Effects of deletion of tropomyosin overlap on regulated actomyosin subfragment 1 ATPase.

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.     

Cooperative binding of myosin subfragment one to regulated actin as measured by fluorescence changes of troponin I modified with different fluorophores

Binding studies of myosin subfragment one (S-1) to regulated actin in the presence and absence of Ca2+ indicate that, as S-1 binds to regulated actin, tropomyosin-actin units undergo a cooperative transition from a weak to a strong S-1-binding form. Trybus and Taylor (Trybus, K.M., and Taylor, E.W. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 7209-7213) suggested that this transition could be measured by the change in fluorescence of troponin I modified with 4-(N-iodoacetoxyethyl-N-methyl)-7-nitrobenz-2-oxa-1,3-diazole (IANBD). In the present study, this was tested by determining whether the change in fluorescence was proportional to the fraction of tropomyosin-actin units in the strong S-1-binding form as predicted by our model on the cooperative binding of S-1 to regulated actin (Hill, T.L., Eisenberg, E., and Greene, L.E. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3186-3190). Experiments were performed both in the presence and absence of Ca2+ by using troponin I modified with either IANBD or 5'-iodoacetamidofluorescein. In the presence of Ca2+, it was found, in agreement with the suggestion of Trybus and Taylor, that the change in fluorescence induced by S-1 was proportional to the fraction of tropomyosin-actin units shifting into the strong S-1 binding form, rather than to the fraction of actin sites having bound S-1. In the absence of Ca2+, the change in fluorescence induced by S-1 also did not reflect the binding of S-1 to regulated actin. However, in contrast to the results in the presence of Ca2+, the change in fluorescence induced by S-1 binding in the absence of Ca2+ was not in agreement with the fraction of tropomyosin-actin units calculated to be in the strong S-1 binding form by the model of Hill et al. Although a more complex model than that of Hill et al. may account for the observed fluorescence changes, it seems equally likely that at least in the absence of Ca2+, the change in fluorescence may be reflecting a more complex behavior than only the transition of tropomyosin-actin units between the weak and strong S-1-binding forms.     

The Hill model for binding myosin S1 to regulated actin is not equivalent to the McKillop-Geeves model

The Hill two-state cooperativity model and the McKillop-Geeves (McK-G) three-state model predict very similar binding traces of myosin subfragment 1 (S1) binding to regulated actin filaments in the presence and absence of calcium, and both fit the experimental data reasonably well [Chen et al., Biophys. J., 80, 2338-2349]. Here, we compared the Hill model and the McK-G model for binding myosin S1 to regulated actin against three sets of experimental data: the titration of regulated actin with S1 and the kinetics of S1 binding of regulated actin with either excess S1 to actin or excess actin to S1. Each data set was collected for a wide range of specified calcium concentrations. Both models were able to generate reasonable fits to the time course data and to titration data. The McK-G model can fit all three data sets with the same calcium-concentration-sensitive parameters. Only K(B) and K(T) show significant calcium dependence, and the parameters have a classic pCa curve. A unique set of the Hill model parameters was extremely difficult to estimate from the best fits of multiple sets of data. In summary, the McK-G cooperativity model more uniquely resolves parameters estimated from kinetic and titration data than the Hill model, predicts a sigmoidal dependence of key parameters with calcium concentration, and is simpler and more suitable for practical use.     

The effects of troponin T fragments T1 and T2 on the binding of nonpolymerizable tropomyosin to F-actin in the presence and absence of troponin I and troponin C

Using a nonpolymerizable form of tropomyosin (NPTM) we have investigated the interactions between the T1 (residues 1-158) and T2 (residues 159-259) regions of troponin T and the other components of the thin filament at 50 mM KCl +/- Ca2+. Under these conditions the binding of NPTM to F-actin is fully restored by whole troponin (+/- Ca2+), and in each case, retains a residual degree of cooperativity as demonstrated by Scatchard and Hill plots. Fragment T2 alone had a small inductive effect on the interaction of NPTM with F-actin. In the presence of troponin I, this interaction is increased to a level which exceeds that observed with either component alone. The effects of T2 and troponin I are moderately (-Ca2+) and markedly (+Ca2+) reduced by troponin C. While fragment T1 alone did not promote induction, it accentuated the effects of T2 and troponin I. Since T1 does not interact with T2 or troponin I but does interact weakly with the NH2 terminus of tropomyosin and can be expected to bind weakly at the residual interaction site(s) at the COOH terminus of NPTM, the observed effects of T1 have been ascribed to the linking of neighboring NPTM molecules at their ends.     

