An actin-dependent conformational change in myosin

Conformational changes within myosin lead to its movement relative to an actin filament. Several crystal structures exist for myosin bound to various nucleotides, but none with bound actin. Therefore, the effect of actin on the structure of myosin is poorly understood. Here we show that the swing of smooth muscle myosin lever arm requires both ADP and actin. This is the first direct observation that a conformation of myosin is dependent on actin. Conformational changes within myosin were monitored using fluorescence resonance energy transfer techniques. A cysteine-reactive probe is site-specifically labeled on a 'cysteine-light' myosin variant, in which the native reactive cysteines were removed and a cysteine engineered at a desired position. Using this construct, we show that the actin-dependent ADP swing causes an 18 A change in distance between a probe on the 25/50 kDa loop on the catalytic domain and a probe on the regulatory light chain, corresponding to a 23 degrees swing of the light-chain domain.     

Direct modeling of x-ray diffraction pattern from skeletal muscle in rigor

Available high-resolution structures of F-actin, myosin subfragment 1 (S1), and their complex, actin-S1, were used to calculate a 2D x-ray diffraction pattern from skeletal muscle in rigor. Actin sites occupied by myosin heads were chosen using a "principle of minimal elastic distortion energy" so that the 3D actin labeling pattern in the A-band of a sarcomere was determined by a single parameter. Computer calculations demonstrate that the total off-meridional intensity of a layer line does not depend on disorder of the filament lattice. The intensity of the first actin layer A1 line is independent of tilting of the "lever arm" region of the myosin heads. Myosin-based modulation of actin labeling pattern leads not only to the appearance of the myosin and "beating" actin-myosin layer lines in rigor diffraction patterns, but also to changes in the intensities of some actin layer lines compared to random labeling. Results of the modeling were compared to experimental data obtained from small bundles of rabbit muscle fibers. A good fit of the data was obtained without recourse to global parameter search. The approach developed here provides a background for quantitative interpretation of the x-ray diffraction data from contracting muscle and understanding structural changes underlying muscle contraction.     

Analysis of equatorial x-ray diffraction patterns from skeletal muscle.

Some of the factors that affect the intensities and the phases of the first five equatorial x-ray reflections from skeletal muscle are studied by simplified models describing axially projected mass distributions in unit cells. Examples of mass distributions that produce various phase combinations and intensities are presented. Effects due to radial movement of crossbridges and those due to mass transfer between the thick filament and the thin filament regions are compared. In addition, the study suggests that some features in the reconstructed filament structures could be due to the consequences of limited resolution.     

Effects of pressure on equatorial x-ray fiber diffraction from skeletal muscle fibers.

When skeletal muscle fibers are subjected to a hydrostatic pressure of 10 MPa (100 atmospheres), reversible changes in tension occur. Passive tension from relaxed muscle is unaffected, rigor tension rises, and active tension falls. The effects of pressure on muscle structure are unknown: therefore a pressure-resistant cell for x-ray diffraction has been built, and this paper reports the first study of the low-angle equatorial patterns of pressurized relaxed, rigor, and active muscle fibers, with direct comparisons from the same chemically skinned rabbit psoas muscle fibers at 0.1 and 10 MPa. Relaxed and rigor fibers show little change in the intensity of the equatorial reflections when pressurized to 10 MPa, but there is a small, reversible expansion of the lattice of 0.7 and 0.4%, respectively. This shows that the order and stability of the myofilament lattice is undisturbed by this pressure. The rise in rigor tension under pressure is thus probably due to axial shortening of one or more components of the sarcomere. Initial results from active fibers at 0.1 MPa show that when phosphate is added the lattice spacing and equatorial intensities change toward their relaxed values. This indicates cross-bridge detachment, as expected from the reduction in tension that phosphate induces. 10 MPa in the presence of phosphate at 11 degrees C causes tension to fall by a further 12%, but not change is detected in the relative intensity of the reflections, only a small increase in lattice spacing. Thus pressure appears to increase the proportion of attached cross-bridges in a low-force state.     

