The Tail Domain of Myosin Va Modulates Actin Binding to One Head

Calcium activates full-length myosin Va steady-state enzymatic activity and favors the transition from a compact, folded "off" state to an extended "on" state. However, little is known of how a head-tail interaction alters the individual actin and nucleotide binding rate and equilibrium constants of the ATPase cycle. We measured the effect of calcium on nucleotide and actin filament binding to full-length myosin Va purified from chick brains. Both heads of nucleotide-free myosin Va bind actin strongly, independent of calcium. In the absence of calcium, bound ADP weakens the affinity of one head for actin filaments at equilibrium and upon initial encounter. The addition of calcium allows both heads of myosin Va·ADP to bind actin strongly. Calcium accelerates ADP binding to actomyosin independent of the tail but minimally affects ATP binding. Although 18O exchange and product release measurements favor a mechanism in which actin-activated Pi release from myosin Va is very rapid, independent of calcium and the tail domain, both heads do not bind actin strongly during steady-state cycling, as assayed by pyrene actin fluorescence. In the absence of calcium, inclusion of ADP favors formation of a long lived myosin Va·ADP state that releases ADP slowly, even after mixing with actin. Our results suggest that calcium activates myosin Va by allowing both heads to interact with actin and exchange bound nucleotide and indicate that regulation of actin binding by the tail is a nucleotide-dependent process favored by linked conformational changes of the motor domain. Calcium activates full-length myosin Va steady-state enzymatic activity and favors the transition from a compact, folded "off" state to an extended "on" state. However, little is known of how a head-tail interaction alters the individual actin and nucleotide binding rate and equilibrium constants of the ATPase cycle. We measured the effect of calcium on nucleotide and actin filament binding to full-length myosin Va purified from chick brains. Both heads of nucleotide-free myosin Va bind actin strongly, independent of calcium. In the absence of calcium, bound ADP weakens the affinity of one head for actin filaments at equilibrium and upon initial encounter. The addition of calcium allows both heads of myosin Va·ADP to bind actin strongly. Calcium accelerates ADP binding to actomyosin independent of the tail but minimally affects ATP binding. Although 18O exchange and product release measurements favor a mechanism in which actin-activated Pi release from myosin Va is very rapid, independent of calcium and the tail domain, both heads do not bind actin strongly during steady-state cycling, as assayed by pyrene actin fluorescence. In the absence of calcium, inclusion of ADP favors formation of a long lived myosin Va·ADP state that releases ADP slowly, even after mixing with actin. Our results suggest that calcium activates myosin Va by allowing both heads to interact with actin and exchange bound nucleotide and indicate that regulation of actin binding by the tail is a nucleotide-dependent process favored by linked conformational changes of the motor domain. Class V myosins participate in a diverse array of cellular functions ranging from organelle transport, mRNA localization and transport, and apoptotic signaling pathways (1Reck-Peterson S.L. Provance Jr., D.W. Mooseker M.S. Mercer J.A. Biochim. Biophys. Acta. 2000; 1496: 36-51Crossref PubMed Scopus (243) Google Scholar, 2Krendel M. Mooseker M.S. Physiology (Bethesda). 2005; 20: 239-251Crossref PubMed Scopus (283) Google Scholar). Vertebrate myosin Va, one of three myosins V expressed in vertebrates, is the best characterized of the Class V myosins. Individual double-headed vertebrate myosin Va molecules are processive and take multiple steps along an actin filament before dissociating (3Mehta A.D. Rock R.S. Rief M. Spudich J.A. Mooseker M.S. Cheney R.E. Nature. 