Subfragment 1 Research Articles

Conformation and dynamics of the SH1-SH2 helix in scallop myosin.

Atomic structures of scallop myosin subfragment 1(S1) with the bound MgADP, MgAMPPNP, and MgADP.BeF(x) provide crystallographic evidence for a destabilization of the helix containing reactive thiols SH1 (Cys703) and SH2 (Cys693). A destabilization of this helix was not observed in previous structures of S1 (from chicken skeletal, Dictyostelium discoideum, and smooth muscle myosins), including complexes for which solution experiments indicated such a destabilization. In this study, the factors that influence the SH1-SH2 helix in scallop S1 were examined using monofunctional and bifunctional thiol reagents. The rate of monofunctional labeling of scallop S1 was increased in the presence of MgADP and MgATPgammaS but was inhibited by MgADP.V(i) and actin. The resulting changes in ATPase activities of S1 were symptomatic of SH2 and not SH1 modification, which was confirmed by mass spectrometry analysis. With bifunctional reagents of various lengths, cross-linking did not occur on a short time scale in the absence of nucleotides. In the presence of MgADP, cross-linking was greatly enhanced for all of the reagents. These reactions, as well as the formation of a disulfide bond between SH1 and SH2, were much faster in scallop S1.ADP than in rabbit skeletal S1.ADP and were rate-limited by the initial attachment of the reagent to scallop S1. The cross-linking sites were mapped by mass spectrometry to SH1 and SH2. These results reveal isoform-specific differences in the conformation and dynamics of the SH1-SH2 helix, providing a possible explanation for destabilization of this helix in some scallop S1 but not in other S1 isoform structures.

Differential Regulation of the Actomyosin Interaction by Skeletal and Cardiac Troponin Isoforms

There are significant isoform differences between the skeletal and cardiac troponin complexes. Studies of the regulatory properties of these proteins have previously shown only significant differences in the calcium dependence of their regulation. Using a sensitive myosin subfragment 1 (S1) binding assay we show that in the presence of calcium, thin filaments reconstituted with either skeletal or cardiac troponin produce virtually identical S1 binding curves. However in the absence of calcium the S1 binding curves differ considerably. Combined with kinetic measurements, curve fitting to the three-state thin filament regulatory model shows the main difference is that calcium produces a 4-fold change in K(T) (the closed-open equilibrium) for the skeletal system but little change in the cardiac system. The results show a significant difference in the range of regulatory effect between the cardiac and skeletal systems that we interpret as effects upon actin-troponin (Tn)I-TnC binding equilibria. As structural data show that the Ca(2+)-bound TnC structures differ, the additional counter-intuitive result here is that with respect to myosin binding the +Ca(2+) state of the two systems is similar whereas the -Ca(2+) state differs. This shows the regulatory tuning of the troponin complex produced by isoform variation is the net result of a complex series of interactions among all the troponin components.

The Regulation of Subtilisin-cleaved Actin by Tropomyosin/Troponin

Vertebrate striated muscle contraction is regulated in a Ca(2+)-dependent fashion by tropomyosin (Tm) and troponin (Tn). This regulation involves shifts in the position of Tm and Tn on actin filaments and may include conformational changes in actin that are then communicated to myosin subfragment 1 (S1). To determine whether subdomain 2 of actin plays a role in this regulation, the DNase-I loop 38-52 of this subdomain was cleaved by subtilisin between residues Met(47) and Gly(48). Despite impaired unregulated function, the potentiation and regulation of cleaved actin movement in the in vitro motility assay was not significantly different from that of uncleaved actin. Stopped-flow measurements of ADP release from regulated and unregulated cleaved acto-S1 showed a marked increase in ADP release from acto-S1 in the presence of the regulatory complex. The enhancement of the actin affinity for S1 in the presence of regulatory proteins was greater for uncleaved than for cleaved F-actin. Finally, both cleaved and uncleaved actins protect myosin loop 1 from papain cleavage equally well. Our results suggest that the potentiation of actin function in the in vitro motility assay by regulatory proteins stems from changes in cross-bridge cycle kinetics. In addition, the unimpaired calcium-sensitive regulation of cleaved actin indicates that subdomain 2 conformation does not play an essential role in the regulation process.