Triple-resonance multidimensional NMR study of calmodulin complexed with the binding domain of skeletal muscle myosin light-chain kinase: indication of a conformational change in the central helix

Heteronuclear 3D and 4D NMR experiments have been used to obtain 1H, 13C, and 15N backbone chemical shift assignments in Ca(2+)-loaded calmodulin complexed with a 26-residue synthetic peptide (M13) corresponding to the calmodulin-binding domain (residues 577-602) of rabbit skeletal muscle myosin light-chain kinase. Comparison of the chemical shift values with those observed in peptide-free calmodulin [Ikura, M., Kay, L. E., & Bax, A. (1990) Biochemistry 29, 4659-4667] shows that binding of M13 peptide induces substantial chemical shift changes that are not localized in one particular region of the protein. The largest changes are found in the first helix of the Ca(2+)-binding site I (E11-E14), the N-terminal portion of the central helix (M72-D78), and the second helix of the Ca(2+)-binding site IV (F141-M145). Analysis of backbone NOE connectivities indicates a change from alpha-helical to an extended conformation for residues 75-77 upon complexation with M13. This conformational change is supported by upfield changes in the C alpha and carbonyl chemical shifts of these residues relative to M13-free calmodulin and by hydrogen-exchange experiments that indicate that the amide protons of residues 75-82 are in fast exchange (kexch greater than 10 s-1 at pH 7, 35 degrees C) with the solvent. No changes in secondary structure are observed for the first helix of site I or the C-terminal helix of site IV. Upon complexation with M13, a significant decrease in the amide exchange rate is observed for residues T110, L112, G113, and E114 at the end of the second helix of site III.     

X-ray study of myosin heads in contracting frog skeletal muscle

Using synchrotron radiation, the behaviour of the diffuse X-ray scatter was investigated in the relaxed and active phases of auxotonic and isometric contractions. Muscles were stimulated tetanically for 0.75 of a second, leaving intervals of three minutes between successive contractions. In isometric contractions the scatter is very asymmetric, which means that the myosin heads have a strongly preferred orientation. During tension rise the scatter expands in the meridional direction and contracts in the equatorial direction, the maximal local intensity change being about 20%. The shape change indicates that on average the myosin heads become oriented more perpendicularly to the fibre axis. The distribution of orientations at peak tension is quite different from that we found previously in X-ray scattering data from rigor muscles. In auxotonic contractions where muscles shorten against an increasing tension the scatter is practically circularly symmetrical. This suggests that during shortening the myosin heads go evenly through a wide range of orientations. It is concluded that the results from both the auxotonic and isometric experiments provide strong support for the rotating myosin head model. In isometric contractions the transition between the relaxed phase and peak tension is accompanied by an overall increase in scattering intensity of about 10%: this corresponds to a relative increase in the fraction of disordered myosin heads by almost 30%.     

Ligand-induced myosin subfragment 1 global conformational change

The effects of selected ligands on the structure of myosin subfragment 1 (S1) were compared by using transient electrical birefringence techniques. With pairs of dilute solutions of S1 at 3.5 degrees C in low ionic strength (mu = 0.020 M) buffers that had matched electrical impedances, S1 with Mg2+, MgADP, or MgADP.Vi bound was subjected to 6-7-microseconds external electrical fields in the Kerr law range. Specific Kerr constants and the rates of rotational Brownian motion after the electric field was removed were measured. Neither Mg2+ nor MgADP had a measurable effect on either observable, but when orthovanadate (Vi) bound S1.MgADP it decreased the rotational correlation coefficient from 267 +/- 6 to 244 +/- 10 ns. Parallel measurements of MgATPase activity indicated that S1.MgADP.Vi was greater than 95% inhibited. These results confirm the conclusion of Aguirre et al. [(1989) Biochemistry 28, 799] that Vi binding to S1.MgADP increases its rate of rotational Brownian motion and provide data that are more quantitatively correlated with S1 structure. The Vi-induced change in the rotational correlation coefficient is consistent with S1 becoming more flexible or more compact when Vi binds. Assuming that S1.MgADP.Vi is an analogue for S1.MgADP.Pi, the structural changes observed for S1-ligand complexes in solution are discussed in relation to possible structural changes of intermediates on the kinetic pathway of ATPase hydrolysis. A new model of force generation by S1 in muscle is hypothesized.     

