scholarly journals The effect of nucleotide upon a specific isomerization of actomyosin subfragment 1

1988 ◽  
Vol 256 (1) ◽  
pp. 41-46 ◽  
Author(s):  
M A Geeves ◽  
T E Jeffries
Keyword(s):  
Bound States ◽  
Bound State ◽  
Second Step ◽  
Weakly Bound ◽  
Binding Model ◽  
Nucleotide Analogues ◽  
A Value ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
Whole Muscle

The binding of actin to myosin subfragment 1 (S1) has been shown to occur as a two-step reaction [Coates, Criddle & Geeves (1985) Biochem. J. 232, 351-356]. In the first step actin is weakly bound and the second step involves the complex isomerizing to a more tightly bound state. This isomerization can be followed specifically by monitoring the fluorescence of actin that has been covalently labelled with N-(pyren-1-yl)-iodoacetamide at Cys-374 [Geeves, Jeffries & Millar (1986) Biochemistry 25, 8454-8458]. We report here that the presence of nucleotides and nucleotide analogues affects the equilibrium between the strongly bound and weakly bound states (referred to as K2). In the presence of ATP, [gamma-thio]ATP or ADP and vanadate a value of approx. less than 10(-2) was estimated for K2. In the presence of PPi or ADP a value of approx. 2.3 or 10 respectively was obtained. An increase in KCl concentration or the presence of 40% ethylene glycol was found to decrease K2 in the presence of ADP. The data presented here are consistent with the two-step binding model proposed by Geeves, Goody & Gutfreund [(1984) J. Muscle Res. Cell Motil. 5, 351-361], where it was suggested that the transition between weakly bound and strongly bound states is closely associated with the force-generating event in whole muscle.

CRYO-Electron Microscopy of the Acto-Myosin-ATP Complex

1990 ◽  
Vol 48 (1) ◽  
pp. 248-249
Author(s):  
John Trinickt ◽  
Howard White
Keyword(s):  
Electron Microscopy ◽  
Atp Hydrolysis ◽  
Myosin Head ◽  
Bound State ◽  
Cross Linking ◽  
Weakly Bound ◽  
Cryo Electron Microscopy ◽  
Myosin Subfragment 1 ◽  
Attached State ◽  
High Fraction

The primary force of muscle contraction is thought to involve a change in the myosin head whilst attached to actin, the energy coming from ATP hydrolysis. This change in attached state could either be a conformational change in the head or an alteration in the binding angle made with actin. A considerable amount is known about one bound state, the so-called strongly attached state, which occurs in the presence of ADP or in the absence of nucleotide. In this state, which probably corresponds to the last attached state of the force-producing cycle, the angle between the long axis myosin head and the actin filament is roughly 45°. Details of other attached states before and during power production have been difficult to obtain because, even at very high protein concentration, the complex is almost completely dissociated by ATP. Electron micrographs of the complex in the presence of ATP have therefore been obtained only after chemically cross-linking myosin subfragment-1 (S1) to actin filaments to prevent dissociation. But it is unclear then whether the variability in attachment angle observed is due merely to the cross-link acting as a hinge.We have recently found low ionic-strength conditions under which, without resorting to cross-linking, a high fraction of S1 is bound to actin during steady state ATP hydrolysis. The structure of this complex is being studied by cryo-electron microscopy of hydrated specimens. Most advantages of frozen specimens over ambient temperature methods such as negative staining have already been documented. These include improved preservation and fixation rates and the ability to observe protein directly rather than a surrounding stain envelope. In the present experiments, hydrated specimens have the additional benefit that it is feasible to use protein concentrations roughly two orders of magnitude higher than in conventional specimens, thereby reducing dissociation of weakly bound complexes.

The dynamics of the interaction between myosin subfragment 1 and pyrene-labelled thin filaments, from rabbit skeletal muscle

1986 ◽  
Vol 229 (1254) ◽  
pp. 85-95 ◽  
Keyword(s):  
Skeletal Muscle ◽  
Binding Affinity ◽  
Kinetic Studies ◽  
Rabbit Skeletal Muscle ◽  
Fluorescent Label ◽  
Weakly Bound ◽  
Thin Filaments ◽  
Fluorescence Titration ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1