Effects of tryptic digestion on myosin subfragment 1 and its actin-activated adenosinetriphosphatase

Myosin subfragment 1 (S-1) was fluorescently labeled at its rapidly reacting thiol ("SH1"). Short exposure to trypsin cuts the S-1 heavy chain into three still-associated fragments (20K, 50K, and 27K) [Balint, M., Wolf, L., Tarcsafalvi, A., Gergely, J., & Sreter, F.A. (1978) Arch. Biochem. Biophys. 190, 793-799] which bind F-actin to the same extent as does the uncut labeled S-1, as indicated by time-resolved fluorescence anisotropy decay (at 4 degrees C, pH 7, in 0.15 M KC1 and 5 mM MgC12, +/- 1 mM ADP). These results are thus in agreement with turbidity measurements on similar systems as reported by Mornet et al. [Mornet, D., Pantel, P., Audemard, E., & Kassab, R. (1979) Biochem. Biophys. Res. Commun. 89, 925-932]. The excited-state lifetime of the fluorescent label on cut S-1 is indistinguishable from that on normal S-1 (+/- ADP, +/- F-actin). F-Actin activation of MgATPase of cut S-1 is lower than that for normal S-1 at moderate concentrations of F-actin, as reported by Mornet et al. (1979). But as the F-actin concentration is increased, the MgATPase activities for cut S-1 approach those for uncut S-1. In terms of an eight-species steady-state kinetics scheme involving actin binding to free S-1, S-1 . ATP, S-1. ADP X P, and S-1 . ADP, actin affinity for the species S-1 . ADP X P was found to be 13.4 times greater for uncut S-1 than for cut S-1 [at 24 degrees C, pH 7.0, in 3 mM KC1, 1 mM ATP, 1 mM MgCl2, and 20 mM N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid].     

Effect of ethylene glycol on the interaction of different myosin subfragment 1.nucleotide complexes with actin

To elucidate the structure of the cross-bridge intermediates in the actomyosin ATPase cycle, several laboratories have added both ethylene glycol and AMP-PNP to muscle fibers. These studies suggested that ethylene glycol shifts the structure of myosin.AMP-PNP toward the weak-binding conformation, i.e., toward the structure of myosin.ATP. Since only the weak-binding conformation of myosin subfragment 1 (S-1) binds with no apparent cooperativity to the troponin-tropomyosin-actin complex (regulated actin), we used this as a probe to examine the conformation of various S-1.nucleotide complexes in ethylene glycol. Our results show that ethylene glycol markedly weakens the binding strength of S-1, S-1.ADP, and S-1.AMP-PNP to actin but has almost no effect on the binding strength of S-1.ATP. As in muscle fibers, at 40% ethylene glycol, the binding strength of S-1.AMP-PNP to actin becomes very similar to the binding strength of S-1.ATP. In the presence of troponin-tropomyosin, the binding of S-1.AMP-PNP to actin shows no apparent cooperativity in 40% ethylene glycol. Therefore, our results confirm that ethylene glycol shifts the structure of the myosin.AMP-PNP toward the weak-binding conformation. However, our results also suggest that ethylene glycol has a direct effect on the regulated actin complex. This is shown by the fact that ethylene glycol markedly increases the cooperative binding of S-1.ADP to regulated actin both in the presence and in the absence of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the binding of L-S- and L-R-diastereoisomers of the substrate analog L-methionine sulfoximine to glutamine synthetase from Escherichia coli