Solution Structure of Heavy Meromyosin by Small-angle Scattering

Elucidation of x-ray crystal structures for the S1 subfragment of myosin afforded atomic resolution of the nucleotide and actin binding sites of the enzyme. The structures have led to more detailed hypotheses regarding the mechanisms by which force generation is coupled to ATP hydrolysis. However, the three-dimensional structure of double-headed myosin consisting of two S1 subfragments has not yet been solved. Therefore, to investigate the overall shape and relative orientations of the two heads of myosin, we performed small-angle x-ray and neutron scattering measurements of heavy meromyosin containing all three light chains (LC(1-3)) in solution. The resulting small-angle scattering intensity profiles were best fit by models of the heavy meromyosin head-tail junction in which the angular separation between heads was less than 180 degrees. The S1 heads of the best fit models are not related by an axis of symmetry, and one of the two S1 heads is bent back along the rod. These results provide new information on the structure of the head-tail junction of myosin and indicate that combining scattering measurements with high resolution structural modeling is a feasible approach for investigating myosin head-head interactions in solution.

Isolation and kinetic characterisation of myosin and myosin S1 from the Drosophila indirect flight muscles.

The potential to explore myosin function through the alternative exons and mutations of the single muscle myosin heavy chain gene, Mhc of Drosophila requires detailed kinetic analysis of the myosins. We have obtained microgram quantities of enzymatically active Drosophila myosin and subfragment 1 (S1) from dissected indirect flight muscles. Using recent developments in stopped-flow and flash-photolysis methods combined with fluorescent/light scattering technologies we have determined some of the key kinetic parameters of actin-myosin and myosin-nucleotide interactions. The rate of ATP-induced dissociation of actin from Drosophila myosin (0.23 microM(-1) s(-1)) and subfragment 1 (S1, 0.82 microM(-1) s(-1)) are both fast and similar to values measured for mammalian skeletal muscle myosins and S1 fragments respectively. The ATP-induced cross bridge dissociation of Drosophila acto.S1 is expected to be fast since, for a rapidly contracting muscle like the Drosophila flight muscle, the post power stroke cross bridge must detach rapidly from actin or become a drag on the contracting filament. ATP-induced detachment is preceded by ADP release and this is proposed as the rate-limiting step that defines muscle shortening velocity. We show that the affinity of ADP for acto.S1 at 400 microM is 2-3 fold weaker than fast vertebrate myosins. This leads to an estimate of the ADP release rate constant of 4000 s(-1). We show that this predicts a maximum shortening velocity very similar to that obtained from in vivo estimates of indirect flight muscle shortening. The data is therefore compatible with ADP dissociation limiting the in vivo shortening velocity.

The 2′- O- and 3′- O-Cy3-EDA-ATP(ADP) Complexes with Myosin Subfragment-1 are Spectroscopically Distinct

Ribose-modified highly-fluorescent sulfoindocyanine ATP and ADP analogs, 2′(3′)- O-Cy3-EDA-AT(D)P, with kinetics similar to AT(D)P, enable myosin and actomyosin ATPase enzymology with single substrate molecules. Stopped-flow studies recording both fluorescence and anisotropy during binding to skeletal muscle myosin subfragment-1 (S1) and subsequent single-turnover decay of steady-state intermediates showed that on complex formation, 2′- O- isomer fluorescence quenched by 5%, anisotropy increased from 0.208 to 0.357, and then decayed with turnover rate k cat 0.07 s −1; however, 3′- O- isomer fluorescence increased 77%, and anisotropy from 0.202 to 0.389, but k cat was 0.03 s −1. Cy3-EDA-ADP·S1 complexes with vanadate (V i) were studied kinetically and by time-resolved fluorometry as stable analogs of the steady-state intermediates. Upon formation of the 3′- O-Cy3-EDA-ADP·S1·V i complex fluorescence doubled and anisotropy increased to 0.372; for the 2′- O- isomer, anisotropy increased to 0.343 but fluorescence only 6%. Average fluorescent lifetimes of 2′- O- and 3′- O-Cy3-EDA-ADP·S1·V i complexes, 0.9 and 1.85 ns, compare with ∼0.7 ns for free analogs. Dynamic polarization shows rotational correlation times higher than 100 ns for both Cy3-EDA-ADP·S1·V i complexes, but the 2′- O-isomer only has also a 0.2-ns component. Thus, when bound, 3′- O-Cy3-EDA-ADP’s fluorescence is twofold brighter with motion more restricted and turnover slower than the 2′- O-isomer; these data are relevant for applications of these analogs in single molecule studies.

