Submillisecond rotational dynamics of spin-labeled myosin heads in myofibrils.

The rotational motion of crossbridges, formed when myosin heads bind to actin, is an essential element of most molecular models of muscle contraction. To obtain direct information about this molecular motion, we have performed saturation transfer EPR experiments in which spin labels were selectively and rigidly attached to myosin heads in purified myosin and in glycerinated myofibrils. In synthetic myosin filaments, in the absence of actin, the spectra indicated rapid rotational motion of heads characterized by an effective correlation time of 10 microseconds. By contrast, little or no submillisecond rotational motion was observed when isolated myosin heads (subfragment-1) were attached to glass beads or to F-actin, indicating that the bond between the myosin head and actin is quite rigid on this time scale. A similar immobilization of heads was observed in spin-labeled myofibrils in rigor. Therefore, we conclude that virtually all of the myosin heads in a rigor myofibril are immobilized, apparently owing to attachment of heads to actin. Addition of ATP to myofibrils, either in the presence or absence of 0.1 mM Ca2+, produced spectra similar to those observed for myosin filaments in the absence of actin, indicating rapid submillisecond rotational motion. These results indicate that either (a) most of the myosin heads are detached at any instant in relaxed or activated myofibrils or (b) attached heads bearing the products of ATP hydrolysis rotate as rapidly as detached heads.     

Measurement of the fraction of myosin heads bound to actin in rabbit skeletal myofibrils in rigor

Tryptic digestion of rabbit skeletal myofibrils at physiological ionic strength and pH results in cleavage of the myosin heavy chain at one site giving two bands (M_r = 200,000 and 26,000) on sodium dodecyl sulfate/polyacrylamide gels. Following addition of sodium pyrophosphate (to 1 mm) to dissociate the myosin heads from actin, tryptic proteolysis results in production of three bands, 160K², 51K and 26K, with a 74K band appearing as a precursor of the 51K and 26K species. Under these conditions, there is insignificant cleavage of heavy chain to the heavy and light meromyosins. Trypsin-digested myofibrils yield the same amount of rod as native myofibrils when digested with papain. These results indicate that actin blocks tryptic cleavage of the myosin heavy chain at a site 74K from the N terminus. From measurements of the amount of 51K species formed by digestion of rigor fibers at various sarcomere lengths, we estimate that at least 95% of the myosin heads are bound to actin at 100% overlap of thick and thin filaments. Hence all myosin molecules can bind to actin, and consequently both heads of a myosin molecule can interact simultaneously with actin filaments under rigor conditions.     

Rotational dynamics of actin-bound intermediates in the myosin ATPase cycle.

We have used saturation-transfer electron paramagnetic resonance (ST-EPR) to detect the microsecond rotational motions of spin-labeled myosin subfragment one (MSL-S1) bound to actin in the presence of the ATP analogues AMPPNP (5'-adenylylimido diphosphate) and ATP gamma S [adenosine 5'-O-(3-thiotriphosphate)], which are believed to trap myosin in strongly and weakly bound intermediate states of the actomyosin ATPase cycle, respectively. Sedimentation binding measurements were used to determine the fraction of myosin heads bound to actin under ST-EPR conditions and the fraction of heads containing bound nucleotide. ST-EPR spectra were then corrected to obtain the spectrum corresponding to the ternary complex (actin.MSL-S1.nucleotide). The ST-EPR spectrum of MSL-S1.AMPPNP bound to actin is identical to that obtained in the absence of nucleotide (rigor complex), indicating no rotational motion of MSL-S1 relative to actin on the microsecond time scale. However, MSL-S1-ATP gamma S bound to actin is rotationally mobile, with an effective rotational correlation time (tau r) of 17 +/- 2 microseconds. This motion is similar to that observed previously for actin-bound MSL-S1 during the steady-state hydrolysis of ATP [Berger et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 8753-8757]. We conclude that, in solution, the weakly bound actin-attached states of the myosin ATPase cycle undergo microsecond rotational motions, while the strongly bound intermediates do not, and that these motions are likely to be involved in the molecular mechanism of muscle contraction.     