Active site ligand interactions with dodecameric glutamine synthetase from Escherichia coli were studied spectrally, using the resolved L-S- and L-R-diastereoisomers of the substrate analog L-methionine-SR-sulfoximine. direct measurements of the reversible binding of the S-isomer to unadenylylated manganese-enzyme show a stoichiometry of 1 eq/subunit and negative cooperativity with a Hill coefficient of 0.7. The affinity of this enzyme complex is greatest for the S-isomer alone ([S]0.5 = 35 microM), least with the R-isomer alone ([S]0.5 = 0.38 mM), and intermediate (but closer to that for the S-isomer) for an equimolar mixture of S- and R-isomers ([S]0.5 = 61 microM). The affinity for the S-isomer is enhanced greater than 35-fold by ADP and is decreased approximately 3-fold by adenylylation of the enzyme. Shrake, A., Whitley, E. J. Jr., and Ginsburg, A. ((1980) J. Biol. Chem. 255, 581-589) reported that UV spectral perturbations markedly differ for binding commercial L-methionine-SR-sulfoximine to unadenylylated and adenylylated manganese enzymes. However, essentially the same saturating protein difference spectrum is produced by binding the resolved S- and R-diastereoisomers, and equimolar mixture of S- and R-isomers, and the commercial S- and R-isomeric mixture to a particular enzyme complex. Since neither the subunit interactions that give rise to the observed negative cooperativity of binding nor the affinity differences in binding the S- and R-isomers are reflected in protein difference spectra, spectral perturbations derive from a conformational change that is solely a marked for the occupancy of the single subunit site by either isomer.     

Fluorescence characterization of structural transitions at the strong actin binding motif in skeletal myosin affinity labeled at cysteine 540 with novel spectroscopic cysteaminyl mixed disulfides

We have synthesized the luminescent and fluorescent lanthanide chelate S-(2-nitro-5-thiobenzoic acid)cysteaminyldiethylenetriaminepentaacetate-5-[(2-aminoethyl)am ino ]naphthalene-1-sulfonic acid as well as the fluorescent analogue S-(2-nitro-5-thiobenzoic acid)cysteaminyl-5-carboxyfluorescein using the procedure we recently described [Bertrand, R., Capony, J.-P., Derancourt, J., and Kassab, R. (1999) Biochemistry 38, 11914-11925]. Both mixed disulfides react with the skeletal myosin motor domain (S-1) as actin site-directed agents and label exclusively and stoichiometrically Cys 540 in the hydrophobic strong actin binding helix-loop-helix motif, causing only a 1.9-2.4-fold decrease in the V(max) for acto-S-1 ATPase. The covalently attached cysteaminyl probe side chain spans maximally 17 and 8 A, respectively, and the fluorophores have different polarity, volume, and flexibility. Thus, they may provide complementary spectroscopic information on the environmental properties of this critical actin binding region. Here, we have analyzed by extrinsic fluorescence spectroscopy S-1 derivatized with the fluorescein label or with the Tb(3+) or Eu(3+) chelate of the other label to assess the conformational transitions precisely occurring at this site upon interaction with F-actin, nucleotides, or phosphate analogues. For either label, specific spectral changes of significant amplitude were obtained, identifying at least two major structural states. One was mediated by rigor binding of F-actin in the absence or presence of MgADP. It was abolished by MgATP, and it was not produced by the binding of nonpolymerizable G-actin. A modeling of the corresponding changes in the intensity and lambda(max) of the fluorescence emission spectra, achieved using the fluorescent adducts of 2-mercaptoethanol in varying concentrations of dimethylformamide, illustrates the predicted apolar nature of the strong acto-S-1 interface. A second state was promoted by the binding of ATP, AMP-PNP, ADP.AlF4, ADP. BeFx, or PP(i). It should be prevalent in the weak acto-S-1 binding complexes. The accompanying fluorescence intensity reduction, observed with each label, in both the absence and presence of F-actin, would result from a specific modification by these ligands of the probe orientation and/or solvent accessibility as suggested by acrylamide quenching experiments. It could represent the spectral manifestation of the predicted allosteric linkage from the ATPase site to the strong actin binding site of S-1 that modulates the acto-S-1 affinity. Our study offers the basis necessary for further detailed spectroscopic investigations on the conformational dynamics in solution of the stereospecific and hydrophobic actin binding motif during the skeletal cross-bridge cycle.     

