Kinetic properties of binding of myosin subfragment-1 with F-actin in the absence of nucleotide

The rate constant for the binding of myosin subfragment-1 (S-1) with F-actin in the absence of nucleotide, k1, and that for dissociation of the F-actin-myosin subfragment-1 complex (acto-S-1), k-1, were measured independently. The rate of S-1 binding with F-actin was measured from the time course of the change in the light scattering intensity after mixing S-1 with various concentrations of F-actin and k1 was found to be 2.55 X 10(6) M-1 X S-1 at 20 degrees C. The dissociation rate of acto-S-1 was determined using F-actin labeled with pyrenyl iodoacetamide (Pyr-FA). Pyr-FA, with its fluorescence decreased by binding with S-1, was mixed with acto-S-1 complex and the rate of displacement of F-actin by Pyr-FA was measured from the decrease in the Pyr-FA fluorescence intensity. The k-1 value was calculated to be 8.5 X 10(-3) S-1 (or 0.51 min-1). The value of the dissociation constant of S-1 from acto-S-1 complex, Kd, was calculated from Kd = k-1/k1 to be 3.3 X 10(-9) M at 20 degrees C. Kd was also measured at various temperatures (0-30 degrees C), and the thermodynamic parameters, delta G degree, delta H degree, and delta S degree, were estimated from the temperature dependence of Kd to be -11.3 kcal/mol, +2.5 kcal/mol, and +47 cal/deg . mol, respectively. Thus, the binding of the myosin head with F-actin was shown to be endothermic and entropy-driven.     

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)     

Interaction of myosin subfragments with F-actin.

The effect of ionic strength, temperature, and divalent cations on the association of myosin with actin was determined in the ultracentrifuge using scanning absorption optics. The association constant (Ka) for the binding of heavy meromyosin (HmM) to F-actin was 1 X 10(7) M-1 at 20 degrees C, in 0.10 M KCl, 0.01 M imidazole (pH 7.0), 5 MM potassium phosphate, 1 mM MgCl2, and 0.3 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. Ka was the same for HMM prepared by trypsin or chymotrypsin. The affinity of subfragment 1 (S1) for actin under the same ionic conditions was 3 X 10(6) M-1. Varying the preparative procedure for S1 had little effect on Ka. The small difference in binding energy between HMM and S1 suggests that either only one head can bind strongly to actin at a time or that free energy is lost during the sterically unfavorable attachment of the two heads to actin.     

Interaction of F-actin-AMPPNP with myosin subfragment-1 and troponin-tropomyosin: influence of an extra phosphate at the nucleotide binding site in F-actin on its function

An unsplitable analogue of ATP (adenylyl imidodiphosphate; AMPPNP) was incorporated into F-actin [Cooke, R. (1975) Biochemistry 14, 3250-3256]. The resulting polymers (F-actin-AMPPNP) activated the ATPase activity of myosin subfragment-1 (S1) as efficiently as normal F-actin; neither the maximum velocity at infinite actin concentration (Vmax) nor the affinity of actin to S1 in the presence of ATP (1/KATPase) changed, which indicates that the terminal phosphate of the bound nucleotide at the cleft region between the two domains of the actin molecule [Kabsch, W., Mannherz, H.G., & Suck, D. (1985) EMBO J. 4, 2113-2118] is not directly involved in a myosin binding site. However, the interaction of F-actin with troponin-tropomyosin was strongly modulated by the replacement of ADP with AMPPNP. The troponin-tropomyosin complex strongly enhanced the activation of S1-ATPase activity by F-actin-AMPPNP in the presence of Ca2+, although it has no effect on the activation by normal F-actin-ADP. KATPase was enhanced about threefold by troponin-tropomyosin in the presence of Ca2+, while Vmax was not markedly changed. F-actin-AMPPNP is highly potentiated by troponin-tropomyosin even with low S1 to actin ratios and at high ATP conditions. In the absence of Ca2+, the activation by F-actin-AMPPNP was inhibited normally by troponin-tropomyosin. The results suggest that the terminal beta-phosphate of the bound nucleotide in F-actin is located in a region which is important for regulation of the interaction with myosin.     