Analysis of nucleotide myosin complexes in skeletal muscle fibres by DSC and EPR

The internal dynamics and thermal unfolding of fibre bundles prepared from rabbit psoas muscle has been studied in the presence of nucleotides by differential scanning calorimetry (DSC) and electron paramagnetic resonance (EPR) spectroscopy. Using ADP, adenosine 5'-triphosphate (ATP), AMP.PNP and inorganic phosphate analogue orthovanadate (V(i)), AlF(4)(-) and BeF(3)(-), three intermediate states of the ATP hydrolysis cycle were simulated in glycerinated muscle fibres. In the main transition of the DSC pattern, three overlapping endotherms were detected in rigor, four in strongly as well as weakly binding state of myosin to actin. Deconvolution procedure showed that the transition temperature of 67.5 degrees C was the same for rigor and strong binding state of myosin. In contrast, nucleotide binding induced shift of the melting temperatures of 52 degrees C and 67.5 degrees C, appeared a new fourth peak at 74 and 77 degrees C and produced changes in the calorimetric enthalpies. The changes of the parameters of the peak functions suggest global rearrangements of the internal structure in myosin heads in the intermediate states. In the presence of ADP or ATP plus phosphate analogue orthovanadate or beryllium fluoride, aluminium fluoride, the conventional EPR spectra of spin-labeled muscle fibres showed large changes in the ordering of the probe molecules, and a new distribution of spin labels appeared. ATP plus orthovanadate induced the orientation disorder of myosin heads; the random population of spin labels gave evidence of large local conformational and motional changes in the internal structure of myosin heads. Saturation transfer EPR measurements reported increased rotational mobility of spin labels in the presence of ATP plus phosphate analogues corresponding to weakly binding state of myosin to actin.     

Tryptic digestion of rabbit skeletal myofibrils: an enzymatic probe of myosin cross-bridges

Tryptic digestion of rabbit skeletal myofibrils under physiological ionic strength and pH conditions was used as a probe of cross-bridge interaction with actin in the presence of nucleotides and pyrophosphate. Under rigor conditions, digestion of myofibrils at 24 degrees C results in the formation of 25K, 110K [heavy meromyosin (HMM)], and light meromyosin (LMM) fragments as the main reaction products. Very little if any 50K peptide is generated in such digestions. In the presence of magnesium pyrophosphate, magnesium 5'-adenylyl imidodiphosphate (MgAMPPNP), and MgATP, the main cleavage proceeds at two positions, 25K and 75K from the N-terminal portion of myosin, yielding the 25K, 50K, and 150K species. The relative amounts of the 50K, 110K, and 150K peptides and the rates of myosin heavy-chain digestion in the presence of pyrophosphate and AMPPNP indicate partial dissociation of myosin from actin. Direct centrifugation measurements of the binding of HMM and subfragment 1 (S-1) to actin in myofibrils confirm that cross-bridges partition between attached and detached states in the presence of these ligands. In the presence of MgADP, HMM and S-1 remain attached to actin at 24 degrees C. However, tryptic digestion of myofibrils containing MgADP is consistent with the existence of a mixed population of attached and detached cross-bridges, suggesting that only one head on each myosin molecule is attached to actin. As shown by tryptic digestion of myofibrils and the measurements of HMM and S-1 binding to actin, nucleotide- and pyrophosphate-induced dissociation of cross-bridges is more pronounced at 4 than at 24 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic rates of tryptic digestion of bovine cardiac myofibrils. An improved measurement of cross-bridge dissociation

The rates of tryptic digestion of the 50/20-kDa junction in myosin in cardiac myofibrils were determined under various solvent conditions. This cleavage reaction is slow in the rigor solvent and proceeds at a fast rate in the presence of MgATP. When the reaction solvent contains 50% ethylene glycol, the digestion of myosin in the presence of MgATP occurs at the same rate as in myofibrils relaxed by Mg adenyl-5'-yl imidodiphosphate (AMP-PNP). It is shown that with the help of two reference rates of digestion, for attached and dissociated myosin heads, the initial cleavage rates of myosin in the presence of nucleotides accurately measure the dissociation of cross-bridges from actin in myofibrils. Under physiological salt conditions and at 24 degrees C, MgADP, MgPPi, and MgAMP-PNP cause only small cross-bridge detachment (less than or equal to 15%) in cardiac myofibrils. The dissociation of myosin from actin is greatly increased by lowering the solvent temperature to 4 degrees C. Lowering the salt concentration of the solvent from 0.1 to 0.01 M NaCl has the most pronounced effect on the rates of myosin digestion in the presence of MgATP. In the low salt medium a substantial fraction of myosin heads (at least 30%) appears to be attached to actin in the presence of 5 mM MgATP.     