Activation of actin-cardiac myosin subfragment 1 MgATPase rate by Ca2+ shows cooperativity intrinsic to the thin filament

The magnesium adenosinetriphosphatase (MgATPase) rate of cardiac myosin subfragment 1 (S-1) was studied in the presence of regulated actin in order to investigate the mechanism by which Ca2+ cooperatively induces cardiac muscle contraction. The MgATPase rate increased cooperatively with Ca2+, exhibiting a Hill coefficient of 1.8 and 50% activation at pCa 5.75. This cooperative response occurred despite an experimental design excluding several potential sources of cooperativity. First, to exclude spurious cooperativity due to erroneous calculation of pCa at low ionic strength, the affinities of Ca2+ and Mg2+ for [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) were measured by a novel method using Quin 2. At pH 7.06, 25 degrees C, and mu = 30 mM, the KD was 140 nM for CaEGTA and 2.7 mM for MgEGTA. Second, the cooperativity was not produced by actin-myosin S-1 binding; myosin S-1 was bound to only 1 of every 300 actin promoters, and earlier work [Tobacman, L. S., & Adelstein, R. S. (1986) Biochemistry 25, 798-802] had shown that cardiac myosin S-1 binds with equal affinity to the thin filament at very low Ca2+ and at saturating Ca2+ concentrations. Furthermore, the adenosine 5'-triphosphate turnover rate of the myosin S-1 was independent of enzyme concentration at low, intermediate, and saturating Ca2+ concentrations. Finally, since cardiac troponin has only one regulatory Ca2+-specific site, cooperative interactions between such sites could not occur. These data suggest that part of the cooperativity conferred by interaction between adjacent troponin-tropomyosin complexes is intrinsic to the thin filament and independent of myosin.     

Effects of the amino-terminal regions of tropomyosin and troponin T on thin filament assembly.

Bacterially expressed alpha-tropomyosin lacks the amino-terminal acetylation present in muscle tropomyosin and binds poorly to actin (Hitchcock-DeGregori, S. E., and Heald, R. W. (1987) J. Biol. Chem. 262, 9730-9735). Using a linear lattice model, we determined the affinity (Ko) of unacetylated tropomyosin or troponin-unacetylated tropomyosin for an isolated site on the actin filament and the fold increase in affinity (y) when binding is to an adjacent site. The absence of tropomyosin acetylation decreased Ko 2 orders of magnitude in the absence of troponin. Tropomyosin acetylation also enhanced troponin-tropomyosin binding to actin, not by increasing cooperativity (y), but rather by increasing Ko. These results suggest that the amino-terminal region of tropomyosin is a crucial actin binding site. Troponin promoted unacetylated tropomyosin binding to actin, increasing Ko more than 1,000-fold. Troponin70-259, which lacks the troponin T peptide (1-69) spanning the overlap between adjacent tropomyosins, behaved similarly to intact troponin. Cooperative interactions between adjacent troponin-tropomyosin complexes remained strong despite the use of a nonpolymerizable tropomyosin and a troponin unable to bridge neighboring tropomyosins physically. The Ko for troponin70-259-unacetylated tropomyosin was 500-fold greater than for troponin159-259-unacetylated tropomyosin, indicating that troponin T residues 70-158 are critical for anchoring troponin-tropomyosin to F-actin. The mechanism of cooperative thin filament assembly is discussed.     

Binding of ADP and 5'-adenylyl imidodiphosphate to rabbit muscle myofibrils

The binding of [3H]ADP and [3H]adenyl-5'-yl-imidodiphosphate ([3H]AMP-PNP) to rabbit skeletal myofibrils was measured at 25 and 7 degrees C, mu = 0.12 M, using [14C]mannitol as a volume marker. We found that ADP bound to myosin heads in overlap with a binding constant of about 10(4) M-1, similar to the value we previously obtained in vitro with acto.S-1. The binding of AMP-PNP to myosin heads was measured both in and out of overlap. The affinity of AMP-PNP to the heads out of overlap was similar to that obtained in vitro with S-1 alone. The binding of AMP-PNP to the myosin heads in overlap was much weaker. We could fit these data with a binding constant of about 1 x 10(3) M-1, assuming a single population of cross-bridges and 1 mol of AMP-PNP bound per mol of myosin head. This value was reduced by a factor of 2 when we corrected for nonspecific binding. It was also possible to fit the data assuming two equal populations of cross-bridges with one of the populations binding AMP-PNP about 5-fold more strongly than the other population. Therefore, for at least half of the cross-bridges in overlap, the binding of AMP-PNP is almost as weak as the value of 3 x 10(2) M-1 we previously measured for the acto.S-1 complex in vitro (Biosca, J. A., Greene, L. E., and Eisenberg, E. (1986) J. Biol. Chem. 261, 9793-9800).     

