid succinimidyl ester (FHS) selectively modifies Lys553, which is part of the strong actin-binding site of myosin subfragment 1 (S1). We found that the reaction of FHS with Lys533 is accompanied by a decrease in the fluorescence intensity of the reagent. The rate of the FHS reaction increased with increasing pH implying that the unprotonated form of the epsilon-amino group of Lys553 reacts with FHS. Addition of 0.4 M KCl reduced the rate of reaction significantly, which indicates ionic strength-dependent changes in the structure of S1. Limited trypsinolysis of S1 before the FHS reaction also decreased the rate of the reaction showing that the structural integrity of S1 is needed for the reactivity of Lys553. ATP, ADP, ADP.BeF(x), ADP.AlF(4), ADP.V(i) and pyrophosphate significantly decreased the rate of Lys553 labelling, suggesting nucleotide-induced conformational changes in the environment of Lys553. The fluorescence emission spectrum of the Lys553-bound FH moiety and the quenching of its fluorescence by nitromethane was not influenced by nucleotides, implying that the chemical reactivity but not the accessibility of Lys553 was decreased by the nucleotide-induced conformational change. In the presence of ATP when the M(**)ADP.P(i) state of the ATPase cycle is predominantly populated, the reaction rate decreased more than in the case of the S1.ADP.AlF(4)(-) and S1.ADP.V(i) complexes, which are believed to mimic the M(**)ADP.P(i) state. This indicates that the conformation of the S1-ADP.AlF(4)(-) and S1.ADP.V(i) complexes in the vicinity of Lys553 does not resemble the structure of the M(**)ADP.P(i) state. The rate of Lys553 labelling decreased strongly in the presence of actin. The nitromethane quenching of the Lys553-bound FHS was not influenced by actin, which indicates that the reduced reaction rate is not due to steric hindrance caused by the bulky protein but by actin induced conformational changes in the vicinity of Lys553.     

Structural and functional impact of site-directed methionine oxidation in myosin

We have examined the structural and functional effects of site-directed methionine oxidation in Dictyostelium (Dicty) myosin II using mutagenesis, in vitro oxidation, and site-directed spin-labeling for electron paramagnetic resonance (EPR). Protein oxidation by reactive oxygen and nitrogen species is critical for normal cellular function, but oxidative stress has been implicated in disease progression and biological aging. Our goal is to bridge understanding of protein oxidation and muscle dysfunction with molecular-level insights into actomyosin interaction. In order to focus on methionine oxidation and to facilitate site-directed spectroscopy, we started with a Cys-lite version of Dicty myosin II. For Dicty myosin containing native methionines, peroxide treatment decreased actin-activated myosin ATPase activity, consistent with the decline in actomyosin function previously observed in biologically aged or peroxide-treated muscle. Methionine-to-leucine mutations, used to protect specific sites from oxidation, identified a single methionine that is functionally sensitive to oxidation: M394, near the myosin cardiomyopathy loop in the actin-binding interface. Previously characterized myosin labeling sites for spectroscopy in the force-producing region and actin-binding cleft were examined; spin-label mobility and distance measurements in the actin-binding cleft were sensitive to oxidation, but particularly in the presence of actin. Overall secondary structure and thermal stability were unaffected by oxidation. We conclude that the oxidation-induced structural change in myosin includes a redistribution of existing structural states of the actin-binding cleft. These results will be applicable to the many biological and therapeutic contexts in which a detailed understanding of protein oxidation as well as function and structure relationships is sought.     

Myosin and Tropomyosin Stabilize the Conformation of Formin-nucleated Actin Filaments

The conformational elasticity of the actin cytoskeleton is essential for its versatile biological functions. Increasing evidence supports that the interplay between the structural and functional properties of actin filaments is finely regulated by actin-binding proteins; however, the underlying mechanisms and biological consequences are not completely understood. Previous studies showed that the binding of formins to the barbed end induces conformational transitions in actin filaments by making them more flexible through long range allosteric interactions. These conformational changes are accompanied by altered functional properties of the filaments. To get insight into the conformational regulation of formin-nucleated actin structures, in the present work we investigated in detail how binding partners of formin-generated actin structures, myosin and tropomyosin, affect the conformation of the formin-nucleated actin filaments using fluorescence spectroscopic approaches. Time-dependent fluorescence anisotropy and temperature-dependent Förster-type resonance energy transfer measurements revealed that heavy meromyosin, similarly to tropomyosin, restores the formin-induced effects and stabilizes the conformation of actin filaments. The stabilizing effect of heavy meromyosin is cooperative. The kinetic analysis revealed that despite the qualitatively similar effects of heavy meromyosin and tropomyosin on the conformational dynamics of actin filaments the mechanisms of the conformational transition are different for the two proteins. Heavy meromyosin stabilizes the formin-nucleated actin filaments in an apparently single step reaction upon binding, whereas the stabilization by tropomyosin occurs after complex formation. These observations support the idea that actin-binding proteins are key elements of the molecular mechanisms that regulate the conformational and functional diversity of actin filaments in living cells.     