1999; 400: 590-593Crossref PubMed Scopus (675) Google Scholar), although not all Class V myosins exhibit processive movement (4Reck-Peterson S.L. Tyska M.J. Novick P.J. Mooseker M.S. J. Cell Biol. 2001; 153: 1121-1126Crossref PubMed Scopus (107) Google Scholar). Calcium enhances the steady-state actin-activated ATPase activity but diminishes the in vitro motility of full-length myosin Va (5Cheney R.E. O'Shea M.K. Heuser J.E. Coelho M.V. Wolenski J.S. Espreafico E.M. Forscher P. Larson R.E. Mooseker M.S. Cell. 1993; 75: 13-23Abstract Full Text PDF PubMed Scopus (380) Google Scholar). This regulation is partially due to calcium binding the calmodulin light chains (6Cameron L.C. Carvalho R.N. Araujo J.R. Santos A.C. Tauhata S.B. Larson R.E. Sorenson M.M. Arch. Biochem. Biophys. 1998; 355: 35-42Crossref PubMed Scopus (12) Google Scholar, 7Wang F. Thirumurugan K. Stafford W.F. Hammer III, J.A. Knight P.J. Sellers J.R. J. Biol. Chem. 2004; 279: 2333-2336Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 8Krementsov D.N. Krementsova E.B. Trybus K.M. J. Cell Biol. 2004; 164: 877-886Crossref PubMed Scopus (175) Google Scholar, 9Li X.D. Mabuchi K. Ikebe R. Ikebe M. Biochem. Biophys. Res. Commun. 2004; 315: 538-545Crossref PubMed Scopus (86) Google Scholar). A recombinant myosin Va dimer missing the tail is fully activated in the absence of calcium, whereas full-length myosin is not, indicating that additional levels of calcium-dependent regulation are mediated by the C-terminal cargo-binding "tail" domain. Cameron et al. (6Cameron L.C. Carvalho R.N. Araujo J.R. Santos A.C. Tauhata S.B. Larson R.E. Sorenson M.M. Arch. Biochem. Biophys. 1998; 355: 35-42Crossref PubMed Scopus (12) Google Scholar) described a calcium-dependent change in intrinsic fluorescence of full-length myosin Va, interpreted to reflect a conformational change in the heavy chain. This prediction has since been confirmed by electron microscopy and analytical ultracentrifugation studies that identified a large conformational change of full-length myosin Va from a compact conformation in the absence of calcium to an extended conformation in the presence of calcium (7Wang F. Thirumurugan K. Stafford W.F. Hammer III, J.A. Knight P.J. Sellers J.R. J. Biol. Chem. 2004; 279: 2333-2336Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 8Krementsov D.N. Krementsova E.B. Trybus K.M. J. Cell Biol. 2004; 164: 877-886Crossref PubMed Scopus (175) Google Scholar, 9Li X.D. Mabuchi K. Ikebe R. Ikebe M. Biochem. Biophys. Res. Commun. 2004; 315: 538-545Crossref PubMed Scopus (86) Google Scholar). The compact conformation, believed to represent an enzymatic "off" state, appears to arise from an interaction of the tail with the N-terminal head or light chain-binding "neck" (10Thirumurugan K. Sakamoto T. Hammer III, J.A. Sellers J.R. Knight P.J. Nature. 2006; 442: 212-215Crossref PubMed Scopus (140) Google Scholar, 11Liu J. Taylor D.W. Krementsova E.B. Trybus K.M. Taylor K.A. Nature. 2006; 442: 208-211Crossref PubMed Scopus (172) Google Scholar). Despite increasing evidence that the tail domain regulates myosin Va motor function, it remains unclear which ATPase cycle transitions are affected in the compact conformation and how calcium activates motor function. Kinetic parameters of key ATPase cycle events have been measured for individual full-length, tissue-purified myosin Va using single-molecule methods (3Mehta A.D. Rock R.S. Rief M. Spudich J.A. Mooseker M.S. Cheney R.E. Nature. 1999; 400: 590-593Crossref PubMed Scopus (675) Google Scholar, 12Rief M. Rock R.S. Mehta A.D. Mooseker M.S. Cheney R.E. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9482-9486Crossref PubMed Scopus (359) Google Scholar, 13Veigel C. Wang F. Bartoo M.L. Sellers J.R. Molloy J.E. Nat. Cell Biol. 2002; 4: 59-65Crossref PubMed Scopus (333) Google Scholar). However, these experiments immobilized myosin Va to a surface or a bead by adsorption through the tail and therefore could not explicitly account for tail regulation. Consequently, little is known about how the tail domain regulates the kinetics of myosin Va. In this study, we have measured actin and nucleotide binding to tissue-purified chick brain myosin Va and have tested the effect of calcium on these parameters. Our results favor a mechanism in which the tail domain of myosin Va interacts with and weakens actin binding to one of the two heads in the absence of calcium and indicate that this interaction is favored by bound ADP. The data suggest that a long lived myosin·ADP state in the absence of actin contributes to the low ATPase activity in the folded "off" state. Reagents—All chemicals and reagents were the highest purity commercially available. ATP was purchased from Roche Applied Science, and ADP was purchased from Sigma. Mant 3The abbreviations used are: mant, 2(3)-O-N-methylanthraniloyl; mantATP, 2(3)-O-N-methylanthraniloyl-ATP; mantADP, 2(3)-O-N-methylanthraniloyl-ADP. -labeled nucleotides were prepared as described (14Hannemann D.E. Cao W. Olivares A.O. Robblee J.P. De La Cruz E.M. Biochemistry. 2005; 44: 8826-8840Crossref PubMed Scopus (71) Google Scholar). Nucleotide concentrations were determined by absorbance (15Robblee J.P. Olivares A.O. De La Cruz E.M. J. Biol. Chem. 2004; 279: 38608-38617Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). A molar equivalent of MgCl2 was added to nucleotides immediately before use. Protein Expression and Purification—Full-length chick brain myosin Va with bound essential light chains and calmodulin was purified from brains of newly hatched chicks (16Cheney R.E. Methods Enzymol. 1998; 298: 3-18Crossref PubMed Scopus (49) Google Scholar). Recombinant double-headed chicken myosin Va missing the C-terminal globular tail (tailless myosin V-HMM) was truncated to include 20 native heptad repeats of predicted coiled-coil, followed by a GCN4 leucine zipper to ensure dimerization and a FLAG tag (17Rock R.S. Rice S.E. Wells A.L. Purcell T.J. Spudich J.A. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13655-13659Crossref PubMed Scopus (320) Google Scholar). Tailless myosin V-HMM with bound essential light chain and calmodulin was purified from Sf9 cells by FLAG affinity chromatography. Purity was >98% for all preparations. Actin was purified from rabbit skeletal muscle, labeled with pyrene, and gel-filtered over Sephacryl S-300HR (18De La Cruz E.M. J. Mol. Biol. 2005; 346: 557-564Crossref PubMed Scopus (122) Google Scholar). Ca2+-actin monomers were converted to Mg2+-actin monomers with 0.2 mm EGTA and 50 μm MgCl2 (excess over actin) immediately prior to polymerization by dialysis against KMg50 buffer (50 mm KCl, 2 mm MgCl2, 0.1 mm EGTA, 2 mm dithiothreitol, and 10 mm imidazole, pH 7.0). Phalloidin (1.1 mol eq) was used to stabilize actin filaments. Equilibrium Titrations—Myosin Va binding to actin filaments was measured from the myosin dependence of pyrene actin fluorescence quenching. Native chick brain myosin Va (with or without 1 mm ADP) containing 3 μm exogenous calmodulin was equilibrated with 60 or 100 nm pyrene actin filaments at 25 ± 1 °C for 40–60 min. Apyrase (0.5 units ml–1) was included to achieve rigor (no nucleotide) conditions where indicated. In samples containing calcium, the free Ca2+ was calculated using the program WinMaxC32 (Chris Patton, Stanford University) (available on the World Wide Web at www.stanford.edu/~cpatton). Steady-state fluorescence intensities were measured at 25 ± 1 °C using a thermostatted Photon Technologies Intl. (New Brunswick, NJ) Alphascan fluorescence spectrometer. Binding stoichiometries were obtained by fitting the fluorescence intensities at 405 nm (λex = 365 nm) to Equation 1,F(r)=Fo+(F∞-Fo)×((r+KdAtot+n)-(r+KdAtot+n)2-4·r·n2·n)(Eq. 1) where F(r) represents the fluorescence intensity (F) as a function of the myosin heads/actin ratio (r), Fo is the fluorescence in the absence of myosin, F∞ is the fluorescence intensity at infinitely high r (i.e. saturating myosin heads/actin), Kd is the apparent dissociation equilibrium constant of actomyosin, Atot is the total actin concentration, and n is the stoichiometry of