In vitro actomyosin motility in deuterium oxide.

Actin filament velocities in an in vitro motility assay system were measured both in heavy water (deuterium oxide, D2O) and water (H2O) to examine the effect of D2O on the actomyosin interaction. The dependence of the sliding velocity on pD of the D2O assay solution showed a broad pD optimum of around pD 8.5 which resembled the broad pH optimum (pH 8.5) of the H2O assay solution, but the maximum velocity (4.1 +/- 0.5 microm/sec, n=11) at pD 8.5 in D2O was about 60% of that (7.1 +/- 1.1 microm/sec, n=11) at pH 8.5 in H2O. The Km values of 95 and 80 microM and Vmax values of 3.2 and 5.1 microm/sec for the D2O and H2O assay were obtained by fitting the ATP concentration dependence of the velocity (at pD and pH 7.5) to the Michaelis-Menten equation. The Km value of actin-activated Mg-ATPase activity of myosin subfragment 1(S1) was decreased from 50 microM[actin] in H2O to 33 microM[actin] in D2O without any significant changes in Vmax (9.4 s(-1) in D2O and 9.3 s(-1) in H2O). The rate constants of ADP release from the acto-S1-ADP complex measured by the stopped flow method were 361 +/- 26 s(-1) (n=27) in D2O and 512 +/- 39 s(-1) (n=27) in H2O at 6 degrees C. These results suggest that the decrease in the in vitro actin-myosin sliding velocity in D2O results from a slowing of the release of ADP from the actomyosin-ADP complex and the increase in the affinity of actin for myosin in the presence of ATP in D2O.

Trifluoperazine inhibits the MgATPase activity and in vitro motility of conventional and unconventional myosins.

Trifluoperazine, a calmodulin antagonist, has recently been shown to inhibit the MgATPase activity of scallop myosin in the absence of light chain dissociation (Patel et al. (2000) J Biol Chem 275: 4880-4888). To investigate the generality of this observation and the mechanism by which it occurs, we have examined the ability of trifluoperazine to inhibit the enzymatic properties of other conventional and unconventional myosins. We show that trifluoperazine can inhibit the actin-activated MgATPase activity of rabbit skeletal muscle myosin II heavy meromyosin (HMM), phosphorylated turkey gizzard smooth muscle myosin II HMM, phosphorylated human nonmuscle myosin IIA HMM and myosin V subfragment-1 (S1). In all cases half maximal inhibition occurred at 50-75 microM trifluoperazine while light chains (myosin II) or calmodulin (myosin V) remained associated with the heavy chains. In vitro motility of all myosins tested was completely inhibited by trifluoperazine. Chymotryptic digestion of baculovirus-expressed myosin V HMM possessing only two calmodulin binding sites yielded a minimal motor fragment with no bound calmodulin. The MgATPase of this fragment was inhibited by trifluoperazine over the same range of concentrations as the S1 fragment of myosin.

Three-dimensional structural analysis of individual myosin heads under various functional states.

Half a century has passed since the dedicated studies on the contraction mechanisms of muscle began, with considerable knowledge on its molecular architecture. Two major hypotheses were raised very early, one, ”sliding filament theory”,1,2 and the other, “crossbridge theory”. 3 The former was readily accepted, because the phenomenon was apparently visible under optical microscope. The latter, however, has been hindered from thorough experimental proof even now, though nothing other than crossbridges connect thick and thin filaments enabling force development. The original idea postulated the rowing movement of actin-bound myosin head coupled with ATP hydrolysis, but it was later replaced by swinging of the “lever-arm” moiety,4 according to the discovery of intramolecular bending by X-ray crystallography.5-8 One of the major reasons for such persistent difficulty to prove this simple hypothesis might be the lack of means to directly observe the actual structural change of working crossbridges with time and spatial resolution enough to visualize the fine details of the molecular nano-machine. Though the crystal structure of each component; actin9, 10 and myosin subfragment-1 (S1) with or without various nucleotides,5-8 was determined ten years ago, none of their complexed form was solved nor might be the subject matter for easy crystallization.KeywordsActin FilamentMyosin HeadEssential Light ChainMyosin Motor DomainScallop MyosinThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Conformational Dynamics of the SH1-SH2 Helix in the Transition States of Myosin Subfragment-1