Orientation changes of the myosin light chain domain during filament sliding in active and rigor muscle

Structural changes in myosin power many types of cell motility including muscle contraction. Tilting of the myosin light chain domain (LCD) seems to be the final step in transducing the energy of ATP hydrolysis, amplifying small structural changes near the ATP binding site into nanometer-scale motions of the filaments. Here we used polarized fluorescence measurements from bifunctional rhodamine probes attached at known orientations in the LCD to describe the distribution of orientations of the LCD in active contraction and rigor. We applied rapid length steps to perturb the orientations of the population of myosin heads that are attached to actin, and thereby characterized the motions of these force-bearing myosin heads. During active contraction, this population is a small fraction of the total. When the filaments slide in the shortening direction in active contraction, the long axis of LCD tilts towards its nucleotide-free orientation with no significant twisting around this axis. In contrast, filament sliding in rigor produces coordinated tilting and twisting motions.     

Functional and ultrastructural effects of a missense mutation in the indirect flight muscle-specific actin gene of Drosophila melanogaster.

A single-site mutation of the flight-muscle-specific actin gene of Drosophila melanogaster causes a substitution of glutamic acid 93 by lysine in all the actin encoded in the indirect flight muscle (IFM). In these Act88FE93K mutants, myofibrillar bundles of thick and thin filaments are present but lack Z-discs and all sarcomeric repeats. Dense filament bundles, which are probably aberrant Z-discs, are seen in myofibrils of pupal flies, but early in adult life these move to the periphery of the fibrils and are not seen in skinned adult fibres. Consistent with this observation, alpha-actinin and other high molecular weight proteins, possibly associated with Z-discs, are not detected on SDS/polyacrylamide gels or Western blots of skinned adult IFM. The mutation lies at the beginning of a loop in the small domain of actin, near the myosin binding region. However, that the mutant actin binds myosin heads is shown by (1) rigor crossbridges in electron micrographs, (2) the appropriate rise in stiffness when ATP is withdrawn in mechanical experiments, and (3) equal protection against tryptic digestion provided by rigor binding between actin and myosin in both wild-type and mutant fibres. Reversal of rigor chevron angle along some thin filaments reflects reversal of thin-filament polarity due to lattice disorder. The absence of Z-discs, alpha-actinin and two high molecular weight proteins, and binding studies by others, suggest that the substitution at residue 93 affects the binding of the mutant actin to a protein, possibly alpha-actinin, which is necessary for Z-disc assembly or maintenance.     

Microsecond rotational motion of spin-labeled myosin heads during isometric muscle contraction. Saturation transfer electron paramagnetic resonance.

We have used saturation transfer electron paramagnetic resonance (ST-EPR) to detect the microsecond rotational motions of spin-labeled myosin heads in bundles of skinned muscle fibers, under conditions of rigor, relaxation, and isometric contraction. Experiments were performed on fiber bundles perfused continuously with an ATP-regenerating system. Conditions were identical to those we have used in previous studies of myosin head orientation, except that the fibers were perpendicular to the magnetic field, making the spectra primarily sensitive to rotational motion rather than to the orientational distribution. In rigor, the high intensity of the ST-EPR signal indicates the absence of microsecond rotational motion, showing that heads are all rigidly bound to actin. However, in both relaxation and contraction, considerable microsecond rotational motion is observed, implying that the previously reported orientational disorder under these conditions is dynamic, not static, on the microsecond time scale. The behavior in relaxation is essentially the same as that observed when myosin heads are detached from actin in the absence of ATP (Barnett and Thomas, 1984), corresponding to an effective rotational correlation time of approximately 10 microseconds. Slightly less mobility is observed during contraction. One possible interpretation is that in contraction all heads have the same mobility, corresponding to a correlation time of approximately 25 microseconds. Alternatively, more than one motional population may be present. For example, assuming that the spectrum in contraction is a linear combination of those in relaxation (mobile) and rigor (immobile), we obtained a good fit with a mole fraction of 78-88% of the heads in the mobile state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microsecond rotational dynamics of phosphorescent-labeled muscle cross-bridges

