Orientation of spin-labeled light chain-2 exchanged onto myosin cross-bridges in glycerinated muscle fibers.

Electron paramagnetic resonance (EPR) spectroscopy has been used to study the angular distribution of a spin label attached to rabbit skeletal muscle myosin light chain 2. A cysteine reactive spin label, 3-(5-fluoro-2,4-dinitroanilino)-2,2,5,5- tetramethyl-1-pyrrolidinyloxy (FDNA-SL) was bound to purified LC2. The labeled LC2 was exchanged into glycerinated muscle fibers and into myosin and its subfragments. Analysis of the spectra of labeled fibers in rigor showed that the probe was oriented with respect to the fiber axis, but that it was also undergoing restricted rotations. The motion of the probe could be modeled assuming rapid rotational diffusion (rotational correlation time faster than 5 ns) within a "cone" whose full width was 70 degrees. Very different spectra of rigor fibers were obtained with the fiber oriented parallel and perpendicular to the magnetic field, showing that the centroid of each cone had the same orientation for all myosin heads, making an angle of approximately 74 degrees to the fiber axis. Binding of light chains or labeled myosin subfragment-1 to ion exchange heads immobilized the probes, showing that most of the motion of the probe arose from protein mobility and not from mobility of the probe relative to the protein. Relaxed labeled fibers produced EPR spectra with a highly disordered angular distribution, consistent with myosin heads being detached from the thin filament and undergoing large angular motions. Addition of pyrophosphate, ADP, or an ATP analogue (AMPPNP), in low ionic strength buffer where these ligands do not dissociate cross-bridges from actin, failed to perturb the rigor spectrum. Applying static strains as high as 0.16 N/mm2 to the labeled rigor fibers also failed to change the orientation of the spin label. Labeled light chain was exchanged into myosin subfragment-1 (S1) and the labeled S1 was diffused into fibers. EPR spectra of these fibers had a component similar to that seen in the spectra of fibers into which labeled LC2 had been exchanged directly. However, the fraction of disordered probes was greater than seen in fibers. In summary, the above data indicate that the region of the myosin head proximal to the thick filament is ordered in rigor, and disordered in relaxation.     

Orientation of the essential light chain region of myosin in relaxed, active, and rigor muscle

The orientation of the ELC region of myosin in skeletal muscle was determined by polarized fluorescence from ELC mutants in which pairs of introduced cysteines were cross-linked by BSR. The purified ELC-BSRs were exchanged for native ELC in demembranated fibers from rabbit psoas muscle using a trifluoperazine-based protocol that preserved fiber function. In the absence of MgATP (in rigor) the ELC orientation distribution was narrow; in terms of crystallographic structures of the myosin head, the LCD long axis linking heavy-chain residues 707 and 843 makes an angle (β) of 120-125° with the filament axis. This is ∼30° larger than the broader distribution determined previously from RLC probes, suggesting that, relative to crystallographic structures, the LCD is bent between its ELC and RLC regions in rigor muscle. The ELC orientation distribution in relaxed muscle had two broad peaks with β ∼70° and ∼110°, which may correspond to the two head regions of each myosin molecule, in contrast with the single broad distribution of the RLC region in relaxed muscle. During isometric contraction the ELC orientation distribution peaked at β ∼105°, similar to that determined previously for the RLC region.     

Intradomain distances in the regulatory domain of the myosin head in prepower and postpower stroke states: fluorescence energy transfer.

The relative movement of the catalytic and regulatory domains of the myosin head (S1) is likely to be the force generating conformational change in the energy transduction of muscle [Rayment, I., Holden, H. M., Whittaker, M., Yohn, C. B., Lorenz, M., Holmes, K. C., and Milligan, R. A. (1993) Science 261, 58-65]. To test this model we have measured, using frequency-modulated FRET, three distances between the catalytic domain and regulatory domains and within the regulatory domain of myosin. The donor/acceptor pairs included MHC cys707 and ELC cys177; ELC cys177 and RLC cys154; and ELC cys177 and gizzard RLC cys108. The IAEDANS (donor) or acceptor (DABMI or IAF) labeled light chains (ELC and RLC) were exchanged into monomeric myosin and the distances were measured in the putative prepower stroke states (in the presence of MgATP or ADP/AlF(4-)) and the postpower stroke states (ADP and the absence of nucleotides). For each of the three distances, the donor/acceptor pairs were reversed to minimize uncertainty in the distance measured, arising from probe orientational factors. The distances obtained from FRET were in close agreement with the distances in the crystal structure. Importantly, none of the measured distances varied by more than 2 A, putting a strong constraint on the extent of conformational changes within S1. The maximum axial movement of the distal part of myosin head was modeled using FRET distance changes within the myosin head reported here and previously. These models revealed an upper bound of 85 A for a swing of the regulatory domain with respect to the catalytic domain during the power stroke. Additionally, an upper bound of 22 A could be contributed to the power stroke by a reorientation of RLC with respect to the ELC during the power stroke.     