Hill coefficient for estimating the magnitude of cooperativity in gating transitions of voltage-dependent ion channels

A frequently used measure for the extent of cooperativity in ligand binding by an allosteric protein is the Hill coefficient, obtained by fitting data of initial reaction velocity (or fractional binding saturation) as a function of substrate concentration to the Hill equation. Here, it is demonstrated that the simple two-state Boltzmann equation that is widely used to fit voltage-activation data of voltage-dependent ion channels is analogous to the Hill equation. A general empiric definition for a Hill coefficient (n(H)) for channel gating transitions that is analogous to the logarithmic potential sensitivity function of Almers is derived. This definition provides a novel framework for interpreting the meaning of the Hill coefficient. In considering three particular and simple gating schemes for a voltage-activated cation channel, the relation of the Hill coefficient to the magnitude and nature of cooperative interactions along the reaction coordinate of channel gating is demonstrated. A possible functional explanation for the low value of the Hill coefficient for gating transitions of the Shaker voltage-activated K(+) channel is suggested. The analogy between the Hill coefficients for ligand binding and for channel gating transitions further points to a unified conceptual framework in analyzing enzymes and channels behavior.     

Molecular cooperativity of drebrin(1-300) binding and structural remodeling of f-actin

Drebrin A, an actin-binding protein, is a key regulatory element in synaptic plasticity of neuronal dendrites. Understanding how drebrin binds and remodels F-actin is important for a functional analysis of their interactions. Conventionally, molecular models for protein-protein interactions use binding parameters derived from bulk solution measurements with limited spatial resolution, and the inherent assumption of homogeneous binding sites. In the case of actin filaments, their structural and dynamic states—as well as local changes in those states—may influence their binding parameters and interaction cooperativity. Here, we probed the structural remodeling of single actin filaments and the binding cooperativity of DrebrinA_1-300 –F–actin using AFM imaging. We show direct evidence of DrebrinA_1-300-induced cooperative changes in the helical structure of F-actin and observe the binding cooperativity of drebrin to F-actin with nanometer resolution. The data confirm at the in vitro molecular level that variations in the F-actin helical structure can be modulated by cooperative binding of actin-binding proteins.     

Comparison of the effects of smooth and skeletal tropomyosin on skeletal actomyosin subfragment 1 ATPase

Chicken gizzard tropomyosin, like rabbit skeletal tropomyosin, inhibits and activates skeletal actomyosin subfragment 1 ATPase at low and high [subfragment 1], respectively, showing that both smooth and skeletal tropomyosin qualitatively produce similar cooperative effects on activity. For gizzard tropomyosin, however, the extent of the inhibition was less, and the activation curve rose more sharply at lower [subfragment 1]. In terms of a two-state cooperative activity model for the actin-tropomyosin filament (Hill, T. L., Eisenberg, E., and Chalovich, J. (1981) Biophys. J. 35, 99-112), these results qualitatively suggest that, for the gizzard tropomyosin system, more units are initially in the active state (in the absence of subfragment 1) and that the switching of units to the active state is more cooperative. The greater cooperativity indicated for the gizzard system may be a consequence of the greater rigidity of gizzard tropomyosin indicated from conformational studies.     

Effect of F-actin upon the binding of ADP to myosin and its fragments.