Instability of F-actin in the absence of ATP: a small amount of myosin destabilizes F-actin.

The effects of the neutral salt concentration, pH, and coexistence of myosin on the denaturation of F-actin without ATP at low temperature were studied using the DNase I inhibition assay. The percent denaturation of F-actin gradually increased with a decrease in pH from 8.0 to 5.2, on incubation for 2 weeks in the presence of 50 mM KCl at 0 degrees C. This change was much faster in 0.5 M KCl and more than 75% of the F-actin became denatured on incubation for 1 week at pH 5.2. The buffer composition was found to exert a strong influence on the denaturation of F-actin. That is, there was a tendency for the denaturation of F-actin at pH 6.0 to be faster in MES[2-(N-morpholino)ethanesulfonic acid]-NaOH buffer than in sodium phosphate buffer, the critical concentrations of actin in 0.5 M KCl being 0.31 mg/ml for MES-NaOH buffer and 0.15 mg/ml for sodium phosphate buffer. A sigmoidal relationship was found between the percent denaturation of F-actin and the KCl concentration added, the greatest change occurring at KCl concentrations between 0.25 and 0.75 M. The time courses of the denaturation of F-actin showed that the percent denaturation rose at first and that in time the rate of the increase decreased. In the case of pH 8.0 and 0.5 M KCl, it took about 1 week for the denaturation rate to begin to drop. The pH of 6.0 further promoted the instability of F-actin exposed to high KCl concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence anisotropy of labeled F-actin: influence of divalent cations on the interaction between F-actin and myosin heads

The interaction between F-actin and soluble proteolytic fragments of myosin, heavy meromyosin and myosin subfragment 1 without ATP, has been studied by measuring the static anisotropy and the transient anisotropy decay of the fluorescent chromophore N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl) ethylenediamine bound to F-actin. In the presence of Ca2+ ions, the mobility of the chromophore was strongly decreased by adding heavy meromyosin or myosin subfragment 1, and this conformation change of F-actin showed a strong cooperativity; that is, a very small amount of myosin heads induced the maximum anisotropy change. On the other hand, in the presence of Mg2+ ions, the addition of a small amount of myosin subfragment 1 or of heavy meromyosin increased the mobility of labeled F-actin that reached a maximum at a molar ratio of about 1/25 or 1/50, respectively. With further addition of myosin heads, the mobility of the labeled actin decreased. From these studies, one concludes that F-actin undergoes a conformation change by interacting with myosin heads, which depends on the nature of the divalent cations present in the solution.     

F-actin and myosin II binding domains in supervillin

Detergent-resistant membranes contain signaling and integral membrane proteins that organize cholesterol-rich domains called lipid rafts. A subset of these detergent-resistant membranes (DRM-H) exhibits a higher buoyant density ( approximately 1.16 g/ml) because of association with membrane skeleton proteins, including actin, myosin II, myosin 1G, fodrin, and an actin- and membrane-binding protein called supervillin (Nebl, T., Pestonjamasp, K. N., Leszyk, J. D., Crowley, J. L., Oh, S. W., and Luna, E. J. (2002) J. Biol. Chem. 277, 43399-43409). To characterize interactions among DRM-H cytoskeletal proteins, we investigated the binding partners of the novel supervillin N terminus, specifically amino acids 1-830. We find that the supervillin N terminus binds directly to myosin II, as well as to F-actin. Three F-actin-binding sites were mapped to sequences within amino acids approximately 280-342, approximately 344-422, and approximately 700-830. Sequences with combinations of these sites promote F-actin cross-linking and/or bundling. Supervillin amino acids 1-174 specifically interact with the S2 domain in chicken gizzard myosin and nonmuscle myosin IIA (MYH-9) but exhibit little binding to skeletal muscle myosin II. Direct or indirect binding to filamin also was observed. Overexpression of supervillin amino acids 1-174 in COS7 cells disrupted the localization of myosin IIB without obviously affecting actin filaments. Taken together, these results suggest that supervillin may mediate actin and myosin II filament organization at cholesterol-rich membrane domains.     