Paramagnetic probes attached to a light chain on the myosin head are highly disordered in active muscle fibers.

We have measured the orientation of a region of the myosin head, close to the junction with the rod, during active force generation. Paramagnetic probes were attached specifically to a reactive cysteine (Cys 125) of purified myosin light chain 2 (LC2) and exchanged into myosin heads in glycerinated rabbit psoas muscle. Electron paramagnetic resonance spectroscopy was used to monitor the orientation of the probes. Previous work has shown that the LC2 bound spin probes are significantly ordered in rigor and muscle in the presence of adenosine diphosphate (ADP). In contrast, there is a nearly random angular distribution in relaxed muscle. We show here that during the generation of isometric tension, all of the LC2 bound spin probes (98 +/- 1.6%) show an angular distribution similar to that of relaxed muscle. These findings contrast with results obtained from probes attached to Cys 707 on the cross-bridge, located close to the actin binding site, where, during active force generation, a proportion of the spin probes were ordered as in rigor, whereas the remaining probes were disordered as in relaxation. To test the hypothesis that this ordered component is due to modification of Cys 707, we measured the spectra obtained from probes attached to LC2 in fibers modified at Cys 707. The modification of Cys 707 did not produce an ordered component in these spectra. The absence of an ordered component at the LC2 site limits the populations of some states in active fibers. An actin/myosin/ADP state is thought to be the major force-producing state. Our present results show that the populations of states with ordered probes on LC2 are < 2% in active fibers; thus, the major force-producing state is different from the one obtained by addition of ADP to rigor fibers.     

Influence of the bound nucleotide on the molecular dynamics of actin

Rotational dynamics of actin spin-labelled with maleimide probes at the reactive thiol Cys-374 were studied. Replacement of the bound nucleotide by Br8ATP in G-actin and Br8ADP in F-actin causes significant increase of the rotational correlation time of the spin probe, indicating reduced motion in both G and F-actin. The orientation dependence of the electron paramagnetic resonance spectra in oriented F-actin filaments revealed an altered molecular order of the probe when the nucleotide was a Br-substituted one. The bound nucleotide affects the myosin S1 ATPase activation by actin; both Vmax and K(actin) decreased significantly when the bound nucleotide of actin was Br8ADP.     

Two new modes of smooth muscle myosin regulation by the interaction between the two regulatory light chains, and by the S2 domain

Previous studies indicated that single-headed smooth muscle myosin and S1 (a single head fragment) are not regulated through phosphorylation of the regulatory light chain (RLC). To investigate the importance of the double-headedness of myosin and of the S2 region for the phosphorylation-dependent regulation, we made three types of recombinant mutant smooth muscle HMMs with one intact head and an N-terminally truncated head. The truncated head of Delta MD lacked the motor domain, that of Delta(MD+ELC) lacked the motor and essential light chain binding domains, and single-headed HMM had one intact head alone. The basal ATPase activities of the three mutants decreased as the KCl concentration became less than 0.1 M. Such a decrease was not observed for S1, which had no S2 region, suggesting that S2 is necessary for this myosin behavior. This activity decrease also disappeared when RLCs of Delta MD and Delta(MD+ELC), but that of single-headed HMM, were phosphorylated. When their RLCs were unphosphorylated, the three mutants exhibited similar actin-activated ATPase levels. However, when they were phosphorylated, the actin-activated ATPase activities of Delta MD and Delta(MD+ELC) increased to the S1 level, while that of single-headed HMM remained unchanged. Even in the phosphorylated state, the actin-activated ATPase activities of the three mutants and S1 were much lower than that of wild-type HMM. We propose that S2 has an inhibitory function that is canceled by an interaction between two phosphorylated RLCs. We also propose that a cooperative interaction between two motor domains is required for a higher level of actin activation.     