The effect of F-actin upon the binding of ADP to rabbit skeletal muscle myosin, heavy meromyosin, and subfragment 1 was studied by equilibrium dialysis, ultracentrifuge transport, and light scattering techniques. Both myosin and H-meromyosin (HMM) bind a maximum of approximately 1.6 mol of ADP/mol of protein, while S-1 binds approximately 0.9 mol of ADP/mol of protein. The affinity for ADP of all three preparations was similar at a given ionic strength (approximately 10(6) M-1 at 0.05 M KCl) and decreased with increasing ionic strength. Under conditions similar to those used for the measurement of ADP binding, the binding sites of myosin, HMM, and subfragment 1 (S-1) are saturated with actin at molar ratios of 2, 2, and 1 mol of actin monomer/mol of protein, respectively, as determined by light scattering, ultracentrifuge transport, and in the case of myosin by ATPase measurements. F-actin was found to inhibit ADP binding, but even at an actin concentration at least twice that required for saturation of myosin, HMM, or S-1, significant ADP binding remained. This ADP binding was inhibited by 10(-4) M pyrophosphate. The observations are consistent with the formation of an actomyosin-ADP complex in which actin and ADP are bound to myosin at distinct but interacting sites.     

Dissociation of the actin.subfragment 1 complex by adenyl-5'-yl imidodiphosphate, ADP, and PPi

The ability of adenyl-5'-yl imidodiphosphate (AMP-PNP), ADP, and PPi to dissociate the actin.myosin subfragment 1 (S-1) complex was studied using an analytical ultracentrifuge with UV optics, which enabled the direct determination of the dissociated S-1. At mu = 0.22 M, pH 7.0, 22 degrees C, with saturating nucleotide present, ADP weakens the binding of S-1 to actin about 40-fold (K congruent to 10(5) M-1), while both AMP-PNP and PPi weakens the binding about 400-fold (K congruent to 10(4) M-1). This 10-fold stronger dissociating effect of AMP-PNP and PPi compared to ADP correlates with our data showing that the binding of AMP-PNP and PPi to S-1 is about 10-fold stronger than the binding of ADP. In contrast, the binding constants of ADP, AMP-PNP, and PPi to acto.S-1 are nearly identical (K congruent to 5 x 10(3) M-1). At 4 degrees C, AMP-PNP has only a 3-fold stronger dissociating effect than ADP and, similarly, our data suggest that the binding of AMP-PNP and ADP to S-1 is quite similar at 4 degrees C. AMP-PNP and PPi are, therefore, somewhat better dissociating agents than ADP, but the difference among these three ligands is quite small. These data also show that actin and nucleotide bind to separate but interacting sites on S-1 and that the S-1 molecules bind independently along the F-actin filament with a binding constant of about 1 x 10(7) M-1 at 22 degrees C and physiological ionic strength.     

Fluorescence probe studies of the state of tropomyosin in reconstituted muscle thin filaments

The monomer fluorescence of N-(1-pyrenyl)maleimide-labeled tropomyosin bound to F-actin (PTm-actin) increases when myosin subfragment 1 (S1) binds to actin and is half complete when only approximately 1 S1 is bound to 7 actin subunits [Ishii, Y., & Lehrer, S. S. (1985) Biochemistry 24, 6631-6638]. Similar studies of the binding of S1 and S1-ADP to fully reconstituted thin filaments [PTm-actin-troponin (Tn)] are now reported. The pyrene monomer fluorescence change was half complete when approximately 0.5 S1/7 actin subunits and approximately 1.5 S1/7 actin subunits were bound in the presence and absence of Ca2+, respectively. In the presence of Mg2+-ADP, when S1 binding is weakened, the S1 binding profiles and fluorescence changes were sigmoidal, with the cooperative transitions occurring at lower [S1] in the presence of Ca2+ as first shown by Greene and Eisenberg for S1 binding [Greene, L., & Eisenberg, E. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2616-2620]. It was possible to fit both the binding and fluorescence data with the same parameters of a two-state (weak and strong S1 binding) cooperative binding model [Hill, T., Eisenberg, E., & Greene, L. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3186-3190] for each Ca2+ situation if the fluorescence change is interpreted as the fraction of tropomyosin (Tm) units in the strong S1 binding state. These data indicate that the fluorescence change is a direct measure of the S1-induced change of state of Tm in the fully reconstituted thin filament.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between regulated actomyosin ATPase activity and cooperative binding of myosin to regulated actin