Light diffraction study of single skeletal muscle fibres.

Light diffraction patterns from isolated frog semitendinosus muscle fibers were examined. When transilluminated by laser light, the muscle striations produce a diffraction pattern consisting of a series of lines that are projected as points onto an optical detector by a lens system. Diffraction data may be sequentially stored every 18 ms for later processing by digital computer systems. First- and second-order diffraction line intensities were examined from intact, chemically skinned, and glycerinated single fibers. The diffraction line intensities demonstrated a strong length dependence upon passive stretch from reference length to 3.6 micrometer. The first-order intensity linearly increased an average of 15-fold over the range examined. The magnitude of the second order intensity was less than the first order and showed an exponential rise with increasing length. Both first- and second-order intensities decreased upon muscle activation. Data from chemically skinned and glycerinated single fibers were not significantly different from intact fibers, indicating that the membrane structure has little effect upon the diffraction phenomenon in muscle. Theoretical model systems are examined in an attempt to find the basis of these results. Neither an analysis based on a diffraction grating with variable spacing nor the unit cell model of Fujime provides an explanation for the observed length dependency of intensity. Though the origin of the intensity decrease upon stimulation is not known, we have suggested that it could result from lateral misalignment of myofibrils and can occur upon activation.     

Time-resolved x-ray diffraction study of the troponin-associated reflexions from the frog muscle.

The vertebrate skeletal muscle gives rise to a series of x-ray reflexions indexed as orders (n) of 77 nm, the even orders being meridional whereas the odd orders being near-meridional. The diffraction intensities associated with these reflexions originate from the axial period of 39 nm attributable to the repeat of troponin-tropomyosin on the thin filament. In the present study, the x-ray intensities of the furthest inner reflexions, A2 (n = 2) reflexion at an axial spacing of 1/39 nm-1 and A4 (n = 4) reflexion at 1/19 nm, of this series were measured with a time resolved manner. Upon activation of the frog striated muscle, the two reflexions underwent biphasic time courses of the intensity changes. With A2 reflexion, a rapid intensity increase by 16%, being completed by the time when tension rises to 5%, was followed by a slow intensity decrease down to 50%, which was associated with the tension rise. In both phases, lateral widths remained unchanged. A4 reflexion also behaves in the same way, although the first phase (the intensity increase) was not clear due to unsatisfactory statistics. We interpret phase one as being caused by conformational change of the troponin-tropomyosin complex upon binding of Ca2+ to troponin, whereas phase two being due to direct contribution of the mass of the myosin heads bound to the thin filament, although possible contribution of conformational changes of the regulatory proteins to phase two is not excluded. The results indicated that the calcium activation of the thin filament leads the onset of the actomyosin interaction.     

Slow myosin in developing rat skeletal muscle

Through S1 nuclease mapping using a specific cDNA probe, we demonstrate that the slow myosin heavy-chain (MHC) gene, characteristic of adult soleus, is expressed in bulk hind limb muscle obtained from the 18-d rat fetus. We support these results by use of a monoclonal antibody (mAb) which is highly specific to the adult slow MHC. Immunoblots of MHC peptide maps show the same peptides, uniquely recognized by this antibody in adult soleus, are also identified in 18-d fetal limb muscle. Thus synthesis of slow myosin is an early event in skeletal myogenesis and is expressed concurrently with embryonic myosin. By immunofluorescence we demonstrate that in the 16-d fetus all primary myotubes in future fast and future slow muscles homogeneously express slow as well as embryonic myosin. Fiber heterogeneity arises owing to a developmentally regulated inhibition of slow MHC accumulation as muscles are progressively assembled from successive orders of cells. Assembly involves addition of new, superficial areas of the anterior tibial muscle (AT) and extensor digitorum longus muscle (EDL) in which primary cells initially stain weakly or are unstained with the slow mAb. In the developing AT and EDL, expression of slow myosin is unstable and is progressively restricted as these muscles specialize more and more towards the fast phenotype. Slow fibers persisting in deep portions of the adult EDL and AT are interpreted as vestiges of the original muscle primordium. A comparable inhibition of slow MHC accumulation occurs in the developing soleus but involves secondary, not primary, cells. Our results show that the fate of secondary cells is flexible and is spatially determined. By RIA we show that the relative proportions of slow MHC are fivefold greater in the soleus than in the EDL or AT at birth. After neonatal denervation, concentrations of slow MHC in the soleus rapidly decline, and we hypothesize that, in this muscle, the nerve protects and amplifies initial programs of slow MHC synthesis. Conversely, the content of slow MHC rises in the neonatally denervated EDL. This suggests that as the nerve amplifies fast MHC accumulation in the developing EDL, accumulation of slow MHC is inhibited in an antithetic fashion. Studies with phenylthiouracil-induced hypothyroidism indicate that inhibition of slow MHC accumulation in the EDL and AT is not initially under thyroid regulation. At later stages, the development of thyroid function plays a role in inhibiting slow MHC accumulation in the differentiating EDL and AT.(ABSTRACT TRUNCATED AT 400 WORDS)     