Phosphorylation regulates the ADP-induced rotation of the light chain domain of smooth muscle myosin.

We have observed the effects of MgADP and thiophosphorylation on the conformational state of the light chain domain of myosin in skinned smooth muscle. Electron paramagnetic resonance (EPR) spectroscopy was used to monitor the orientation of spin probes attached to the myosin regulatory light chain (RLC). Two spectral states were seen, termed here "intermediate" and "final", that are distinguished by a approximately 24 degrees axial rotation of spin probes attached to the RLC. The two observed conformations are similar to those found previously for smooth muscle myosin S1; the final state corresponds to the major conformation of S1 in the absence of ADP, while the intermediate state corresponds to the conformation of S1 with ADP bound. Light chain domain orientation was observed as a function of the MgADP concentration and the extent of RLC thiophosphorylation. In rigor (no MgADP), LC domains were distributed equally between the intermediate state and the final state; upon addition of saturating (3.5 mM) MgADP, about one-third of the LC domains in the final state rotated approximately 20 degrees axially to the intermediate state. The progression of the change in populations was fit to a simple binding equation, yielding an apparent dissociation constant of approximately 110 microM for skinned smooth muscle fibers and approximately 730 microM for thiophosphorylated, skinned smooth muscle fibers. These observations suggest a model that explains the behavior of "latch bridges" in smooth muscle.     

Caldesmon freezes the structure of actin filaments during the actomyosin ATPase cycle

Hybrid contractile apparatus was reconstituted in skeletal muscle ghost fibers by incorporation of skeletal muscle myosin subfragment 1 (S1), smooth muscle tropomyosin and caldesmon. The spatial orientation of FITC-phalloidin-labeled actin and IAEDANS-labeled S1 during sequential steps of the acto-S1 ATPase cycle was studied by measurement of polarized fluorescence in the absence or presence of nucleotides conditioning the binding affinity of both proteins. In the fibers devoid of caldesmon addition of nucleotides evoked unidirectional synchronous changes in the orientation of the fluorescent probes attached to F-actin or S1. The results support the suggestion on the multistep rotation of the cross-bridge (myosin head and actin monomers) during the ATPase cycle. The maximal cross-bridge rotation by 7 degrees relative to the fiber axis and the increase in its rigidity by 30% were observed at transition between A**.M**.ADP.Pi (weak binding) and A--.M--.ADP (strong binding) states. When caldesmon was present in the fibers (OFF-state of the thin filament) the unidirectional changes in the orientation of actin monomers and S1 were uncoupled. The tilting of the myosin head and of the actin monomer decreased by 29% and 90%, respectively. It is suggested that in the "closed" position caldesmon "freezes" the actin filament structure and induces the transition of the intermediate state of actomyosin towards the weak-binding states, thereby inhibiting the ATPase activity of the actomyosin.     

Effects of AMPPNP on the orientation and rotational dynamics of spin-labeled muscle cross-bridges.

We have used electron paramagnetic resonance (EPR) to investigate the orientation, rotational motion, and actin-binding properties of rabbit psoas muscle cross-bridges in the presence of the nonhydrolyzable nucleotide analogue, 5'-adenylylimido-diphosphate (AMPPNP). This analogue is known to decrease muscle tension without affecting its stiffness, suggesting an attached cross-bridge state different from rigor. We spin-labeled the SH1 groups on myosin heads and performed conventional EPR to obtain high-resolution information about the orientational distribution, and saturation transfer EPR to measure microsecond rotational motion. At 4 degrees C and 100 mM ionic strength, we find that AMPPNP increases both the orientational disorder and the microsecond rotational motion of myosin heads. However, computer analysis of digitized spectra shows that no new population of probes is observed that does not match either rigor or relaxation in both orientation and motion. At 4 degrees C, under nearly saturating conditions of 16 mM AMPPNP (Kd = 3.0 mM, determined from competition between AMPPNP and an ADP spin label), 47.5 +/- 2.5% of myosin heads are dynamically disoriented (as in relaxation) without a significant decrease in rigor stiffness, whereas the remainder are rigidly oriented as in rigor. The oriented heads correspond to actin-attached heads in a ternary complex, and the disoriented heads correspond to detached heads, as indicated by EPR experiments with spin-labeled subfragment 1 (S1) that provide independent measurements of orientation and binding. We take these findings as evidence for a single-headed cross-bridge that is as stiff as the double-headed rigor cross-bridge. The data are consistent with a model in which, in the presence of saturating AMPPNP, one head of each cross-bridge binds actin about 10 times more weakly, whereas the remaining head binds at least 10 times more strongly, than extrinsic S1. Thus, although there is no evidence for heads being attached at nonrigor angles, the attached cross-bridge differs from that of rigor. The heterogeneous behavior of heads is probably due to steric effects of the filament lattice.     