The α-helix containing the thiols, SH1 (Cys-707) and SH2 (Cys-697), has been proposed to be one of the structural elements responsible for the transduction of conformational changes in the myosin head (subfragment-1 (S1)). Previous studies, using a method that isolated and measured the rate of the SH1-SH2 cross-linking step, showed that this helix undergoes ligand-induced conformational changes. However, because of long incubation times required for the formation of the transition state complexes (S1.ADP.BeF x, S1.ADP.AlF 4−, and S1.ADP.V i), this method could not be used to determine the cross-linking rate constants for such states. In this study, kinetic data from the SH1-SH2 cross-linking reaction were analyzed by computational methods to extract rate constants for the two-step mechanism. For S1.ADP.BeF x, the results obtained were similar to those for S1.ATP γS. For reactions involving S1.ADP.AlF 4− and S1.ADP.V i, the first step (SH1 modification) is rate limiting; consequently, only lower limits could be established for the rate constants of the cross-linking step. Nevertheless, these results show that the cross-linking rate constants in the transition state complexes are increased at least 20-fold for all the reagents, including the shortest one, compared with nucleotide-free S1. Thus, the SH1-SH2 helix appears to be destabilized in the post-hydrolysis state.

Differential scanning calorimetric study of myosin subfragment 1 with tryptic cleavage at the N-terminal region of the heavy chain.

The thermal unfolding of myosin subfragment 1 (S1) cleaved by trypsin was studied by differential scanning calorimetry. In the absence of nucleotides, trypsin splits the S1 heavy chain into three fragments (25, 50, and 20 kDa). This cleavage has no appreciable influence on the thermal unfolding of S1 examined in the presence of ADP, in the ternary complexes of S1 with ADP and phosphate analogs, such as orthovanadate (Vi) or beryllium fluoride (BeFx), and in the presence of F-actin. In the presence of ATP and in the complexes S1.ADP.Vi or S1.ADP.BeFx, trypsin produces two additional cleavages in the S1 heavy chain: a faster cleavage in the N-terminal region between Arg23 and Ile24, and a slower cleavage at the 50 kDa fragment. It has been shown that the N-terminal cleavage strongly decreases the thermal stability of S1 by shifting the maximum of its thermal transition by about 7 degrees C to a lower temperature, from 50 degrees C to 42.4 degrees C, whereas the cleavage at both these sites causes dramatic destabilization of the S1 molecule leading to total loss of its thermal transition. Our results show that S1 with ATP-induced N-terminal cleavage is able, like uncleaved S1, to undergo global structural changes in forming the stable ternary complexes with ADP and Pi analogs (Vi, BeFx). These changes are reflected in a pronounced increase of S1 thermal stability. However, S1 cleaved by trypsin in the N-terminal region is unable, unlike S1, to undergo structural changes induced by interaction with F-actin that are expressed in a 4-5 degrees C shift of the S1 thermal transition to higher temperature. Thus, the cleavage between Arg23 and Ile24 does not significantly affect nucleotide-induced structural changes in the S1, but it prevents structural changes that occur when S1 is bound to F-actin. The results suggest that the N-terminal region of the S1 heavy chain plays an important role in structural stabilization of the entire motor domain of the myosin head, and a long-distance communication pathway may exist between this region and the actin-binding sites.