Use of stable analogs of myosin ATPase intermediates for kinetic studies of the "weak" binding of myosin heads to F-actin.

It is known that ternary complexes of myosin subfragment 1 (S1) with ADP and the Pi analogs beryllium fluoride (BeFx) and aluminum fluoride (AlF4-) are stable analogs of the myosin ATPase intermediates M* x ATP and M** x ADP x Pi, respectively. Using kinetic approaches, we compared the rate of formation of the complexes S1 x ADP x BeFx and S1 x ADP x AlF4- in the absence and in the presence of F-actin, as well as of the interaction of these complexes with F-actin. We show that in the absence of F-actin the formation of S1 x ADP x BeFx occurs much faster (3-4 min) than that of S1 x ADP x AlF4- (hours). The formation of these complexes in the presence of F-actin led to dissociation of S1 from F-actin, this process being monitored by a decrease in light scattering. The light scattering decrease of the acto-S1 complex occurred much faster after addition of BeFx (during 1 min) than after addition of AlF4- (more than 20 min). In both cases the light scattering of the acto-S1 complex decreased by 40-50%, but it remained much higher than that of F-actin measured in the absence of S1. The interaction of the S1 x ADP x BeFx and S1 x ADP x AlF4- complexes with F-actin was studied by the stopped-flow technique with high time resolution (no more than 0.6 sec after mixing of S1 with F-actin). We found that the binding of S1 x ADP x BeFx or S1 x ADP x AlF4- to F-actin is accompanied by a fast increase in light scattering, but it does not affect the fluorescence of a pyrene label specifically attached to F-actin. We conclude from these data that within this time range a "weak" binding of the S1 x ADP x BeFx and S1 x ADP x AlF4- complexes to F-actin occurs without the subsequent transition of the "weak" binding state to the "strong" binding state. Comparison of the light scattering kinetic curves shows that S1 x ADP x AlF4- binds to F-actin faster than S1 x ADP x BeFx does: the second-order rate constants for the "weak" binding to F-actin are (62.8 +/- 1.8) x 10(6) M-1 x sec-1 in the case of S1 x ADP x AlF4- and (22.6 +/- 0.4) x 10(6) M-1 x sec-1 in the case of S1 x ADP x BeFx. We conclude that the stable ternary complexes S1 x ADP x BeFx and S1 x ADP x AlF4- can be successfully used for kinetic studies of the "weak" binding of the myosin heads to F-actin.     

Reaction of two heads of gizzard myosin with ATP

The reaction intermediates formed by the two heads of smooth muscle myosin were studied. The amount of myosin-phosphate-ADP complex, MPADP, formed was measured from the Pi-burst size over a wide range of ATP concentrations. At low concentrations of ATP, the Pi-burst size was 0.5 mol/mol myosin head, and the apparent Kd value was about 0.15 microM. However, at high ATP concentrations, the Pi burst size increased from 0.5 to 0.75 mol/mol myosin head with an observed Kd value of 15 microM. The binding of nucleotides to gizzard myosin during the ATPase reaction was directly measured by a centrifugation method. Myosin bound 0.5 mol of nucleotides (ATP and ADP) with high affinity (Kd congruent to 1 microM) and 0.35 mol of nucleotides with low affinity (Kd = 24 microM) for ATP. These results indicate that gizzard myosin has two kinds of nucleotide binding sites, one of which forms MPADP with high affinity for ATP while the other forms MPADP and MATP with low affinity for ATP. We studied the correlation between the formation of MPADP and the dissociation of actomyosin. The amount of Pi-burst size was not affected by the existence of F-actin, and when 0.5 mol of ATP per mol of myosin head was added to actomyosin (1 mg/ml F-actin, 5 microM myosin at 0 degrees C) most (93%) of the added ATP was hydrolyzed in the Pi-burst phase. All gizzard actomyosin dissociated when 1 mol of ATP per mol myosin head was added to actomyosin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of myosin with F-actin: time-dependent changes at the interface are not slow

