Preparation and characterization of frog muscle myosin subfragment 1 and actin.
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ABSTRACT:
SUBMITTER: Ferenczi MA
PROVIDER: S-EPMC1184145 | biostudies-other | 1978 Apr
REPOSITORIES: biostudies-other
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Project description:The data presented here confirm and provide further experimental evidence that rabbit skeletal-muscle myosin subfragment-1 (S-1) binds to the postulated actin-(338-348) hydrophobic segment [Kabsch, Mannherz, Suck, Pai and Holmes (1990) Nature (London) 347, 37-44] with high affinity in the absence and presence of MgATP. The apparent dissociation constant of the S-1 interaction (5.5 x 10(-7) M) with the actin-(338-348) peptide was of the same order of magnitude as that of the actin-(18-28) binding site (2 x 10(-6) M). In similar conditions, fragmented (27 kDa-50 kDa-20 kDa) S-1 also bound to the peptide. Antibodies directed to the vicinal sequence 348-358 were rapidly eliminated from actin by S-1 interaction and weakened S-1 binding to monomeric or filamentous actin. The antigenic site (348-358) is located very close to the C-terminal S-1-binding site (360-369) and encompasses some residues (Leu-349 and Phe-352) included in the hydrophobic S-1-binding region [Schröder, Manstein, Jahn, Holden, Rayment, Holmes and Spudich (1993) Nature (London) 364, 171-174]. It was observed that anti-[actin-(348-358)] antibodies were also unable to decrease actomyosin ATPase activity, in contrast with previous results obtained with anti-[actin-(18-28)] antibodies [Adams and Reisler (1993) Biochemistry 32, 5051-5056]. The hydrophobic actin-(338-348) peptide used in considerable excess was unable to perturb acto-S-1 and S-1 activities in contrast with results obtained with the N-terminal actin peptide [Kôgler, Moir, Trayer and Ruegg (1991) FEBS Lett. 294, 31-34].
| S-EPMC1138102 | biostudies-other
Project description:The Mg2+-dependent ATPase (adenosine 5'-triphosphatase) mechanism of myosin and subfragment 1 prepared from frog leg muscle was investigated by transient kinetic technique. The results show that in general terms the mechanism is similar to that of the rabbit skeletal-muscle myosin ATPase. During subfragment-1 ATPase activity at 0-5 degrees C pH 7.0 and I0.15, the predominant component of the steady-state intermediate is a subfragment-1-products complex (E.ADP.Pi). Binary subfragment-1-ATP (E.ATP) and subfragment-1-ADP (E.ADP) complexes are the other main components of the steady-state intermediate, the relative concentrations of the three components E.ATP, E.ADP.Pi and E.ADP being 5.5:92.5:2.0 respectively. The frog myosin ATPase mechanism is distinguished from that of the rabbit at 0-5 degrees C by the low steady-state concentrations of E.ATP and E.ADP relative to that of E.ADP.Pi and can be described by: E + ATP k' + 1 in equilibrium k' - 1 E.ATP k' + 2 in equilibrium k' - 2 E.ADP.Pi k' + 3 in equilibrium k' - 3 E.ADP + Pi k' + 4 in equilibrium k' - 4 E + ADP. In the above conditions successive forward rate constants have values: k' + 1, 1.1 X 10(5)M-1.S-1; k' + 2 greater than 5s-1; k' + 3, 0.011 s-1; k' + 4, 0.5 s-1; k'-1 is probably less than 0.006s-1. The observed second-order rate constants of the association of actin to subfragment 1 and of ATP-induced dissociation of the actin-subfragment-1 complex are 5.5 X 10(4) M-1.S-1 and 7.4 X 10(5) M-1.S-1 respectively at 2-5 degrees C and pH 7.0. The physiological implications of these results are discussed.