myosin Va head binding to actin subunits in a filament (15Robblee J.P. Olivares A.O. De La Cruz E.M. J. Biol. Chem. 2004; 279: 38608-38617Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The stoichiometry (n), initial (Fo) and final (F∞) fluorescence were allowed to float when fitting. Values of Kd were constrained to the values calculated from the ratio of the experimentally determined association and dissociation rate constants, except where indicated. Kinetic Measurements and Analysis—All kinetic experiments were performed at 25 ± 0.1 °C in KMg50 buffer with an Applied Photophysics (Surrey, UK) SX.18MV-R stopped flow apparatus. Concentrations stated are final after mixing. Pyrene (λex = 365 nm) and mant nucleotide fluorescence (λex = 297 nm) were monitored at 90° from the incident light source through a 400-nm long pass glass filter. Long time courses were corrected for contributions from photobleaching. Most time courses shown are of individual, unaveraged, 1000-point transients collected with the instrument in "oversampling" mode, where the intrinsic time constant for data acquisition is ∼30 μs. Typically, multiple (two or three) time courses were averaged before analysis. Time courses of fluorescence change were fitted to a sum of exponentials using software provided with the instrument. Fitting was limited to data beyond 3 ms to account for the instrument dead time and to exclude data acquired during the continuous flow phase of mixing. Uncertainties are reported as S.E. of the best fits. Time courses of myosin Va and myosin Va·ADP binding to pyrene actin filaments were measured under pseudo-first order conditions with actin ≫ myosin heads (15Robblee J.P. Olivares A.O. De La Cruz E.M. J. Biol. Chem. 2004; 279: 38608-38617Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Myosin Va dissociation from pyrene actin filaments was measured by competition with a large molar excess of unlabeled actin (15Robblee J.P. Olivares A.O. De La Cruz E.M. J. Biol. Chem. 2004; 279: 38608-38617Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Time courses of nucleotide (ATP, ADP, mant-ATP, or mant-ADP) binding were acquired under pseudo-first order conditions with nucleotide ≫ myosin or actomyosin. Actomyosin samples were prepared at a binding density of 0.1–0.2 myosin heads/actin. ADP binding to actomyosin was measured by kinetic competition with ATP (15Robblee J.P. Olivares A.O. De La Cruz E.M. J. Biol. Chem. 2004; 279: 38608-38617Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 19Robblee J.P. Cao W. Henn A. Hannemann D.E. De La Cruz E.M. Biochemistry. 2005; 44: 10238-10249Crossref PubMed Scopus (46) Google Scholar). The ADP dependence of the observed fast phase rate constant was fitted to Equation 2,kfast=K1T'k+2T'ATP+K1D'k+2D'ADP1+K1T'ATP+K1D'ADP(Eq. 2) where K1T′ and K1D′ are the equilibrium constants for actomyosin·ATP and actomyosin·ADP collision complex formation, respectively, K+2T′ is the isomerization rate constant defining the strong (low pyrene fluorescence) to weak (high pyrene fluorescence) actin binding transition, and k+2D′ is the isomerization rate constant defining the weak to strong ADP binding transition, as defined by the following two-step nucleotide (N) binding mechanism.AM+N⇄k1NAM(N)⇄k+2N'k-2N'AM·N(Eq. 3) Measurement of Oxygen Isotopic Exchange during ATP Hydrolysis—Hydrolysis of ATP was performed in KMg50 buffer containing 50–55% 18Owater supplemented with 2 mm MgATP, 2–4 mm phosphoenolpyruvate (PEP), and 100 units/ml pyruvate kinase to regenerate ATP and prevent accumulation of ADP (20De La Cruz E.M. Sweeney H.L. Ostap E.M. Biophys. J. 2000; 79: 1524-1529Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). The reactions were quenched with acid, and the Pi was isolated and analyzed for 18O content (21Hackney D.D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 18338-18343Crossref PubMed Scopus (81) Google Scholar). Equilibrium Titration of Pyrene Actin and Myosin Va—Myosin Va binding quenches the fluorescence of pyrene actin filaments in the presence and absence of calcium (22Tauhata S.B. dos Santos D.V. Taylor E.W. Mooseker M.S. Larson R.E. J. Biol. Chem. 2001; 276: 39812-39818Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), allowing for "strong" actin binding, using the definition of Geeves (23Geeves M.A. Goody R.S. Gutfreund H. J. Muscle Res. Cell Motil. 