Glycine 699 is pivotal for the motor activity of skeletal muscle myosin

Myosin couples ATP hydrolysis to the translocation of actin filaments to power many forms of cellular motility. A striking feature of the structure of the muscle myosin head domain is a 9-nm long "lever arm" that has been postulated to produce a 5-10-nm power stroke. This motion must be coupled to conformational changes around the actin and nucleotide binding sites. The linkage of these sites to the lever arm has been analyzed by site-directed mutagenesis of a conserved glycine residue (G699) found in a bend joining two helices containing the highly reactive and mobile cysteine residues, SH1 and SH2. Alanine mutagenesis of this glycine (G699A) dramatically alters the motor activity of skeletal muscle myosin, inhibiting the velocity of actin filament movement by > 100-fold. Analysis of the defect in the G699A mutant myosin is consistent with a marked slowing of the transition within the motor domain from a strong binding to a weak binding interaction with actin. This result is interpreted in terms of the role of this residue (G699) as a pivot point for motion of the lever arm. The recombinant myosin used in these experiments has been produced in a unique expression system. A shuttle vector containing a regulated muscle-specific promoter has been developed for the stable expression of recombinant myosin in C2C12 cells. The vector uses the promoter/enhancer region, the first two and the last five exons of an embryonic rat myosin gene, to regulate the expression of an embryonic chicken muscle myosin cDNA. Stable cell lines transfected with this vector express the unique genetically engineered myosin after differentiation into myotubes. The myosin assembles into myofibrils, copurifies with the endogenous myosin, and contains a complement of muscle-specific myosin light chains. The functional activity of the recombinant myosin is readily analyzed with an in vitro motility assay using a species-specific anti-S2 mAb to selectively assay the recombinant protein. This expression system has facilitated manipulation and analysis of the skeletal muscle myosin motor domain and is also amenable to a wide range of structure-function experiments addressing questions unique to the muscle-specific cytoarchitecture and myosin isoforms.     

Selective cleavage at lysine of the 50 kDa-20 kDa connector loop segment of skeletal myosin S-1 by endoproteinase Arg-C

The reaction of endoproteinase Arg-C on the skeletal myosin head heavy chain was investigated through characterization of peptides and amino acid sequence analysis. The protease splits exclusively the 50 kDa-20 kDa junction at the lysine cluster spanning residues 639-641 and does not affect any other protease-sensitive region of the entire myosin heavy chain. The sensitivity of the cleavage to actin and nucleotide binding makes this protease a very specific conformational probe of S-1. The nicked S-1 derivative, containing an intact NH2-terminal 75 kDa fragment, may serve as a tool for gaining further insights into the domain structure and function of the myosin head.     

Mechanism of blebbistatin inhibition of myosin II

Blebbistatin is a recently discovered small molecule inhibitor showing high affinity and selectivity toward myosin II. Here we report a detailed investigation of its mechanism of inhibition. Blebbistatin does not compete with nucleotide binding to the skeletal muscle myosin subfragment-1. The inhibitor preferentially binds to the ATPase intermediate with ADP and phosphate bound at the active site, and it slows down phosphate release. Blebbistatin interferes neither with binding of myosin to actin nor with ATP-induced actomyosin dissociation. Instead, it blocks the myosin heads in a products complex with low actin affinity. Blind docking molecular simulations indicate that the productive blebbistatin-binding site of the myosin head is within the aqueous cavity between the nucleotide pocket and the cleft of the actin-binding interface. The property that blebbistatin blocks myosin II in an actin-detached state makes the compound useful both in muscle physiology and in exploring the cellular function of cytoplasmic myosin II isoforms, whereas the stabilization of a specific myosin intermediate confers a great potential in structural studies.     

Muscle contraction: a mechanism of energy transduction

A mechanism of muscle contraction is presented in which energy from the hydrolysis of MgATP is transferred directly to conformational strain in a flexible segment of the myosin head. That segment is proximal to both the active site and the subfragment 1—subfragment 2 hinge (the portion of the myosin molecule that connects each of its two enzymatically active globular heads to the long thin helical body). This proximity allows configurational changes at the active site, which are an intrinsic part of the enzymatic mechanism, to impose a localized strain, or distortion, near the hinge. The energy, trapped in the protein this way, is subsequently used for mechanical work when other enzymatically-induced conformational changes free the strained segment of the myosin head to unbend. As this happens, the head rotates and the distal end (opposite the hinge) attaches to the actin filament and pulls on it. In this mechanism, actin interacts with myosin in two different ways: (1) at the active site where it activates a step in the hydrolysis of MgATP that frees the head to rotate; (2) at the distal end of myosin, where it forms the grip through which the rotating head pulls on the actin filament. The first interaction allows actin to initiate primary movement of the myosin head; the second directs the force and allows the movement of the head to be used for the sliding motion of the actin and myosin filaments during contraction. In this model, there are also two different energy transfers: one occurs in the transduction process itself when energy from hydrolysis is trapped as conformational distortion in the hinge region; the other occurs, reversibly, when actin and myosin form and then break the distal grip; in this second transfer there is no net energy change in the course of a cycle. A chemical mechanism is suggested to explain actin-activation of hydrolysis at the active site-hinge region.     

Cooperativity in F-actin filaments on binding of myosin subfragments, demonstrated by fluorescence of 1, N6- ethenoadenosine diphosphate.

The fluorescence lifetime of 1,N⁶-ethenoadenosine diphosphate (?-ADP) is 33 ns when bound to F-actin at 4 °C. When heavy meromyosin or myosin subfragment-1 binds to the F-actin filament, the lifetime of ?-ADP drops, reaching 29 ns when every actin monomer is bound to a myosin head. The change in lifetime is a consequence of cooperative conformational changes among the actin monomers. The results of these experiments support the contention that there are differences in the ways in which the two heads of the myosin molecule interact with the actin filament.