| S-EPMC1184146 | biostudies-other
Project description:Hydration water is essential for a protein to perform its biological function properly. In this study, the dynamics of hydration water around F-actin and myosin subfragment-1 (S1), which are the partner proteins playing a major role in various cellular functions related to cell motility including muscle contraction, was characterized by incoherent quasielastic neutron scattering (QENS). The QENS measurements on the D2O- and H2O-solution samples of F-actin and S1 provided the spectra of hydration water, from which the translational diffusion coefficient (DT), the residence time (τT), and the rotational correlation time (τR) were evaluated. The DT value of the hydration water of S1 was found to be much smaller than that of the hydration water of F-actin while the τT values were similar between S1 and F-actin. On the other hand, the τR values of the hydration water of S1 was found to be larger than that of the hydration water of F-actin. It was also found that the DT and τR values of the hydration water of F-actin are similar to those of bulk water. These results suggest a significant difference in mobility of the hydration water between S1 and F-actin: S1 has the typical hydration water, the mobility of which is reduced compared with that of bulk water, while F-actin has the unique hydration water, the mobility of which is close to that of bulk water rather than the typical hydration water around proteins.
| S-EPMC5600338 | biostudies-literature
Project description:We have used actin labelled at Cys-374 with N-(1-pyrenyl)iodoacetamide [Kouyama & Mihashi (1981) Eur. J. Biochem. 114, 33-38] to monitor pressure-induced relaxations of acto-myosin subfragment 1. This label greatly increases the sensitivity of measurement of dissociated actin and reveals the presence of two relaxations. The experimental data can be fitted by a model in which actin binds subfragment 1 relatively weakly (K = 5.9 X 10(4) M-1) and then isomerizes to a more tightly bound complex (K = 1.7 X 10(7) M-1). This directly observed isomerization supports the model of Geeves, Goody & Gutfreund [(1984) J. Muscle Res. Cell. Motil. 5, 351-361]. The rate of the isomerization is too high to be observed in the pressure-jump apparatus (less than 200 microseconds), but analysis of the amplitudes allows estimation of the equilibrium constant of the isomerization as 280 (20 degrees C, 0.1 M-KCl, pH 7). The equilibrium is sensitive to temperature, pressure, ionic strength and the presence of ethylene glycol. The pressure-sensitivity of the isomerization suggests a significant conformational change of the acto-myosin subfragment 1 complex.
| S-EPMC1152886 | biostudies-other
Project description:Hydration structures around F-actin and myosin subfragment-1 (S1), which play central roles as counterparts in muscle contraction, were investigated by small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS). The radius of gyration of chymotryptic S1 was evaluated to be 41.3±1.1 Å for SAXS, 40.1±3.0 Å for SANS in H2O, and 37.8±0.8 Å for SANS in D2O, respectively. The values of the cross-sectional radius of gyration of F-actin were 25.4±0.03 Å for SAXS, 23.4±2.4 Å for SANS in H2O, and 22.6 ± 0.6 Å for SANS in D2O, respectively. These differences arise from different contributions of the hydration shell to the scattering curves. Analysis by model calculations showed that the hydration shell of S1 has the average density 10-15% higher than bulk water, being the typical hydration shell. On the other hand, the hydration shell of F-actin has the average density more than 19% higher than bulk water, indicating that F-actin has a denser, unusual hydration structure. The results indicate a difference in the hydration structures around F-actin and S1. The unusual hydration structure around F-actin may have the structural property of so-called "hyper-mobile water" around F-actin.
| S-EPMC4629667 | biostudies-literature
Project description:Structural studies of myosin have indicated some of the conformational changes that occur in this protein during the contractile cycle, and we have now observed a conformational change in a bound nucleotide as well. The 3.1-A x-ray structure of the scallop myosin head domain (subfragment 1) in the ADP-bound near-rigor state (lever arm =45 degrees to the helical actin axis) shows the diphosphate moiety positioned on the surface of the nucleotide-binding pocket, rather than deep within it as had been observed previously. This conformation strongly suggests a specific mode of entry and exit of the nucleotide from the nucleotide-binding pocket through the so-called "front door." In addition, using a variety of scallop structures, including a relatively high-resolution 2.75-A nucleotide-free near-rigor structure, we have identified a conserved complex salt bridge connecting the 50-kDa upper and N-terminal subdomains. This salt bridge is present only in crystal structures of muscle myosin isoforms that exhibit a strong reciprocal relationship (also known as coupling) between actin and nucleotide affinity.