1H NMR study of rabbit skeletal muscle troponin C: Mg2(+)-induced conformational change

Binding of Mg2+ to rabbit skeletal muscle troponin C (TnC) is studied by means of two-dimensional (2D) 1H NMR spectroscopy. Using the sequence-specific resonance assignment method we assign several resonances of TnC in the Mg2(+)-saturated state. Assigned resonances are used as probes of the following titration experiments: (1) Mg2+ titration of apo-TnC, (2) Mg2+ titration of Ca2TnC, and (3) Mg2+ titration of Ca4TnC. In experiment 1, the slow-exchange behavior is observed for resonances of Phe99, Asp107, Gly108, Tyr109, Ile110, Asp111, His125, Gly144, Arg145, Ile146, Asp147, and Phe148 located at the high-affinity Ca2(+)-binding sites in the C-terminal-half domain. In experiments 1 and 2, the fast-exchange behavior is observed for resonances of Gly32, Asp33, Ser35, Gly68, Thr69, and Asp71 located at the low-affinity Ca2(+)-binding sites in the N-terminal-half domain. These results suggest that Mg2+ ions bind to the N domain as well as the C domain. In experiment 3, no spectral change is observed for all above-mentioned residues in the C domain and also for Gly32 and Gly68 in the N domain. It can be concluded that all Ca2(+)-binding sites in both the N and C domains can bind Mg2+ ions. No significant change is observed for resonances of Phe23, Ile34, Val68, and Phe72 in experiments 1 and 2. These results suggest that Mg2+ binding to the N domain does not induce conformational change in the hydrophobic region of the N domain. 2D-NMR spectra and Mg2(+)-titration data suggest that the antiparallel beta-sheet conformation is formed in both the N and C domains when Mg2+ ions bind to the two domains.     

"Crystalline" myosin cross-bridge array in relaxed bony fish muscle. Low-angle x-ray diffraction from plaice fin muscle and its interpretation

Detailed structural analysis of muscles normally used to study myosin cross-bridge behavior (e.g., frog sartorius muscle, insect flight muscle) is extremely difficult due to the statistical disorder inherent in their myosin filament arrays. Bony fish muscle is different from all other muscle types in having a myosin filament (A-Band) array with good three-dimensional (crystalline) regularity that is coherent right across each myofibril. Rigorous structure analysis is feasible with fish muscle. We show that low-angle x-ray diffraction patterns from plaice fin muscle contain characteristic vertebrate layer lines at orders of 429 (+/- 0.2) A, that these layer lines are well sampled by row-lines from a simple hexagonal lattice of a-spacing 470 (+/- 2.0) A at rest length and that there are meridional reflections, due to axial perturbations of the basic helix of myosin heads, similar in position to those from frog muscle but differing in relative intensities. Clear trends based on modeling to a resolution of 130 A of the observed intensities in the low angle x-ray diffraction pattern from relaxed plaice fin muscle suggest that: (a) the pattern out to 130 A is more sensitive to the distribution of the two heads than it is to details of the head shape, (b) both heads in one myosin molecule probably tilt axially in the same direction by approximately 20-40 degrees relative to a normal to the thick filament backbone, (c) the center of mass of the heads is at 145 to 160 A radius, and (d) the two heads form a compact structure by lying closely adjacent to each other and almost parallel. Little rotational disorder of the heads can occur. Because of its crystallinity, bony fish muscle provides a uniquely useful structural probe of myosin cross-bridge behavior in other muscle states such as rigor and active contraction.     