We have used actin labelled in Cys–374 with N -(1-pyrenyl)iodoacetamide to monitor the dynamics and equilibria of the interaction between myosin subfragment 1 and the actin–troponin–tropomyosin complex in the presence of calcium. These results are compared with those obtained for pure actin and myosin subfragment 1. The sensitivity of this fluorescent label allowed us to measure the binding affinity of myosin subfragment 1 for actin directly by fluorescence titration. The affinity of subfragment 1 for actin is increased sixfold by troponin–tropomyosin in the presence of calcium. Kinetic studies of the interaction of subfragment 1 and actin have revealed an isomerization of the actin–subfragment 1 complex from a state in which actin is weakly bound ( K a = 5.9 x 10 4 M -1 ) to a more tightly bound complex ( K a = 1.7 x 10 7 M -1 ) (Coates, Criddle & Geeves (1985) Biochem. J. 232, 351). Results in the presence of troponin–tropomyosin show the same isomerization. The sixfold increase in affinity of subfragment 1 for actin is shown to be due to a decrease in the rate of dissociation of actin from the weakly bound complex.

scholarly journals Effects of deletion of tropomyosin overlap on regulated actomyosin subfragment 1 ATPase

1989 ◽  
Vol 258 (3) ◽  
pp. 831-836 ◽  
Author(s):  
D H Heeley ◽  
L B Smillie ◽  
E M Lohmeier-Vogel
Keyword(s):  
Ionic Strength ◽  
Binding Model ◽  
Thin Filaments ◽  
System A ◽  
Atpase Assay ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
Low Ionic Strength ◽  
Atpase Inhibition

The role of the overlap region at the ends of tropomyosin molecules in the properties of regulated thin filaments has been investigated by substituting nonpolymerizable tropomyosin for tropomyosin in a reconstituted troponin-tropomyosin-actomyosin subfragment 1 ATPase assay system. A previous study [Heeley, Golosinka & Smillie (1987) J. Biol. Chem. 262, 9971-9978] has shown that at an ionic strength of 70 mM, troponin will induce full binding of nonpolymerizable tropomyosin to F-actin both in the presence and absence of calcium. At a myosin subfragment 1-to-actin ratio of 2:1 ([actin] = 4 microM) and an ionic strength of 50 mM, comparable levels of ATPase inhibition were observed with increasing levels of tropomyosin or the truncated derivative in the presence of troponin (-Ca2+). Large differences were noted, however, in the activation by Ca2+. Significantly lower ATPase activities were observed with nonpolymerizable tropomyosin and troponin (+Ca2+) over a range of subfragment 1-to-actin ratios from 0.25 to 2.5. The concentration of subfragment 1 required to generate ATPase activities exceeding those seen with actomyosin subfragment 1 alone under these conditions was 3-4-fold greater when nonpolymerizable tropomyosin was used. Similar effects were seen at the much lower ionic strength of 13 mM and are consistent with the reduced ATPase activity with nonpolymerizable tropomyosin observed previously [Walsh, Trueblood, Evans & Weber (1985) J. Mol. Biol. 182, 265-269] at low ionic strength and a subfragment 1-to-actin ratio of 1:100. Little cooperativity in activity as a function of subfragment 1 concentration with either intact tropomyosin or its truncated derivative was observed under the present conditions. Further studies are directed towards an understanding of these effects in terms of the two-state binding model for the attachment of myosin heads to regulated thin filaments.

18O-Exchange by Hydrolyzing Enzymes: Extension of the Model to Pi Molecules with Inequivalent Oxygen Atoms in the Bound State

1981 ◽  
Vol 36 (7-8) ◽  
pp. 539-544 ◽  
Author(s):  
Paul Rösch
Keyword(s):  
Statistical Model ◽  
Data Fitting ◽  
Order Rate Constant ◽  
Bound State ◽  
Oxygen Exchange ◽  
Surrounding Water ◽  
Fitting Procedure ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
Hydrolyzing Enzymes

Abstract Enzymes causing an exchange of oxygens from P\ with the surrounding water oxygens are very common. A statistical model for the data evaluation for an observation of this oxygen exchange by isotope methods is presented. It is shown how different cases of inequivalence of the four Pi oxygens may be uncovered. The number of reversals of the oxygen exchange step on the enzyme and the apparent second order rate constant for the binding of Pi to the enzyme are obtained as a result of the data fitting procedure. Cobalt phosphatase, zinc phosphatase, and myosin subfragment 1 are treated as examples.

A spin-lattice relaxation study of the molecular reorientation of 2-methylindole as a function of viscosity and complex formation

1984 ◽  
Vol 62 (8) ◽  
pp. 1618-1621 ◽  
Author(s):  
Kenneth J. Friesen ◽  
Barry J. Blackburn
Keyword(s):  
Bound States ◽  
Lattice Relaxation ◽  
Solvent System ◽  
Bound State ◽  
Lattice Relaxation Time ◽  
Spin Lattice Relaxation ◽  
Molecular Reorientation ◽  
Spin Lattice ◽  
Slip Boundary ◽  
A Value