| S-EPMC428449 | biostudies-literature
Project description:We have determined the specificity of sera elicited by glutaraldehyde-stabilized bovine aortic actin. This modification induces a high titre of antibodies directed against the N-terminal (residues 1-39) and the C-terminal region of smooth-muscle actins. The crude antisera were purified on peptide (corresponding to the 1-9 or 1-8 N-terminal sequences of smooth-muscle isoactins)-polyacrylic-resin columns. By fractionating the antisera we obtained oligoclonal antibody populations specific for each isoactin.
| S-EPMC1147662 | biostudies-other
Project description:Calponin is a thin-filament-associated protein that has been implicated in the regulation of smooth-muscle contractility. It binds to F-actin and inhibits the MgATPase activity of actomyosin. In the present work we have examined the effect of recombinant chicken gizzard alpha-calponin (R alpha CaP) on the binding of rabbit skeletal-muscle myosin subfragment 1 (S1) to F-actin and on the inhibition of its actin-activated MgATPase. We have found that binding of one R alpha CaP molecule to every three to four actin monomers is sufficient for maximal inhibition of acto-S1 ATPase. At this R alpha CaP/actin ratio R alpha CaP does not interfere with S1 binding to F-actin. At higher concentrations, R alpha CaP displaces S1 from F-actin and a 1:1 R alpha CaP-actin monomer complex is formed. R alpha CaP is also able to displace troponin I from its complex with F-actin which may reflect the amino acid sequence similarity between R alpha CaP and troponin I in their actin-binding regions.
| S-EPMC1218099 | biostudies-other
Project description:The actin-myosin head complex in the rigor state reveals several high-affinity sites on the actin molecule in sequences 18-28 and 40-113. In the presence of Mg(2+)-ATP, participation of the actin N-terminal 1-7 sequence is known to occur. The proximity of the C-terminal region of actin to the A1 light chain of the myosin head [S-1(A1)] (where S-1 is myosin subfragment-1) was described previously. We observed that C-terminal antigenic structures located near Met-305, Met-325 and Met-355 and the C-terminal end (Cys-374) of actin are markedly modified in the presence of S-1(A1), S-1(A2) and scallop S-1 and in the absence of Mg(2+)-ATP. This seems to rule out any important specific involvement of the A1 light chain in the described conformational changes. An S-1-binding site was located in this actin C-terminal region by testing the tryptic CB9 peptide (360-372 sequence) previously implicated in the A1 light chain interaction. This peptide was able to bind well to S-1(A1), S-1(A2) and scallop S-1, but not in the presence of Mg(2+)-pyrophosphate. These results strengthen the hypothesis of a multisite interface between S-1 and actin located in the actin subdomain I.
| S-EPMC1132699 | biostudies-other
Project description:Much has been learned about the interaction between myosin and actin through biochemistry, in vitro motility assays and cryo-electron microscopy (cryoEM) of F-actin, decorated with myosin heads. Comparatively less is known about actin-myosin interactions within the filament lattice of muscle, where myosin heads function as independent force generators and thus most measurements report an average signal from multiple biochemical and mechanical states. All of the 3D imaging by electron microscopy (EM) that has revealed the interplay of the regular array of actin subunits and myosin heads within the filament lattice has been accomplished using the flight muscle of the large water bug Lethocerus sp. The Lethocerus flight muscle possesses a particularly favorable filament arrangement that enables all the myosin cross-bridges contacting the actin filament to be visualized in a thin section. This review covers the history of this effort and the progress toward visualizing the complex set of conformational changes that myosin heads make when binding to actin in several static states, as well as the fast frozen actively contracting muscle. The efforts have revealed a consistent pattern of changes to the myosin head structures as determined by X-ray crystallography needed to explain the structure of the different actomyosin interactions observed in situ.
| S-EPMC6479483 | biostudies-literature