Role of the essential light chain in the activation of smooth muscle myosin by regulatory light chain phosphorylation

The activity of smooth and non-muscle myosin II is regulated by phosphorylation of the regulatory light chain (RLC) at serine 19. The dephosphorylated state of full-length monomeric myosin is characterized by an asymmetric intramolecular head–head interaction that completely inhibits the ATPase activity, accompanied by a hairpin fold of the tail, which prevents filament assembly. Phosphorylation of serine 19 disrupts these head–head interactions by an unknown mechanism. Computational modeling (Tama et al., 2005. J. Mol. Biol. 345, 837–854) suggested that formation of the inhibited state is characterized by both torsional and bending motions about the myosin heavy chain (HC) at a location between the RLC and the essential light chain (ELC). Therefore, altering relative motions between the ELC and the RLC at this locus might disrupt the inhibited state. Based on this hypothesis we have derived an atomic model for the phosphorylated state of the smooth muscle myosin light chain domain (LCD). This model predicts a set of specific interactions between the N-terminal residues of the RLC with both the myosin HC and the ELC. Site directed mutagenesis was used to show that interactions between the phosphorylated N-terminus of the RLC and helix-A of the ELC are required for phosphorylation to activate smooth muscle myosin.     

Myosin essential light chain in health and disease

The essential light chain of myosin (ELC) is known to be important for structural stability of the alpha-helical lever arm domain of the myosin head, but its function in striated muscle contraction is poorly understood. Two ELC isoforms are expressed in fast skeletal muscle, a long isoform and its NH(2)-terminal approximately 40 amino acid shorter counterpart, whereas only the long ELC is observed in the heart. Biochemical and structural studies revealed that the NH(2)-terminus of the long ELC can make direct contacts with actin, but the effects of the ELC on the affinity of myosin for actin, ATPase, force, and the kinetics of force generating myosin cross-bridges are inconclusive. Myosin containing the long ELC has been shown to have slower cross-bridge kinetics than myosin with the short isoform. A difference was also reported among myosins with long isoforms. Increased shortening velocity was observed in atrial compared with ventricular muscle fibers. The common findings suggest that ELC provides the fine tuning of the myosin motor function, which is regulated in an isoform and tissue-dependent manner. The functional importance of the ELC is further implicated by the discovery of ELC mutations associated with Familial Hypertrophic Cardiomyopathy. The pathological phenotypes vary in severity, but more notably, almost all ELC mutations result in sudden cardiac death at a young age. This review summarizes the functional roles of striated muscle ELC in normal healthy muscle and in disease. Transgenic animal models and phenotypic characterization of ELC-mediated remodeling of the heart are also discussed.     

Essential light chain modulates phosphorylation-dependent regulation of smooth muscle myosin

To examine the functional role of the essential light chain (ELC) in the phosphorylation-dependent regulation of smooth muscle myosin, we replace the native light chain in smooth muscle myosin with bacterially expressed chimeric ELCs in which one or two of the four helix-loop-helix domains of chicken gizzard ELC were substituted by the corresponding domains of scallop (Aquipecten irradians) ELC. All of these myosins, regardless of the ELC mutations or regulatory light chain (RLC) phosphorylation, showed normal subunit constitutions and NH(4)(+)/EDTA-ATPase activities, both of which were similar to those of native myosin. None of the ELC mutations changed the actin-activated ATPase activity of myosin in the absence of RLC phosphorylation. However, in the presence of RLC phosphorylation, the substitution of domain 1 or 2 in the ELC significantly decreased the actin-activated ATPase activity, whereas the substitution of both of these domains did not change the activity. In contrast to myosin, the domain 2 substitution in the ELC did not affect the actin-activated ATPase activity of single-headed myosin subfragment 1. These results suggest an interhead interaction between domains 1 and 2 of ELCs which is required to attain the full actin-activated ATPase activity of smooth muscle myosin in the presence of RLC phosphorylation.     

