Thermal denaturation of the alkali light chain-20 kDa fragment complex obtained from myosin subfragment 1.

The thermal denaturation of the myosin subfragment 1 (S1) from rabbit skeletal muscle and of its derivatives obtained by tryptic digestion has been studied by means of differential scanning calorimetry. Two distinct thermal transitions were revealed in the isolated complex of the C-terminal 20 kDa fragment of the S1 heavy chain with the alkali light chain. These transitions were identified by means of a thermal gel analysis method. It has been shown that the thermal denaturation of the 20 kDa fragment of the S1 heavy chain correlates with the melting of the most thermostable domain in the S1 molecule. It is concluded that this domain is located in the C-terminal 20 kDa segment of the S1 heavy chain.     

Interaction of alkali light chain 1 with the isolated 20-kilodalton fragment of myosin subfragment-1 heavy chain and F-actin

The binding of one of the alkali light chains of myosin, A1, with the isolated renatured 20-kDa fragment of myosin subfragment-1 heavy chain was demonstrated by means of difference UV absorption spectroscopy. The difference spectrum with either rabbit or chicken A1 showed two characteristic peaks at 279 and 287 nm indicating a perturbation of tyrosyl chromophores by the association with the 20-kDa fragment. The delta epsilon at 287 nm increased with an increase in the molar ratio of A1/20-kDa fragment and reached a maximum value at around equimolar ratio. The maximum delta epsilon value was approximately three times larger with rabbit A1 than with chicken A1. Based on the positions of Tyr residues in the amino acid sequences, the contact surface of A1 with myosin heavy chain was concluded to be spread over a large area of A1. The binding of 20-kDa fragment with F-actin was measured by following the increase in turbidity. The affinity appeared to increase several times in the presence of A1. A1 may possibly control the affinity of myosin for actin.     

Arrangement of myosin heads in relaxed thick filaments from frog skeletal muscle

The distribution of myosin heads on the surface of frog skeletal muscle thick filaments has been determined by computer processing of electron micrographs of isolated filaments stained with tannic acid and uranyl acetate. The heads are arranged in three strands but not in a strictly helical manner and so the structure has cylindrical symmetry. This accounts for the "forbidden" meridional reflections seen in diffraction patterns. Each layer-line therefore represents the sum of terms of Bessel orders 0, +/- 3, +/- 6, +/- 9 and so on. These terms interact so that, unlike a helical object without terms from overlapping Bessel orders, as the azimuth is changed, the amplitude on a layer-line at a particular radius varies substantially and its phase does not alter linearly. Consequently, a three-dimensional reconstruction cannot be produced from a single view. We have therefore used tilt series of three individual filaments to decompose the data on layer-lines 0 to 6 into terms of Bessel orders up to +/- 9 using a least-squares procedure. These data had a least-squares residual of 0.32 and enabled a three-dimensional reconstruction to be obtained at a nominal resolution of 6 nm. This showed, at a radius of about 10 nm, three strands of projecting morphological units with three units spaced along each strand every 42.9 nm axially. We have identified these units with pairs of myosin heads. Successive units along a strand are perturbed axially, azimuthally and radially from the positions expected if the structure was perfectly helical. This may simply be a consequence of steric restrictions in packing the heads on the thick filament surface, but could also reflect an underlying non-helical arrangement of myosin tails, which would be consistent with the thick filament shaft being constructed from three subfilaments in which the tails were arranged regularly. There was also material at a radius of about 6 nm spaced 42.9 nm axially, which we tentatively identified with accessory proteins. The filament shaft had a pronounced pattern of axial staining.     