The protein complex, troponin-tropomyosin, which is bound to the thin actin filament, regulates muscle contraction and relaxation. In the absence of Ca²⁺ the troponin-tropomyosin complex causes muscle to relax, whereas in the presence of Ca²⁺, contraction occurs. Biochemical studies have shown that the troponin-tropomyosin complex has a dual effect on the interaction of the myosin cross-bridge with actin. In the presence of ATP, troponin-tropomyosin strongly inhibits the actomyosin ATPase activity, whereas in the absence of ATP, troponin-tropomyosin confers positive cooperativity on the binding of myosin to actin. We have proposed a simple model [Hill, T. L., Greene, L. E., and Eisenberg, E. (1980)Proc. Natl. Acad. Sci. USA 77, 3186–3190] that accounts for these biochemical observations by postulating that the troponin-tropomyosin-actin complex (regulated actin) can occur in two forms, a turned-on form and a turned-off form. This model defines several cooperativity parameters that describe the behavior of regulated actin. In previous studies we have determined the values of these parameters by studying the cooperative binding of myosin to regulated actin in the absence of ATP. In the present study we also used ATPase and fluorescence measurements to determine these cooperativity parameters. Assuming that the fluorescence change occurs only when two adjacent tropomyosin units shift into the turned-on form, our results show that all three methods give the same values for the cooperativity parameters. These results confirm the prediction of our model that a regulated actin unit that is turned off not only binds S-1 weakly but is also unable to activate the actomyosin ATPase activity.     

The binding of [3H]R-PIA to A1 adenosine receptors produces a conversion of the high- to the low-affinity state

Kinetic evidence for negative cooperativity on the binding of [3H]R-PIA to A1 adenosine receptors was obtained from dissociation experiments at different ligand concentrations and from the equilibrium isotherm. The dissociation curves indicate that there is an apparent ligand-induced transformation of high- to low-affinity states of the receptor. At concentrations of 18.2 nM R-PIA or higher there was only found the low-affinity state of the receptor. In view of these results equilibrium binding data were analyzed by the usual two-state model (assuming that there is an interconversion between them) and by the negative cooperativity model employing the Hill equation.     

Structural insight into the cooperativity between catalytic and noncatalytic sites of F1-ATPase

F1-ATPase, the catalytic sector of Fo-F1 ATPases-ATPsynthases, displays an apparent negative cooperativity for ATP hydrolysis at high ATP concentrations which involves noncatalytic and catalytic nucleotide binding sites. The molecular mechanism of such cooperativity is currently unknown. To get further insights, we have investigated the structural consequences of the single mutation of two residues: Q173L in the alpha-subunit and Q170Y in the beta-subunit of the F1-ATPase of the yeast Schizosaccharomyces pombe. These residues are localized in or near the Walker-A motifs of each subunit and their mutation produces an opposite effect on the negative cooperativity. The betaQ170 residue (M167 in beef heart) is located close to the binding site for the phosphate-Mg moiety of the nucleotide. Its replacement by tyrosine converts this site into a close state with increased affinity for the bound nucleotide and leads to an increase of negative cooperativity. In contrast, the alphaQ173L mutation (Q172 in beef heart) abolishes negative cooperativity due to the loss of two H-bonds: one stabilizing the nucleotide bound to the noncatalytic site and the other linking alphaQ173 to the adjacent betaT354, localized at the alpha(DP)-beta(TP) interface. The properties of these mutants suggest that negative cooperativity occurs through interactions between neighbor alpha- and beta-subunits. Indeed, in the beef heart enzyme, (i) the alpha(DP)-beta(TP) interface is stabilized by a vicinal alphaR171-betaD352 salt bridge (ii) betaD352 and betaT354 belong to a short peptidic stretch close to betaY345, the aromatic group of which interacts with the adenine moiety of the nucleotide bound to the catalytic site. We therefore propose that the betaY345-betaT354 stretch (beef heart numbering) constitutes a short link that drives structural modifications from a noncatalytic site to the neighbor catalytic site in which, as a result, the affinity for ADP is modulated.