Essential Light Chains of Myosin and Their Role in Functioning of the Myosin Motor

This review summarizes current data on the structure and functions of myosin essential light chains (ELCs) and on their role in functioning of the myosin head as a molecular motor. The data on structural and functional features of the N-terminal extension of myosin ELC from skeletal and cardiac muscles are analyzed; the role of this extension in the ATP- dependent interaction of myosin heads with actin in the molecular mechanism of muscle contraction is discussed. The data on possible interactions of the ELC N-terminal extension with the myosin head motor domain in the myosin ATPase cycle are presented, including the results of the authors’ studies that are in favor of such interactions.     

Saturation transfer electron paramagnetic resonance of spin-labeled muscle fibers. Dependence of myosin head rotational motion on sarcomere length

We have investigated the orientation and rotational mobility of spin-labeled myosin heads in muscle fibers as a function of the sarcomere length in the absence of ATP. An iodoacetamide spin label was used to label selectively two-thirds of the sulfhydryl-1 groups in glycerinated rabbit psoas muscle. Conventional electron paramagnetic resonance experiments were used to determine the orientation distribution of the probes relative to the fiber axis, and saturation transfer experiments were used to detect sub-millisecond rotational motion. When fibers are at sarcomere length 2.3 microns (full overlap), spin-labeled heads have a high degree of orientational order. The probes are in a single, narrow orientation distribution (full width 15 degrees), and they exhibit no detectable sub-millisecond rotational motion. When fibers are stretched (sarcomere length increased), either before or after labeling, disorder and microsecond mobility increase greatly, in proportion to the fraction of myosin heads that are no longer in the overlap zone between the thick and thin filaments. Saturation transfer difference spectra show that a fraction of myosin heads equal to the fraction outside the overlap zone have much more rotational mobility than those in fibers at full overlap, and almost as much as in synthetic myosin filaments. The most likely interpretation is that some of the probes, corresponding approximately to the fraction of heads in the overlap zone, remain oriented and immobile, while the rest are highly disordered (angular spread greater than 90 degrees) and mobile (microsecond rotational motion). Thus, it appears that myosin heads are rigidly immobilized by actin, but they rotate through large angles on the microsecond time-scale when detached from actin, even in the absence of ATP.     

High flexibility of the actomyosin crossbridge resides in skeletal muscle myosin subfragment-2 as demonstrated by a new single molecule assay

Popular views of force generation in muscle indicate that a lever arm in the myosin head initiates displacement of the thin filament. However, this lever arm is attached to the thick filament backbone by a flexible combination of coiled coils and hinges in the myosin subfragment-2 (S2); therefore, efficient force generation depends on tension development in this linking structure. Herein, a single molecule assay is developed to examine the flexibility of the intact S2 relative to that of the myosin head. Fluorescently labeled myosin rod is polymerized onto a single myosin molecule that is bound to actin, and the resulting Brownian motion of the rod is analyzed at video rates by digital image processing. Complete rotations of the rod suggest significant amounts of random coil in the linking structure. The close similarity of twist rates for double-headed and single-headed myosin indicates that most of the flexibility originates at or beyond the first pitch of coiled coil in S2 and most likely at the hinge connecting S2 and the light meromyosin. The myosin head has a smaller but still detectable impact on this flexibility, since the addition of ADP to the rigor crossbridge produces differential effects on the torsional characteristics of double-headed versus single-headed myosin.     