Calponin binds to the 20-kilodalton regulatory light chain of myosin

Calponin (CaP) is a 34 kDa smooth muscle-specific protein that has been implicated in regulation of smooth muscle contractility. Two CaP binding sites on smooth muscle myosin rod have been recently described [Szymanski and Tao (1997) J.Biol.Chem. 272, 11142-11146]. We used a combination of cosedimentation, overlay, and fluorescence assays to determine the interaction between CaP and both subfragment 1 of myosin and isolated 20 kDa regulatory light chain of myosin (RLC). Subfragment 1, which was generated by cleavage of myosin with Staphylococcus aureus protease (myosin S1SA) inhibits cosedimentation of CaP with myosin filaments. Fluorescence assay showed that CaP labeled with fluorescent label (DAN-CaP) interacts with myosin S1SA in solution via a single class of binding sites. The binding constant (kaff) of this interaction at 50 mM NaCl is (2. 1 +/- 0.2) x 10(6) M-1 (n = 3). The interaction between DAN-CaP and myosin S1SA depends on ionic strength, and the EC50 of inhibition of this interaction occurs at about 130 mM NaCl. In contrast, the subfragment 1 that was generated by papain digestion (myosin S1PA), which cleaves RLC 4 kDa away from the NH2-terminal end of the molecule, does not interact with DAN-CaP. Overlay and fluorescent assay in solution showed that CaP binds to isolated RLC, suggesting that the interaction between CaP and subfragment 1 of myosin is due to a direct binding of CaP to RLC. CaP binding to myosin S1SA is stronger than to subfragment 2 in physiological salt concentrations. CaP binding to myosin head strengthened upon phosphorylation of RLC by Ca2+/calmodulin-dependent myosin light chain kinase. We suggest that CaP binds to subfragment 1 of myosin, exclusively via the NH2-terminal end of RLC, and this interaction could play a role in regulation of the actin-myosin interaction in smooth muscle contractility.     

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%.     

Cross-linking of the 25- and 20-kilodalton fragments of skeletal myosin subfragment 1 by a bifunctional ATP analogue

The bifunctional photoreactive ATP analogue azidonitrobenzoyl-8-azido-ATP (ANB-8-N3-ATP) was synthesized. This ATP analogue carriers photoreactive azido groups at the eighth position of the adenine ring and at the 3' position of ribose. Photolysis of this analogue in the presence of skeletal muscle alpha-chymotryptic subfragment 1 (S-1) resulted in a new 120-kDa band, while photolysis in the presence of the tryptic S-1 produced a new 45-kDa band. The 45-kDa peptide was shown to be combined with the 25-kDa N-terminal and 20-kDa C-terminal fragments since it was labeled with a monoclonal antibody specific for the N-terminal 25-kDa segment of the S-1 heavy chain, and it was also found to retain the fluorescence of (iodoacetamido)fluorescein attached specifically to the SH-1 thiol of the C-terminal 20-kDa segment. These results indicate that the 25- and 20-kDa peptides are in close contact with the ATPase active site.     

Structural characteristics and aggregation of myosin heads in skeletal muscles

Native conformational modifications of rabbit skeletal muscle myosin and its subfragment-1 (S-1) within the temperature range of 0-40 degrees C and irreversible unfolding of these proteins structure at temperatures 40-70 degrees C have been studied by the fluorescence and light scattering methods. The results obtained permit stating that myosin and its active subfragments form associates at the concentrations above 0.3 microM. Hydrophobic interactions between definite sites of S-1 are likely to be primarily responsible for the association. The complex profile of S-1 melting curve at high ionic strength indicates the existence of three structural domains in the heavy chain of the myosin head.     

Isolation and characterization of the N-terminal 23-kilodalton fragment of myosin subfragment 1

The 23-kDa N-terminal tryptic fragment was isolated from the heavy chain of rabbit skeletal myosin subfragment 1 (S-1). The heavy-chain fragments were dissociated by guanidine hydrochloride following limited trypsinolysis, and the 23-kDa fragment was isolated by gel filtration and ion-exchange chromatography. Finally, the fragment was renatured by removing the denaturants. The CD spectrum of the renatured fragment shows the presence of ordered structure. The tryptophan fluorescence emission spectrum of the fragment is considerably shifted to the red upon adding guanidine hydrochloride which indicates that the tryptophans are located in relatively hydrophobic environments. The two 23-kDa tryptophans, unlike the rest of the S-1 tryptophans, are fully accessible to acrylamide as indicated by fluorescence quenching. The isolated 23-kDa fragment cosediments with F-actin in the ultracentrifuge and significantly increases the light scattering of actin in solution which indicates actin binding. The binding is rather tight (Kd = 0.1 microM) and ionic strength dependent (decreasing with increasing ionic strength). ATP, pyrophosphate, and ADP dissociate the 23-kDa-actin complex with decreasing effectiveness. The isolated 23-kDa fragment does not have ATPase activity; however, it inhibits the actin-activated ATPase activity of S-1 by competing presumably with S-1 for binding sites on actin. F-Actin binds to the 23-kDa fragment immobilized on the nitrocellulose membrane. The fragment was further cleaved, and one of the resulting peptides, containing the 130-204 stretch of residues, was found to bind actin on the nitrocellulose membrane, indicating that this region of the 23-kDa fragment participates in forming an actin binding site.     