Effect of nucleotides and their analogues on essential light chains in myosin head

The domain movement in myosin head plays a decisive role in the energy transduction process of the muscle contraction. During hydrolysis of ATP, the specific formation of strong binding of myosin head for actin causes conformational changes. As a consequence, the light chain-binding motif generates the powerstroke. In our work maleimide spin labels were covalently attached to Cys-177 residue of ELC in subfragment-1 (S1). Our goal was to study the orientation dependence and the motion of S1, which were incorporated into glycerinated skeletal muscle fibres. The electron paramagnetic resonance spectroscopy (EPR) spectra of the probes depended strongly on the orientation of the fibre axis relative to the magnetic field, indicating that the essential light chain (ELC) and the neck were ordered. The probes were undergoing rapid motion within a cone. The half-width of the cone was estimated to be 65±5° (SD, n=8). Addition of ADP affected little the hyperfine splitting and the angular spread of the probe distribution. In the presence of ADP and orthovanadate the intensity of the spectra decreased, which showed the dissociation of S1 and this was accompanied with the disappearance of the orientation dependence.

A novel pressure-jump apparatus for the microvolume analysis of protein-ligand and protein-protein interactions: its application to nucleotide binding to skeletal-muscle and smooth-muscle myosin subfragment-1.

Reactions involving proteins frequently involve large changes in volume, which allows the equilibrium position to be perturbed by changes in pressure. Rapid changes in pressure can thus be used to initiate relaxation in pressure; however, this approach is seldom used, because it requires specialized equipment. We have built a microvolume (50 microl) pressure-jump apparatus, powered by a piezoelectric actuator, based on the original design of Clegg and Maxfield [(1976) Rev. Sci. Instrum. 47, 1383-1393]. This equipment can apply pressure changes of +/-20 MPa (maximally) in time periods as short as 80 micros and follow the resulting change in fluorescence signals. The system is relatively simple to use with fast (approx. 1 min) exchange of samples. In the present study, we show that this system can perturb the binding of 2'(3')-O-(N-methylanthraniloyl)-ADP to myosin subfragment-1(S1) from skeletal and smooth muscles. The kinetic data are consistent with previous work, and in addition show that, although 2'(3')-O-(N-methylanthraniloyl)-ADP binds with a similar affinity to both proteins, the increase in molar volume for the skeletal-muscle S1 binding to ADP is half of that for the smooth-muscle protein. This high-volume change for smooth-muscle S1 may be related to the ability of ADP to induce a 23 degrees tilt in the tail of S1 bound to actin.

Active site control of myosin cross-bridge zeta potential.

The electrical properties of contractile proteins contribute to muscle structure and perhaps function but have not been characterized adequately. Electrophoretic mobility, mu(e), is sensitive to the net electric charge and hydrodynamic size of a molecule in solution. Zeta potential, zeta, particle charge, Q(e), and particle charge-to-mass ratio are proportional to mu(e). We measured mu(e) for nucleotide complexes of skeletal muscle heavy meromyosin (HMM) and subfragment 1 (S1). The results indicate that mu(e) for HMM changes depending on the ligand bound in the active site. The changes in electric charge appear to occur mainly on the S1 moieties. For HMM(MgATPgammaS)(2) and HMM(MgADP.P(i))(2) the values of mu(e) are -0.077 and -0.17 (microm/s)/(V/cm), respectively. For these complexes, mu(e) is independent of [ATP], [ADP], and [P(i)]. When P(i) dissociates from HMM(MgADP.P(i))(2) to form HMM(MgADP)(2), mu(e) decreases to -0.61 (microm/s)/(V/cm). This large decrease in mu(e) is independent of free [ADP] or [ATP]. Increasing [P(i)], on the other hand, increases mu(e) for HMM(MgADP)(2) to values near those observed for the steady-state intermediate. For HMM, mu(e) = -0.34 and is independent of P(i). MgADP binding to HMM decreases mu(e) to -0.57 (microm/s)/(V/cm), and the dissociation constant is 9 microM. Taken together, these data indicate that mu(e) and, thus, zeta are controlled by ligand binding to the active site. The magnitudes of the particle charge-to-mass ratios for the HMM complexes are all in a range that falls within published values determined for a variety of other proteins. Possible roles that the observed nucleotide-dependent changes in cross-bridge electric charge might have in the contractile cycle in muscle are considered.

Troponin-Tropomyosin: An Allosteric Switch or a Steric Blocker?