We have measured the microsecond rotational motions of myosin heads in muscle cross-bridges under physiological ionic conditions at 4 degrees C, by detecting the time-resolved phosphorescence of eosin-maleimide covalently attached to heads in skeletal muscle myofibrils. The anisotropy decay of heads in rigor (no ATP) is constant over the time range from 0.5 to 200 microsecond, indicating that they do not undergo rotational motion in this time range. In the presence of 5 mM MgATP, however, heads undergo complex rotational motion with correlation times of about 5 and 40 microsecond. The motion of heads in relaxed myofibrils is restricted out to 1 ms, as indicated by a nonzero value of the residual anisotropy. The anisotropy decay of eosin-labeled myosin, extracted from labeled myofibrils, also exhibits complex decay on the 200-microsecond time scale when assembled into synthetic thick filaments. The correlation times and amplitudes of heads in filaments (under the same ionic conditions as the myofibril experiments) are unaffected by MgATP and very similar to the values for heads in relaxed myofibrils. The larger residual anisotropy and longer correlation times seen in myofibrils are consistent with a restriction of rotational motion in the confines of the myofibril protein lattice. These are the first time-resolved measurements under physiological conditions of the rotational motions of cross-bridges in the microsecond time range.     

Characteristics of troponin C binding to the myofibrillar thin filament: extraction of troponin C is not random along the length of the thin filament.

Troponin C (TnC) is the Ca(2+)-sensing subunit of troponin responsible for initiating the cascade of events resulting in contraction of striated muscle. This protein can be readily extracted from myofibrils with low-ionic-strength EDTA-containing buffers. The properties of TnC extraction have not been characterized at the structural level, nor have the interactions of TnC with the native myofibrillar thin filament been studied. To address these issues, fluorescein-labeled TnC, in conjunction with high-resolution digital fluorescence microscopy, was used to characterize TnC binding to myofibrils and to determine the randomness of TnC extraction. Fluorescein-5-maleimide TnC (F5M TnC) retained biological activity, as evidenced by reconstitution of Ca(2+)-dependent ATPase activity in extracted myofibrils and binding to TnI in a Ca(2+)-sensitive manner. The binding of F5M TnC to highly extracted myofibrils at low Ca2+ was restricted to the overlap region under rigor conditions, and the location of binding was not influenced by F5M TnC concentration. The addition of myosin subfragment 1 to occupy all actin sites resulted in F5M TnC being bound in both the overlap and nonoverlap regions. However, very little F5M TnC was bound to myofibrils under relaxing conditions. These results suggest that strong binding of myosin heads enhances TnC binding. At high Ca2+, the pattern of F5M TnC binding was concentration dependent: binding was restricted to the overlap region at low F5M TnC concentration, whereas the binding propagated into the nonoverlap region at higher levels. Analysis of fluorescence intensity showed the greatest binding of F5M TnC at high Ca2+ with S1, and these conditions were used to characterize partially TnC-extracted myofibrils. Comparison of partially extracted myofibrils showed that low levels of extraction were associated with greater F5M TnC being bound in the nonoverlap region than in the overlap region relative to higher levels of extraction. These results show that TnC extraction is not random along the length of the thin filament, but occurs more readily in the nonoverlap region. This observation, in conjunction with the influence of rigor heads on the pattern of F5M TnC binding, suggests that strong myosin binding to actin stabilizes TnC binding at low Ca2+.     

Mechanism of nucleotide binding to actomyosin VI: evidence for allosteric head-head communication

We have examined the kinetics of nucleotide binding to actomyosin VI by monitoring the fluorescence of pyrene-labeled actin filaments. ATP binds single-headed 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 approximately five 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.     

ATP induces microsecond rotational motions of myosin heads crosslinked to actin.