The viscosity (η) dependence of the 13C spin-lattice relaxation time (T1) of C-3 of 2-methylindole was investigated by changing the temperature (T) and by changing the solvent system. The observed η/T dependence of the effective reorientation correlation time (calculated from T1) is compared with those derived from theory based on hydrodynamic rotation with "stick" and with "slip" boundary conditions. This data implies that the reorientation of 2-methylindole obeys a near-slip condition in 1,2-dichloroethane. The 13C T1 of C-3 of 2-methylindole was also determined as a function of the fraction complexed with 1,3,5-trinitrobenzene (TNB) at 35 °C in 1,2-dichloroethane. The observed T1 in this case, depends on the rates of reorientation of the molecule in the free and the bound states, the rate of association of the molecules, the rate of dissociation of the complex, as well as increases in viscosity brought about by increases in the concentration of TNB. Analysis of the data, by application of the Anderson–Fryer formulation, gives a rate of reorientation in the bound state of 55 ns−1 at 35 °C and 0.69 cP as compared to a value of 150 ns−1 in the free state under the same conditions.

scholarly journals Time-resolved electron microscopic analysis of the behavior of myosin heads on actin filaments after photolysis of caged ATP.

1993 ◽  
Vol 121 (5) ◽  
pp. 1053-1064 ◽  
Author(s):  
T Funatsu ◽  
E Kono ◽  
S Tsukita
Keyword(s):  
Actin Filaments ◽  
Double Helix ◽  
Microscopic Analysis ◽  
Electron Microscopic ◽  
Weakly Bound ◽  
Time Resolved ◽  
Caged Atp ◽  
Dissociation Equilibrium ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1

The interaction between myosin subfragment 1 (S1) and actin filaments after the photolysis of P3-1-(2-nitrophenyl)ethyl ester of ATP (caged ATP) was analyzed with a newly developed freezing system using liquid helium. Actin and S1 (100 microM each) formed a ropelike double-helix characteristic of rigor in the presence of 5 mM caged ATP at room temperature. At 15 ms after photolysis, the ropelike double helix was partially disintegrated. The number of S1 attached to actin filaments gradually decreased up to 35 ms after photolysis, and no more changes were detected from 35 to 200 ms. After depletion of ATP, the ropelike double helix was reformed. Taking recent analyses of actomyosin kinetics into consideration, we concluded that most S1 observed on actin filaments at 35-200 ms are so called "weakly bound S1" (S1.ATP or S1.ADP.Pi) and that the weakly bound S1 under a rapid association-dissociation equilibrium with actin filaments can be captured by electron microscopy by means of our newly developed freezing system. This enabled us to directly compare the conformation of weakly and strongly bound S1. Within the resolution of deep-etch replica technique, there were no significant conformational differences between weakly and strongly bound S1, and neither types of S1 showed any positive cooperativity in their binding to actin filaments. Close comparison revealed that the weakly and strongly bound S1 have different angles of attachment to actin filaments. As compared to strongly bound S1, weakly bound S1 showed a significantly broader distribution of attachment angles. These results are discussed with special reference to the molecular mechanism of acto-myosin interaction in the presence of ATP.

Structure of Myosin Subfragment-1 from Electron Microscopy and Crystallography

1988 ◽  
Vol 46 ◽  
pp. 156-157
Author(s):  
Donald A. Winkelmann
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Actin Filaments ◽  
Light Chains ◽  
Myosin Filament ◽  
Primary Role ◽  
Heavy Chains ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1 ◽  
Globular Head

The primary role of the interaction of actin and myosin is the generation of force and motion as a direct consequence of the cyclic interaction of myosin crossbridges with actin filaments. Myosin is composed of six polypeptides: two heavy chains of molecular weight 220,000 daltons and two pairs of light chains of molecular weight 17,000-23,000. The C-terminal portions of the myosin heavy chains associate to form an α-helical coiled-coil rod which is responsible for myosin filament formation. The N-terminal portion of each heavy chain associates with two different light chains to form a globular head that binds actin and hydrolyses ATP. Myosin can be fragmented by limited proteolysis into several structural and functional domains. It has recently been demonstrated using an in vitro movement assay that the globular head domain, subfragment-1, is sufficient to cause sliding movement of actin filaments.The discovery of conditions for crystallization of the myosin subfragment-1 (S1) has led to a systematic analysis of S1 structure by x-ray crystallography and electron microscopy. Image analysis of electron micrographs of thin sections of small S1 crystals has been used to determine the structure of S1 in the crystal lattice.

scholarly journals Studies of Ligand-induced Conformational Perturbations in Myosin Subfragment 1

1989 ◽  
Vol 264 (18) ◽  
pp. 10810-10819
Author(s):  
K N Rajasekharan ◽  
M Mayadevi ◽  
M Burke
Keyword(s):  
Myosin Subfragment ◽  
Myosin Subfragment 1
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