The interaction of myosin subfragment 1 (S1) with actin-tropomyosin-troponin (regulated actin) is highly nucleotide dependent. The binding of S1 or S1-ADP (but not S1-ATP nor N, N′- ρ-phenylenedimaleimide-modified S1-ATP) to regulated actin activates ATP hydrolysis even in the absence of Ca 2+. Investigations with S1 and S1-ADP have led to the idea that some actin sites are directly blocked toward the binding of S1 either by tropomyosin or troponin. The blocked state is thought to occur only at ionic strengths greater than 50 mM. The question is whether nonactivating S1 binding is blocked under the same conditions. We show that troponin inhibits binding of the nonactivating state, N, N′- ρ-phenylenedimaleimide-S1-ATP, to actin but only when tropomyosin is absent. A lag in the rate of binding of activating S1 to actin (an indicator of the blocked state) occurs only in the presence of tropomyosin. Thus, tropomyosin inhibits binding of rigor S1 but not S1-ATP-like states. No evidence for an ionic strength-dependent change in the mechanism of regulation was observed either from measurements of the rate of activating S1 binding or from the equilibrium binding of nonactivating S1 to actin. At all conditions examined, N, N′- ρ-phenylenedimaleimide-S1-ATP bound to regulated actin in the absence of Ca 2+. These results support the view of regulation in which tropomyosin movement is an allosteric switch that is modulated by activating myosin binding but that does not function solely by regulating myosin binding.

Ca(2+)- and s1-induced movement of troponin T on mutant thin filaments reconstituted with functionally deficient mutant tropomyosin.

The deletion mutant (D234Tm) of rabbit skeletal muscle alpha-tropomyosin, in which internal actin-binding pseudo-repeats 2, 3, and 4 are missing, inhibits the thin filament activated myosin-ATPase activity whether Ca(2+) ion is present or not [Landis et al. (1997) J. Biol. Chem. 272, 14051-14056]. Fluorescence resonance energy transfer (FRET) showed substantial changes in distances between Cys-60 or 250 of troponin T (TnT) and Gln-41 or Cys-374 of actin on wild-type thin filaments corresponding to three states of thin filaments [Kimura et al. (2002) J. Biochem. 132, 93-102]. Troponin T movement on mutant thin filaments reconstituted with D234Tm was compared with that on wild-type thin filaments to understand from which the functional deficiency of mutant thin filaments derives. The Ca(2+)-induced changes in distances between Cys-250 of TnT and Gln-41 or Cys-374 of F-actin were smaller on mutant thin filaments than on wild-type thin filaments. On the other hand, the distances between Cys-60 of TnT and Gln-41 or Cys-374 of F-actin on mutant thin filaments did not change at all regardless of whether Ca(2+) was present. Thus, FRET showed that the Ca(2+)-induced movement of TnT was severely impaired on mutant thin filaments. The rigor binding of myosin subfragment 1 (S1) increased the distances when the thin filaments were fully decorated with S1 in the presence and absence of Ca(2+). However, plots of the extent of S1-incuced movement of TnT against molar ratio of S1 to actin in the presence and absence of Ca(2+) showed that the S1-induced movement of TnT was also impaired on mutant thin filaments. The deficiency of TnT movement on mutant thin filaments causes the altered S1-induced movement of TnI, and mutant thin filaments consequently fail to activate the myosin-ATPase activity even in the presence of Ca(2+).

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.

Temperature plasticity of contractile proteins in fish muscle.