We have used saturation transfer electron paramagnetic resonance (ST-EPR) to study the effect of ATP on the rotational dynamics of spin-labeled myosin heads crosslinked to actin (XLAS1). We have previously shown that ATP induces microsecond rotational motions in activated myofibrils or muscle fibers, but the possibility remained that the motion occurred only in the detached phase of the cross-bridge cycle. The addition of ATP to the crosslinked preparation has been shown to be a model system for active cross-bridges, presumably providing an opportunity to measure the motion in the attached state, without interference from unattached heads. In the absence of ATP, XLAS1 had very little microsecond rotational mobility, yielding a spectrum identical to that observed for uncrosslinked acto-S1. This suggests that all of the labeled S1 forms normal rigor complexes when crosslinked to actin. The addition of 5 mM ATP greatly increased the microsecond rotational mobility of XLAS1, and the effects were reversed upon depletion of ATP. The most plausible explanation for these results is that myosin heads undergo microsecond rotational motion while attached actively to actin during steady state ATPase activity. These results have important implications for the interpretation of spectroscopic data obtained during muscle contraction.     

Cooperative actions between myosin heads bring effective functions

A recent study with single molecule measurements has reported that muscle myosin, a molecular motor, stochastically generates multiple steps along an actin filament associated with the hydrolysis of a single ATP molecule [Kitamura, K., Tokunaga, M., Esaki, S., Iwane, A.H., Yanagida, T., 2005. Mechanism of muscle contraction based on stochastic properties of single actomyosin motors observed in vitro. Biophysics 1, 1-19]. We have built a model reproducing such a stochastic movement of a myosin molecule incorporated with ATPase reaction cycles and demonstrated that the thermal fluctuation was a key for the function of myosin molecules [Esaki, S., Ishii, Y., Yanagida, T., 2003. Model describing the biased Brownian movement of myosin. Proc. Jpn. Acad. 79 (Ser B), 9-14]. The size of the displacement generated during the hydrolysis of single ATP molecules was limited within a half pitch of an actin filament when a single myosin molecules work separately. However, in muscle the size of the displacement has been reported to be greater than 60 nm [Yanagida, T., Arata, T., Oosawa, F., 1985. Sliding distance of actin filament induced by a myosin crossbridge during one ATP hydrolysis cycle. Nature 316, 366-369; Higuchi et al., 1991]. The difference suggests cooperative action between myosin heads in muscle. Here we extended the model built for an isolated myosin head to a system in which myosin heads are aligned in muscle arrangement to understand the cooperativity between heads. The simulation showed that the rotation of the actin filament [Takezawa, Y., Sugimoto, Y., Wakabayashi, K., 1998. Extensibility of the actin and myosin filaments in various states of skeletal muscles as studied by X-ray diffraction. Adv. Exp. Med. Biol. 453, 309-317; Wakabayashi, K., Ueno, Y., Takezawa, Y., Sugimoto, Y., 2001. Muscle contraction mechanism: use of X-ray synchrotron radiation. Nat. Enc. Life Sci. 1-11] associated with the release of ATPase products and binding of ATP as well as interaction between myosin heads allowed the myosin filament to move greater than a half pitch of the actin filament while a single ATP molecule is hydrolyzed. Our model demonstrated that the movement is loosely coupled to the ATPase cycle as observed in muscle.     

Kinetics of the initial steps of rabbit psoas myofibrillar ATPases studied by tryptophan and pyrene fluorescence stopped-flow and rapid flow-quench. Evidence that cross-bridge detachment is slower than ATP binding

The kinetics of the tryptophan fluorescence enhancement that occurs when myofibrils (rabbit psoas) are mixed with Mg-ATP were studied by stopped-flow in different solvents (water, 40% ethylene glycol, 20% methanol) at 4 degrees C. Under relaxing conditions (low Ca(2+)) in water (mu = 0.16 M, pH 7.4) and at high ATP concentrations, the transient was biphasic, giving a k(fast)(max) of 230 s(-)(1) and a k(slow)(max) of 15 s(-)(1). The kinetics of the two phases were compared with those obtained by chemical sampling using [gamma-(32)P]ATP and quenching in acid (P(i) burst experiments: these give unambiguously the ATP cleavage kinetics), or cold Mg-ATP (cold ATP chase: ATP binding kinetics). k(slow) is due to ATP cleavage, as with S1. Interestingly, k(fast) is slower than the ATP binding kinetics. Instead, this constant appears to report ATP-induced cross-bridge detachment from actin because (1) it was identical to the fluorescence transient obtained on addition of ATP to pyrene-labeled myofibrils; (2) when the initial filament overlap in the myofibrils was decreased, the amplitude of the fast phase decreased; (3) there was no fluorescent enhancement upon the addition of ADP to myofibrils. This is different from the situation with S1 or actoS1 where there was also a fast fluorescent ATP-induced transient but whose kinetics were identical to those of the tight ATP binding. To increase the time resolution and to confirm our results, we also carried out transient kinetics in ethylene glycol and methanol. We interpret our results by a scheme in which a rapid equilibrium between attached (AM.ATP) and detached (M.ATP) states is modulated by the fraction of myosin heads in rigor (AM) during the time of experiment.     