Interaction of the heavy chain of gizzard myosin heads with skeletal F-actin

To probe the molecular properties of the actin recognition site on the smooth muscle myosin heavy chain, the rigor complexes between skeletal F-actin and chicken gizzard myosin subfragments 1 (S1) were investigated by limited proteolysis and by chemical cross-linking with 1-ethyl-3-[3-(dimethyl-amino)propyl]carbodiimide. Earlier, these approaches were used to analyze the actin site on the skeletal muscle myosin heads [Mornet, D., Bertrand, R., Pantel, P., Audemard, E., & Kassab, R. (1981) Biochemistry 20, 2110-2120; Labbé, J.P., Mornet, D., Roseau, G., & Kassab, R. (1982) Biochemistry 21, 6897-6902]. In contrast to the case of the skeletal S1, the cleavage with trypsin or papain of the sensitive COOH-terminal 50K-26K junction of the head heavy chain had no effect on the actin-stimulated Mg2+-ATPase activity of the smooth S1. Moreover, actin binding had no significant influence on the proteolysis at this site whereas it abolished the scission of the skeletal S1 heavy chain. The COOH-terminal 26K segment of the smooth papain S1 heavy chain was converted by trypsin into a 25K peptide derivative, but it remained intact in the actin-S1 complex. A single actin monomer was cross-linked with the carbodiimide reagent to the intact 97K heavy chain of the smooth papain S1. Experiments performed on the complexes between F-actin and the fragmented S1 indicated that the site of cross-linking resides within the COOH-terminal 25K fragment of the S1 heavy chain. Thus, for both the striated and smooth muscle myosins, this region appears to be in contact with F-actin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular movements promoted by metal nucleotides in the heavy-chain regions of myosin heads from skeletal muscle

Molecular movements generated in the heavy-chain regions (27-50-20(X 10(3)) Mr) of myosin S1 on interaction with nucleotides ATP, AMPPNP, ADP and PPi were investigated by limited proteolysis of several enzyme-metal nucleotide complexes in the absence and presence of reversibly bound and crosslinked F-actin. The rate and extent of the nucleotide-promoted conversion of the NH2-terminal 27 X 10(3) Mr and 50 X 10(3) Mr segments into products of 22 X 10(3) Mr and 45 X 10(3) Mr, respectively, were estimated to determine the amplitude of the molecular movements. The 22 X 10(3) Mr peptide was identified by amino acid sequence studies as being derived from cleavage of the peptide bond between Arg and Ile (at position 23 to 24). The 45 X 10(3) Mr peptide, previously shown to represent the NH2-terminal part of the 50 X 10(3) Mr region, would be connected to the adjacent C-terminal 20 X 10(3) Mr region by a pre-existing loop segment of about 5 X 10(3) Mr; the proteolytic sensitivity of the latter region is increased particularly by nucleotide binding. The tryptic reaction proved to be a sensitive indicator of the conformational state of the liganded heavy chain as the rate of peptide bond cleavage in the two regions is dependent on the nature of the bound ligand; it decreases in the order: ATP greater than AMPPNP greater than ADP greater than PPi. It depends also on the nature of the metal present, Mg2+ and Ca2+ being much more effective than K+. Binding of F-actin to the S1-MgAMPPNP complex affords significant protection against breakdown of 27 X 10(3) Mr and 50 X 10(3) Mr peptides, but with concomitant hydrolysis of the 50 X 10(3) Mr-20 X 10(3) Mr junction. Additionally, interaction of MgATP with HMM modulates the tryptic fission of the S1-S2 region. The overall data provide a molecular support for the two-state model of the myosin head and emphasize the involvement of the 50 X 10(3) Mr unit in the mechanism of coupling between the actin and nucleotide binding sites.     