Structure and dynamics of the force-generating domain of myosin probed by multifrequency electron paramagnetic resonance

Spin-labeling and multifrequency EPR spectroscopy were used to probe the dynamic local structure of skeletal myosin in the region of force generation. Subfragment 1 (S1) of rabbit skeletal myosin was labeled with an iodoacetamide spin label at C707 (SH1). X-and W-band EPR spectra were recorded for the apo state and in the presence of ADP and nucleotide analogs. EPR spectra were analyzed in terms of spin-label rotational motion within myosin by fitting them with simulated spectra. Two models were considered: rapid-limit oscillation (spectrum-dependent on the orientational distribution only) and slow restricted motion (spectrum-dependent on the rotational correlation time and the orientational distribution). The global analysis of spectra obtained at two microwave frequencies (9.4 GHz and 94 GHz) produced clear support for the second model and enabled detailed determination of rates and amplitudes of rotational motion and resolution of multiple conformational states. The apo biochemical state is well-described by a single structural state of myosin (M) with very restricted slow motion of the spin label. The ADP-bound biochemical state of myosin also reveals a single structural state (M*, shown previously to be the same as the post-powerstroke ATP-bound state), with less restricted slow motion of the spin label. In contrast, the extra resolution available at 94 GHz reveals that the EPR spectrum of the S1.ADP.V_i-bound biochemical state of myosin, which presumably mimics the S1.ADP.P_i state, is resolved clearly into three spectral components (structural states). One state is indistinguishable from that of the ADP-bound state (M*) and is characterized by moderate restriction and slow motion, with a mole fraction of 16%. The remaining 84% (M**) contains two additional components and is characterized by fast rotation about the x axis of the spin label. After analyzing EPR spectra, myosin ATPase activity, and available structural information for myosin II, we conclude that post-powerstroke and pre-powerstroke structural states (M* and M**) coexist in the S1.ADP.V_i biochemical state. We propose that the pre-powerstroke state M** is characterized by two structural states that could reflect flexibility between the converter and N-terminal domains of myosin.     

Transients in orientation of a fluorescent cross-bridge probe following photolysis of caged nucleotides in skeletal muscle fibres.

In muscle fibres labelled with iodoacetamidotetramethylrhodamine at Cys707 of the myosin heavy chain, the probes have been reported to change orientation when the fibre is activated, relaxed or put into rigor. In order to test whether these motions are indications of the cross-bridge power stroke, we monitored tension and linear dichroism of the probes in single glycerol-extracted fibres of rabbit psoas muscle during mechanical transients initiated by laser pulse photolysis of caged ATP and caged ADP. In rigor dichroism is negative, indicating average probe absorption dipole moments oriented more than 54.7 degrees away from the fibre axis. During activation from rigor induced by photoliberation of ATP from caged ATP in the presence of calcium, the dichroism reversed sign promptly (half-time 12.5 ms for 500 microM-ATP) upon release of ATP, but then changed only slightly during tension development 20 to 100 milliseconds later. During the onset of rigor following transfer of the fibre from an ATP-containing relaxing solution to a rigor medium lacking ATP, force generation preceded the change in dichroism. The dichroism change occurred slowly (half-time 47 s), because binding of ADP to sites within the muscle fibre limited its rate of diffusion out of the fibre. When ADP was introduced or removed, the dichroism transient was similar in time course and magnitude to that obtained after the introduction or removal of ATP. Neither adding nor removing ADP produced substantial changes in force. These results demonstrate that orientation of the rhodamine probes on the myosin head reflects mainly structural changes linked to nucleotide binding and release, rather than rotation of the cross-bridge during force generation.     