The kinetics of formation of the actin-myosin complex have been reinvestigated on the minute and second time scales in sedimentation and chemical cross-linking experiments. With the sedimentation method, we found that the binding of the skeletal muscle myosin motor domain (S1) to actin filament always saturates at one S1 bound to one actin monomer (or two S1 per actin dimer), whether S1 was added slowly (17 min between additions) or rapidly (10 s between additions) to an excess of F-actin. The carbodiimide (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, EDC)-induced cross-linking of the actin-S1 complex was performed on the subsecond time scale by a new approach that combines a two-step cross-linking protocol with the rapid flow-quench technique. The results showed that the time courses of S1 cross-linking to either of the two actin monomers are identical: they are not dependent on the actin/S1 ratio in the 0.3-20-s time range. The overall data rule out a mechanism by which myosin rolls from one to the other actin monomer on the second or minute time scales. Rather, they suggest that more subtle changes occur at the actomyosin interface during the ATP cycle.     

Theoretical considerations in the equilibrium binding of myosin fragments on F-actin

In a previous paper, equilibrium constants for the binding of myosin fragments onto F-actin were assumed known and the statistical problems encountered when the actin sites are occupied to an arbitrary fractional extent were analyzed. The object of the present paper is to attempt to understand the observed order of magnitude of these equilibrium constants in terms of the statistical mechanical degrees of freedom involved. That is, we examine here the equilibrium constants them- selves rather than the statistical consequences of the equilibrium constants. The treatment given amounts to a semi-quantitative sketch or outline of the problem. Structural details are much too uncertain to warrant a careful and rigorous treatment at this time. But the discussion suffices to establish the essential qualitative features of the problem. The procedure used is to exa- mine the important equilibrium constants, one at a time, in terms of the factors (partition functions) that contribute to each constant, together with numerical estimates for these factors.     

Structure and function of the two heads of the myosin molecule. III. Cooperativity of the two heads of the myosin molecule, shown by the effect of modification of head A with rho-chloromercuribenzoate on the interaction of head B with F-actin.

Subfragment-1 of HMM was prepared by tryptic [EC 3.4.21.4] digestion of HMM, which had been modified with 1 mole of CMB per mole of HMM at a specific SH group, SHr. S-1(T) obtained from CMB-HMM retained almost all the CMB, and the amount of bound CMB was about 0.8-0.9 mole per 2 moles of S-1(T). S-2 of CMB-HMM contained no bound CMB. The ATPase [EC 3.6.1.3] activity of HMM increased gradually with increase in the concentration of FA, and the acto-HMM ATPase was inhibited by excess substrate or removal of Ca2+ ions in the presence of RP. The ATPase activity of CMB-HMM increased to a maximum level on adding a small amount of FA, and the acto-CMB-HMM ATPase showed neither substrate inhibition nor Ca2+ sensitivity in the presence of RP. On the other hand, the dependence on the concentration of FA of the ATPase activity of acto-S-1(T) was unaffected by modification of S-1 with CMB. The Ca2+ sensitivity of the ATPase activity of acto-S-1(T) in the presence of RP was also unaffected by the modification. Acto-S-1(T) dissociated almost completely, while acto-CMB-S-1(T) was only 50% dissociated on adding ATP. More than 80% of the bound CMB was contained in S-1(T) undissociated from FA. Furthermore, superprecipitation of actomyosin induced by ATP was completely inhibited by adding about 2 moles of CMB-S-1(T) per mole of actin monomer. On the other hand, about 90% of the burst size of Pi liberation was retained in S-1(T) dissociated from FA. It was concluded that the two heads of the myosin molecule are different: one shows the initial burst of Pi liberation, and does not contain the SHr group which binds CMB (head B), and the other does not show the initial burst and contains the SHr group (head A). It was also concluded that modification of head A of HMM or myosin with CMB increases its binding strength to FA, and consequently the substrate inhibition and Ca2+ sensitivity of acto-HMM or actomyosin ATPase at head B are lost on modification of head A with CMB. CMB-S-1(CT) was prepared by chymotryptic [EC 3.4.21.1] digestion of CMB-myosin, and separated into two fractions by ultracentrifugation of acto-CMB-S-1(CT) in the presence of ATP. Three components of CMB-S-1(CT) with molecular weights of 9, 2.4, and 1.2 X 10(4) were separated by SDS-polyacrylamide gel electrophoresis. The ratios of the peak areas of the three components in electrophoretograms were the same in CMB-S-1(CT) and in the two fractions (1 : 0.18 : 0.09), indicating that heads A and B have the same subunit structure.     