The myosin SH2-50-kilodalton fragment cross-link: location and consequences

Some of us recently described a new interthiol cross-link which occurs in the skeletal myosin subfragment 1-MgADP complex between the reactive sulfhydryl group "SH2" (Cys-697) and a thiol (named SH chi) of the 50-kilodalton (kDa) central domain of the heavy chain; this link leads to the entrapment of the nucleotide at the active site [Chaussepied, P., Mornet, D., & Kassab, R. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2037-2041]. In the present study, we identify SH chi as Cys-540 of the 50-kDa fragment. The portion of the heavy chain including this residue and also extending to Cys-522 that is cross-linkable to the "SH1" thiol [Ue, K. (1987) Biochemistry 26, 1889-1894] is near the SH2-SH1 region. Furthermore, various spectral and enzymatic properties of the (Cys697-Cys540)-N,N'-p-phenylenedimaleimide (pPDM)-cross-linked myosin chymotryptic subfragment 1 (S-1) were established and compared to those for the well-known (SH1-SH2)-pPDM-cross-linked S-1. The circular dichroism spectra of the new derivative were similar to those of native S-1 complexed to MgADP. At 15 mM ionic strength, (Cys697-Cys540)-S-1 binds very strongly to unregulated actin (Ka = 7 X 10(6) M-1), and the actin binding is very weakly affected by ionic strength. Joining actin with the (Cys697-Cys540)-S-1 heavy chain, using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, produces different species than does joining unmodified S-1 with actin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of myosin from starfish sperm heads.

Myosin was extracted and partially purified from the head portion of spermatozoa of the starfish, Asterias amurensis. The sperm myosin showed a specific Ca2+-activated ATPase [EC 3.6.1.3] activity of 0.2 mumoles Pi/min/mg at high ionic strength and pH 6.5. It resembled egg myosin in forming thick filaments, becoming attached to actin filaments. subunit composition, and serological properties.     

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.     

Two non-identical heads of myosin molecules

Four different preparations of skeletal subfragment-1, denoted in this report as S1(Aa), S1(Ab), S1(Ba), and S1(Bb), and two different preparations of cardiac subfragment-1, denoted as S1(A) and S1(B), were obtained as described in our recent report (J. Biochem. 97, 965, 1985). (i) The four preparations were obtained from chicken breast myosin trinitrophenylated with 2,4,6-trinitrobenzene sulfonate in the absence of inorganic pyrophosphate (-PPi), and they were all shown to be trinitrophenylated. Addition of PPi caused change in the absorption spectra of trinitrophenyl(TNP)-S1(Aa) and TNP-S1(Ba), but not in those of TNP-S1(Ab) and TNP-S1(Bb). (ii) The two preparations of S1 were obtained from cardiac myosin trinitrophenylated either in the absence (-) or presence (+) of PPi. S1(B) was trinitrophenylated, whereas S1(A) was not. Specifically emphasized is the observation that the yield of cardiac S1(A) was practically equal to that of cardiac S1(B). On the basis of these results, we propose the hypothesis of "two iso-myosins with non-identical heads," which is essentially a combination of the hypothesis of isoenzymes and that of non-identical heads.     

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.     

Calcium functionally uncouples the heads of myosin VI

This study examines the steady state activity and in vitro motility of single-headed (S1) and double-headed (HMM) myosin VI constructs within the context of two putative modes of regulation. Phosphorylation of threonine 406 does not alter either the rate of actin filament sliding or the maximal actin-activated ATPase rate of S1 or HMM constructs. Thus, we do not observe any regulation of myosin VI by phosphorylation within the motor domain. Interestingly, in the absence of calcium, the myosin VI HMM construct moves in an in vitro motility assay at a velocity that is twice that of S1 constructs, which may be indicative of movement that is not based on a "lever arm" mechanism. Increasing calcium above 10 microm slows both the rate of ADP release from S1 and HMM actomyosin VI and the rates of in vitro motility. Furthermore, high calcium concentrations appear to uncouple the two heads of myosin VI. Thus, phosphorylation and calcium are not on/off switches for myosin VI enzymatic activity, although calcium may alter the degree of processive movement for myosin VI-mediated cargo transport. Lastly, calmodulin mutants reveal that the calcium effect is dependent on calcium binding to the N-terminal lobe of calmodulin.     

Unshadowed myosin molecules: STEM mass-maps of myosin heads.

Myosin molecules were directly visualized without heavy metal shadowing by scanning transmission electron microscopy (STEM) under low dose conditions. The general appearance and dimensions of heavy metal-free molecules were similar to those of shadowed myosin, either after freeze-drying without or air-drying with glycerol. Two characteristic configurations of myosin head regions were found, a first type showing two pear-shaped heads with narrow necks and a second type showing two heads connected by an extra mass in the central regulatory domain where the light chains are located. The mass of the latter type (mol. wt. = 265 +/- 39 kd) is in excellent accordance with biochemical data whereas the mass of the first type is somewhat lower (mol. wt. 219 +/- 44 kd).