Three myosin heavy chain isoforms with different actin-activated Mg(2+)-ATPase activities were found in the fast skeletal muscle from carp (Cyprinus carpio) acclimated to 10 and 30 degrees C. The composition of three types of myosin heavy chain was dependent on acclimation temperature, demonstrating the presence of temperature-specific myosin isoforms in carp. Subsequently, the temperature-dependence of the sliding velocity of fluorescent F-actin in myosins isolated from 10 degrees C- and 30 degrees C-acclimated carp was measured. At 8 degrees C, the filament velocity was three times higher for myosin from 10 degrees C- than from 30 degrees C-acclimated fish. Activation energies (E(a)) for the sliding velocity of F-actin were 63 and 111 kJ mol(-1) for myosins from 10 degrees C- and 30 degrees C-acclimated fish, respectively. Activation energy for actin-activated Mg(2+)-ATPase activity was 0.46 kJ mol(-1) in myosin from 10 degrees C-acclimated fish and 0.54 kJ mol(-1) in myosin from 30 degrees C-acclimated fish. The inactivation rate constant (K(D)) of Ca(2+)-ATPase was 7.5x10(-4)s(-1) at 30 degrees C for myosin from 10 degrees C-acclimated fish, which was approximately twice that for myosin from 30 degrees C-acclimated fish. It is suggested that these differences in thermostability reflect a more flexible structure of the myosin molecule in cold-acclimated carp, which results in a reduced activation enthalpy for contraction and, hence, a higher sliding velocity at low temperatures. Structural analysis of cDNAs encoding the carp myosin heavy chain demonstrated striking differences in two surface loops of myosin subfragment-1 (S1), loops 1 and 2, between the 10 degrees C and 30 degrees C types, which were predominantly expressed in carp acclimated to 10 degrees C and 30 degrees C, respectively. Chimeric myosins composed of Dictyostelium discoideum myosin backbones with loop sequences of carp S1 heavy chain isoforms demonstrated that the diversity of the loop 2 sequence of carp S1 affected the V(max) of actin-activated Mg(2+)-ATPase activity.

Effect of high hydrostatic pressure on chicken myosin subfragment-1

Hydrostatic pressure-induced structural changes in subfragment-1 (S1) of myosin molecule were studied. ATP-induced emission spectra of S1 were used to detect global structural change of S1 by pressure treatment. The fluorescence intensity of unpressurized S1 increased by addition of ATP. The increment of fluorescence of pressurized S1 up to 150 MPa was almost the same as control, whereas it became smaller above 200 MPa. ATP binding ability of S1 examined using 1, N 6-ethenoadenosine 5′-diphosphate (ε-ADP) indicated that the binding of ε-ADP to S1 decreased in the range of 250–300 MPa. S1 pressurized below 250 MPa and unpressurized S1 similarly bound to F-actin, although binding of S1 pressurized above 250 MPa decreased. Electron microscopic observation revealed arrowhead structure in control acto-S1, while disordered arrowhead structure was observed in acto-S1 prepared from pressurized S1 at 300 MPa. S1 pressurized below 250 MPa retained the same actin activated ATPase activity as the control, whereas the activity decreased to 60% at 300 MPa. Pressure treated S1 was easily cleaved by tryptic digestion into three domains, i.e. 27 kDa (N-terminal), 50 and 20 kDa (C-terminal) fragments, which were the same as those in unpressurized one. It is concluded that pressure-induced global structural changes of S1 begin to occur about 150 MPa, and the local structural changes in ATPase and actin binding sites followed with elevating pressure to 250–300 MPa.

Changes in the [formula omitted] NMR spectrum of rabbit muscle myosin subfragment 1 ·MgADP with temperature

In pioneering studies on the 31 P NMR spectra of MgADP bound to the “molecular motor” myosin subfragment 1 (S1) in the temperature range of 0 to 25 °C, Shriver and Sykes [Biochemistry 20 (1981) 2004–2012/6357–6362; Biochemistry 21 (1982) 3022–3028], proposed that MgADP binds to myosin S1 as a mixture of two interconvertible conformers with different chemical shifts for the β-P resonance of the S1-bound MgADP and that the concentrations of these conformers are related by an equilibrium constant K( T). Their model implied that the weighted average of the chemical shifts of the β-P(MgADP) for S1-bound MgADP asymptotically approaches a high temperature limit. Here, and in our earlier paper [K. Konno, K. Ue, M. Khoroshev, H., Martinez, B.D. Ray, M.F. Morales, Proc. Natl. Acad. Sci. USA 97 (2000) 1461–1466], we report experimental similarities to Shriver and Sykes, but diverge from them (especially at 0 °C) in not finding two distinct peaks and in finding that the average chemical shift does not change with temperature. Our observations can be explained by chemical exchange of β-P(MgADP) of S1-bound MgADP between two nearly energetically equivalent environments.