Rigor-force producing cross-bridges in skeletal muscle fibers activated by a substoichiometric amount of ATP

Isometric skinned muscle fibers were activated by the photogeneration of a substoichiometric amount of ATP and their cross-bridge configurations examined during the development of the rigor force by x-ray diffraction and electron microscopy. By the photogeneration of approximately 100 microM ATP, approximately 2/3 of the concentration of the myosin heads in a muscle fiber, muscle fibers originally in the rigor state showed a transient drop of the force and then produced a long-lasting rigor force (approximately 50% of the maximal active force), which gradually recovered to the original force level with a time constant of approximately 4 s. Associated with the photoactivation, muscle fibers revealed small but distinct changes in the equatorial x-ray diffraction that run ahead of the development of force. After reaching a plateau of force, long-lasting intensity changes in the x-ray diffraction pattern developed in parallel with the force decline. Two-dimensional x-ray diffraction patterns and electron micrographs of the sectioned muscle fibers taken during the period of 1-1.9 s after the photoactivation were basically similar to those from rigor preparations but also contained features characteristic of fully activated fibers. In photoactivated muscle fibers, some cross-bridges bound photogenerated ATP and underwent an ATP hydrolysis cycle whereas a significant population of the cross-bridges remained attached to the thin actin filaments with no available ATP to bind. Analysis of the results obtained indicates that, during the ATP hydrolysis reaction, the cross-bridges detached from actin filaments and reattached either to the same original actin monomers or to neighboring actin monomers. The latter cross-bridges contribute to produce the rigor force by interacting with the actin filaments, first producing the active force and then being locked in a noncycling state(s), transforming their configuration on the actin filaments to stably sustain the produced force as a passive rigor force.     

Structural characterization of the binding of Myosin*ADP*Pi to actin in permeabilized rabbit psoas muscle

When myosin is attached to actin in a muscle cell, various structures in the filaments are formed. The two strongly bound states (A*M*ADP and A*M) and the weakly bound A*M*ATP states are reasonably well understood. The orientation of the strongly bound myosin heads is uniform ("stereospecific" attachment), and the attached heads exhibit little spatial fluctuation. In the prehydrolysis weakly bound A*M*ATP state, the orientations of the attached myosin heads assume a wide range of azimuthal and axial angles, indicating considerable flexibility in the myosin head. The structure of the other weakly bound state, A*M*ADP*P(i), however, is poorly understood. This state is thought to be the critical pre-power-stroke state, poised to make the transition to the strongly binding, force-generating states, and hence it is of particular interest for understanding the mechanism of contraction. However, because of the low affinity between myosin and actin in the A*M*ADP*P(i) state, the structure of this state has eluded determination both in isolated form and in muscle cells. With the knowledge recently gained in the structures of the weakly binding M*ATP, M*ADP*P(i) states and the weakly attached A*M*ATP state in muscle fibers, it is now feasible to delineate the in vivo structure of the attached state of A*M*ADP*P(i). The series of experiments presented in this article were carried out under relaxing conditions at 25 degrees C, where approximately 95% of the myosin heads in the skinned rabbit psoas muscle contain the hydrolysis products. The affinity for actin is enhanced by adding polyethylene glycol (PEG) or by lowering the ionic strength in the bathing solution. Solution kinetics and binding constants were determined in the presence and in the absence of PEG. When the binding between actin and myosin was increased, both the myosin layer lines and the actin layer lines increased in intensity, but the intensity profiles did not change. The configuration (mode) of attachment in the A*M*ADP*P(i) state is thus unique among the intermediate attached states of the cross-bridge ATP hydrolysis cycle. One of the simplest explanations is that both myosin filaments and actin filaments are stabilized (e.g., undergo reduced spatial fluctuations) by the attachment. The alignment of the myosin heads in the thick filaments and the alignment of the actin monomers in the thin filaments are improved as a result. The compact atomic structure of M*ADP*P(i) with strongly coupled domains may contribute to the unique attachment configuration: the "primed" myosin heads may function as "transient struts" when attached to the thin filaments.     