Orientation of spin-labeled light chain 2 of myosin heads in muscle fibers

Electron paramagnetic resonance (e.p.r.) spectroscopy has been used to monitor the orientation of spin labels attached rigidly to a reactive SH residue on the light chain 2 (LC2) of myosin heads in muscle fibers. e.p.r. spectra from spin-labeled myosin subfragment-1 (S1), allowed to diffuse into unlabeled rigor (ATP-free) fibers, were roughly approximated by a narrow angular distribution of spin labels centered at 66 degrees relative to the fiber axis, indicating a uniform orientation of S1 bound to actin. On the other hand, spectra from spin-labeled heavy meromyosin (HMM) were roughly approximated by two narrow angular distributions centered at 42 degrees and 66 degrees, suggesting that the LC2 domains of the two HMM heads have different orientations. In contrast to S1 or HMM, the spectra from rigor fibers, in which LC2 of endogenous myosin heads was labeled, showed a random orientation which may be due to distortion imposed by the structure of the filament lattice and the mismatch of the helical periodicities of the thick and thin filaments. However, spectra from the fibers in the presence of ATP analog 5'-adenylyl imidodiphosphate (AMPPNP) were approximated by two narrow angular distributions similar to those obtained with HMM. Thus, AMPPNP may cause the LC2 domain to be less flexible and/or the S2 portion to be more flexible, so as to release the distortion of the LC2 domain and make it return to its natural position. At high ionic strength, AMPPNP disoriented the spin labels as ATP did under relaxing conditions, suggesting that the myosin head is detached from and/or weakly (flexibly) attached to a thin filament.     

Saturation transfer electron parametric resonance of an indane-dione spin-label. Calibration with hemoglobin and application to myosin rotational dynamics.

We have used a recently synthesized indane-dione spin label (2-[-oxyl-2,2,5,5-tetramethyl-3-pyrrolin-3-yl)methenyl]in dane-1,3-dione (InVSL) to study the rotational dynamics of myosin, with saturation-transfer electron paramagnetic resonance (ST-EPR). To determine effective rotational correlation times (tau effr) from InVSL spectra, reference spectra corresponding to known correlation times (tau r) were obtained from InVSL-hemoglobin undergoing isotropic rotational motion in aqueous glycerol solutions. These spectra were used to generate plots of spectral parameters vs. tau r. These plots should be used to analyze ST-EPR spectra of InVSL bound to other proteins, because the spectra are different from those of tempo-maleimide-spin-labeled hemoglobin, which have been used previously as ST-EPR standards. InVSL was covalently attached to the head (subfragment-1; S1) of myosin. EPR spectra and K/EDTA-ATPase activity showed that 70-95% of the heads were labeled, with > or = 90% of the label bound to either cys 707 (SH1) or cys 697 (SH2). ST-EPR spectra of InVSL-S1 attached to glass beads, bound to actin in myofibrils, or precipitated with ammonium sulfate indicated no submillisecond rotational motion. Therefore, InVSL is rigidly immobilized on the protein so that it reports the global rotation of the myosin head. The ST-EPR spectra of InVSL-myosin monomers and filaments indicated tau effr values of 4 and 13 microseconds, respectively, showing that myosin heads undergo microsecond segmental rotations that are more restricted in filaments than in monomers. The observed tau effr values are longer than those previously obtained with other spin labels bound to myosin heads, probably because InVSL binds more rigidly to the protein and/or with a different orientation. Further EPR studies of InVSL-myosin in solution and in muscle fibers should prove complementary to previous work with other labels.     

A model of cross-bridge attachment to actin in the A*M*ATP state based on x-ray diffraction from permeabilized rabbit psoas muscle

A model of cross-bridges binding to actin in the weak binding A*M*ATP state is presented. The modeling was based on the x-ray diffraction patterns from the relaxed skinned rabbit psoas muscle fibers where ATP hydrolysis was inhibited by N-phenylmaleimide treatment (S. Xu, J. Gu, G. Melvin, L. C. Yu. 2002. Biophys. J. 82:2111-2122). Calculations included both the myosin filaments and the actin filaments of the muscle cells, and the binding to actin was assumed to be single headed. To achieve a good fit, considerable flexibility in the orientation of the myosin head and the position of the S1-S2 junction is necessary, such that the myosin head can bind to a nearby actin whereas the tail end was kept in the proximity of the helical track of the myosin filament. Hence, the best-fit model shows that the head binds to actin in a wide range of orientations, and the tail end deviates substantially from its lattice position in the radial direction (approximately 60 A). Surprisingly, the best fit model reveals that the detached head, whose location thus far has remained undetected, seems to be located close to the surface of the myosin filament. Another significant requirement of the best-fit model is that the binding site on actin is near the N terminus of the actin subunit, a position distinct from the putative rigor-binding site. The results support the idea that the essential role played by the weak binding states M*ATP <--> A*M*ATP for force generation lies in its flexibility, because the probability of attachment is greatly increased, compared with the weak binding M*ADP*P(i) <--> A*M*ADP*P(i) states.