Nuclear magnetic resonance study on rabbit skeletal troponin C: calcium-induced conformational change

Rabbit skeletal muscle troponin C (TnC) was investigated by means of 1H NMR in the presence of dithiothreitol that prevents dimerization of the protein. Two-dimensional (2D) 1H NMR spectra were observed in order to assign resonances to specific amino acids. One-dimensional 1H NMR spectra were observed as a function of Ca2+ concentration. The Ca2+-induced spectral change is categorized into two types: type 1 corresponds to the conformational change of the C-terminal-half domain (Ca2+ high-affinity sites) and type 2 to that of the N-terminal-half domain (Ca2+ low-affinity sites). From the 2D NMR spectra and Ca2+ titration data, it was suggested that (1) amide protons of Gly-108, Ile-110, Gly-144, and Ile-146 are hydrogen-bonded when the C-terminal-half domain binds 2 mol of Ca2+ and (2) hydrogen bonds of Gly-108, Ile-110, Gly-144, and Ile-146 are destroyed or weakened when the C-terminal-half domain releases 2 mol of Ca2+. Nuclear Overhauser enhancement difference spectra as well as the Ca2+ titration data suggested that a hydrophobic cluster is formed in the C-terminal-half domain when the C-terminal-half domain binds 2 mol of Ca2+. A hydrophobic cluster exists in the N-terminal-half domain without regard to Ca2+ binding to the N-terminal-half domain. The spectra of Tyr-10 showed both types of spectral change during the Ca2+ titration. The results suggested that Tyr-10 of apo-TnC interacts with the C-terminal-half domain.     

Incorporation of an azobenzene derivative into the energy transducing site of skeletal muscle myosin results in photo-induced conformational changes

The region containing reactive cysteines, Cys 707 (SH1)-Cys 697 (SH2), of skeletal muscle myosin is thought to play a key role in the conformational changes of the myosin head during force generation coupled to ATP hydrolysis. In the present study, we synthesized a photochromic crosslinker, 4,4'-azobenzene-dimaleimide (ABDM), that undergoes reversible cis-trans isomerization upon ultra violet (UV) and visible (VIS) light irradiation resulting in a change in the crosslinking length from 5 to 17 A. The reactive cysteines, SH1 and SH2, of myosin subfragment 1 (S1) were crosslinked with ABDM, yielding an ABDM-S1 complex. The changes in absorbance induced by UV/VIS light irradiation of the complex were similar to those of free ABDM indicating that the incorporation of ABDM at the SH1 and SH2 sites did not disrupt the isomerization of crosslinked ABDM. Small-angle synchrotron X-ray scattering analysis of the ABDM-S1 complex in solution suggested that the localized conformational changes resulting from the cis to trans isomerization on ABDM crosslinking of SH1 and SH2 induced a small but significant swing in the lever arm portion of S1 in the opposite direction from that induced by ATP.     

Structural changes of actin-bound myosin heads after a quick length change in frog skeletal muscle

Changes in the x-ray diffraction pattern from a frog skeletal muscle were recorded after a quick release or stretch, which was completed within one millisecond, at a time resolution of 0.53 ms using the high-flux beamline at the SPring-8 third-generation synchrotron radiation facility. Reversibility of the effects of the length changes was checked by quickly restoring the muscle length. Intensities of seven reflections were measured. A large, instantaneous intensity drop of a layer line at an axial spacing of 1/10.3 nm(-1) after a quick release and stretch, and its partial recovery by reversal of the length change, indicate a conformational change of myosin heads that are attached to actin. Intensity changes on the 14.5-nm myosin layer line suggest that the attached heads alter their radial mass distribution upon filament sliding. Intensity changes of the myosin reflections at 1/21.5 and 1/7.2 nm(-1) are not readily explained by a simple axial swing of cross-bridges. Intensity changes of the actin-based layer lines at 1/36 and 1/5.9 nm(-1) are not explained by it either, suggesting a structural change in actin molecules.