Caldesmon restricts the movement of both C- and N-termini of tropomyosin on F-actin in ghost fibers during the actomyosin ATPase cycle

New data on the movements of tropomyosin singly labeled at alpha- or beta-chain during the ATP hydrolysis cycle in reconstituted ghost fibers have been obtained by using the polarized fluorescence technique which allowed us following the azimuthal movements of tropomyosin on actin filaments. Pronounced structural changes in tropomyosin evoked by myosin heads suggested the "rolling" of the tropomyosin molecule on F-actin surface during the ATP hydrolysis cycle. The movements of actin-bound tropomyosin correlated to the strength of S1 to actin binding. Weak binding of myosin to actin led to an increase in the affinity of the tropomyosin N-terminus to actin with simultaneous decrease in the affinity of the C-terminus. On the contrary, strong binding of myosin to actin resulted in the opposite changes of the affinity to actin of both ends of the tropomyosin molecule. Caldesmon inhibited the "rolling" of tropomyosin on the surface of the thin filament during the ATP hydrolysis cycle, drastically decreased the affinity of the whole tropomyosin molecule to actin, and "freezed" tropomyosin in the position characteristic of the weak binding of myosin to actin.     

Ca(2+)-activated myofibrillar ATPase: transient kinetics and the titration of its active sites.

The transient kinetics of rabbit psoas Ca(2+)-activated myofibrillar Mg(2+)-ATPase were studied in a buffer of near physiological ionic strength at 4 degrees C by the rapid flow quench technique. The initial ATP binding steps were studied by the ATP chase and the cleavage and release of products steps were studied by the Pi burst method. The data obtained were interpreted by the simple scheme [formula; see text] represents the myosin heads with or without actin interaction. The constants obtained with myofibrils (where the molecules are highly organized) were compared with those with myosin subfragment 1 (S1) and cross-linked acto-S1 (where the molecules are dispersed in solution). Myofibrils appear to bind ATP as tightly as do S1 and cross-linked acto-S1. This suggests that with them k-2 less than kcat much less than k2, and it is proposed that the ATP chase method can be used to titrate the ATPase sites in myofibrils. The results of titration and single-turnover experiments revealed that myofibrils may contain partially active myosin heads. It is proposed that these heads bind ATP loosely without hydrolysis, as found with S1 [Tesi, C., N. Bachouchi, N., Barman, T., & Travers, F. (1989) Biochimie 71, 363-372]. There were large Pi bursts with the three preparations, showing that with all of them the release of products step (k4) is rate limiting.(ABSTRACT TRUNCATED AT 250 WORDS)     

An ionic–chemical–mechanical model for muscle contraction

The dynamic process underlying muscle contraction is the parallel sliding of thin actin filaments along an immobile thick myosin fiber powered by oar‐like movements of protruding myosin cross bridges (myosin heads). The free energy for functioning of the myosin nanomotor comes from the hydrolysis of ATP bound to the myosin heads. The unit step of translational movement is based on a mechanical‐chemical cycle involving ATP binding to myosin, hydrolysis of the bound ATP with ultimate release of the hydrolysis products, stress‐generating conformational changes in the myosin cross bridge, and relief of built‐up stress in the myosin power stroke. The cycle is regulated by a transition between weak and strong actin–myosin binding affinities. The dissociation of the weakly bound complex by addition of salt indicates the electrostatic basis for the weak affinity, while structural studies demonstrate that electrostatic interactions among negatively charged amino acid residues of actin and positively charged residues of myosin are involved in the strong binding interface. We therefore conjecture that intermediate states of increasing actin–myosin engagement during the weak‐to‐strong binding transition also involve electrostatic interactions. Methods of polymer solution physics have shown that the thin actin filament can be regarded in some of its aspects as a net negatively charged polyelectrolyte. Here we employ polyelectrolyte theory to suggest how actin–myosin electrostatic interactions might be of significance in the intermediate stages of binding, ensuring an engaged power stroke of the myosin motor that transmits force to the actin filament, and preventing the motor from getting stuck in a metastable pre‐power stroke state. We provide electrostatic force estimates that are in the pN range known to operate in the cycle.