Intermonomer cross-linking of F-actin alters the dynamics of its interaction with H-meromyosin in the weak-binding state

Previous cross-linking studies [Kim E, Bobkova E, Hegyi G, Muhlrad A & Reisler E (2002) Biochemistry 41, 86-93] have shown that site-specific cross-linking among F-actin monomers inhibits the motion and force generation of actomyosin. However, it does not change the steady-state ATPase parameters of actomyosin. These apparently contradictory findings have been attributed to the uncoupling of force generation from other processes of actomyosin interaction as a consequence of reduced flexibility at the interface between actin subdomains-1 and -2. In this study, we use EPR spectroscopy to investigate the effects of cross-linking constituent monomers upon the molecular dynamics of the F-actin complex. We show that cross-linking reduces the rotational mobility of an attached probe. It is consistent with the filaments becoming more rigid. Addition of heavy meromyosin (HMM) to the cross-linked filaments further restricts the rotational mobility of the probe. The effect of HMM on the actin filaments is highly cooperative: even a 1 : 10 molar ratio of HMM to actin strongly restricts the dynamics of the filaments. More interesting results are obtained when nucleotides are also added. In the presence of HMM and ADP, similar strongly reduced mobility of the probe was found than in a rigor state. In the presence of adenosine 5'[betagamma-imido] triphosphate (AMPPNP), a nonhydrolyzable analogue of ATP, weak binding of HMM to either cross-linked or native F-actin increases probe mobility. By contrast, weak binding by the HMM/ADP/AlF4 complex has different effects upon the two systems. This protein-nucleotide complex increases probe mobility in native actin filaments, as does HMM + AMPPNP. However, its addition to cross-linked filaments leaves probe mobility as constrained as in the rigor state. These findings suggest that the dynamic change upon weak binding by HMM/ADP/AlF4 which is inhibited by cross-linking is essential to the proper mechanical behaviour of the filaments during movement.     

Strong binding of myosin heads stretches and twists the actin helix

Calculation of the size of the power stroke of the myosin motor in contracting muscle requires knowledge of the compliance of the myofilaments. Current estimates of actin compliance vary significantly introducing uncertainty in the mechanical parameters of the motor. Using x-ray diffraction on small bundles of permeabilized fibers from rabbit muscle we show that strong binding of myosin heads changes directly the actin helix. The spacing of the 2.73-nm meridional x-ray reflection increased by 0.22% when relaxed fibers were put into low-tension rigor (<10 kN/m(2)) demonstrating that strongly bound myosin heads elongate the actin filaments even in the absence of external tension. The pitch of the 5.9-nm actin layer line increased by approximately 0.62% and that of the 5.1-nm layer line decreased by approximately 0.26%, suggesting that the elongation is accompanied by a decrease in its helical angle (approximately 166 degrees) by approximately 0.8 degrees. This effect explains the difference between actin compliance revealed from mechanical experiments with single fibers and from x-ray diffraction on whole muscles. Our measurement of actin compliance obtained by applying tension to fibers in rigor is consistent with the results of mechanical measurements.