1984; 5: 351-361Crossref PubMed Scopus (116) Google Scholar), to be monitored from changes in fluorescence. At equilibrium, both heads of tissue-purified myosin Va (no bound nucleotide) bind actin strongly (Fig. 1A) with Kd values of ≤1 nm. With bound ADP (≥1 mm MgADP), both heads bind actin strongly as indicated by the levels of pyrene fluorescence quenching (Fig. 1B). However, the titrations are best described by two approximately equal phases with different apparent actin affinities: one that binds strongly (Kdapp 100 The two apparent affinities observed in the equilibrium binding isotherms could arise if one head of myosin Va bound actin strongly (Kd 100 nm) can be eliminated, since this would not yield a biphasic titration. We therefore favor a mechanism in which the two heads of myosin Va·ADP bind actin with different affinities in the absence of calcium; one is strongly bound (quenched pyrene), and the other is weakly bound or detached (unquenched pyrene). The ADP-dependent head asymmetry occurs only in the absence of calcium. When calcium is present, both heads of myosin Va bind actin strongly in rigor and in the presence of ADP (Fig. 1A; 1/KAapp=1 nm in rigor and 1/KADapp<1 nm with ADP). Both heads of recombinant myosin V-HMM missing the C-terminal cargo-binding tail bind actin strongly (Fig. 1, A and B, insets) in the presence and absence of ADP and in the absence of calcium (1/KAapp=1 nm in rigor and 1/KADapp<1 nm with ADP), indicating that the effect of ADP on two-headed actin binding is unique to full-length myosin Va and is presumably mediated through the tail domain. Kinetics of Myosin Va Binding to Actin Filaments—Time courses of fluorescence quenching after mixing pyrene actin filaments with myosin Va or myosin Va·ADP follow double exponentials both in the presence and absence of calcium (Fig. 2, A and B) with fast phase observed rate constants that depend linearly on actin (Fig. 2, C and D), as described for myosin (14Hannemann D.E. Cao W. Olivares A.O. Robblee J.P. De La Cruz E.M. Biochemistry. 2005; 44: 8826-8840Crossref PubMed Scopus (71) Google Scholar) and recombinant myosin V-HMM Sweeney H.L. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). The phases of the total in the absence of calcium and were in the presence of calcium (Fig. 2, A and In this we kinetic only on the fast the data a mechanism (14Hannemann D.E. Cao W. Olivares A.O. Robblee J.P. De La Cruz E.M. Biochemistry. 2005; 44: 8826-8840Crossref PubMed Scopus (71) Google Scholar) or a mixed population of myosin or conformational The Ca2+ dependence of the favors a calcium-dependent equilibrium as reported for myosin (24Nyitrai M. Szent-Gyorgyi A.G. Geeves M.A. Biochem. J. 2003; 370: 839-848Crossref PubMed Scopus (14) Google Scholar, M. Szent-Gyorgyi A.G. Geeves M.A. Biochem. J. 2002; PubMed Scopus Google Scholar). The apparent association rate constants for actin filament binding were obtained from the of the by the observed pseudo-first order rate constant actin (Fig. 2, C and rigor binding was in the absence of calcium and as fast when calcium was ADP the association rate constant about from to in the absence of calcium 1 and ADP has a effect on the association kinetics when calcium is 1 and Tailless myosin V with bound ADP binds actin filaments with an association rate constant of in the absence of calcium Sweeney H.L. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar), that the tail domain actin binding only when ADP is of kinetic and equilibrium constants for nucleotide and actin binding to native actomyosin myosin Va mm imidazole, pH 50 mm KCl, 2 mm MgCl2, mm EGTA, 2 mm 25 myosin Va mm imidazole, pH 50 mm KCl, 2 mm MgCl2, 0.1 mm EGTA, 0.2 mm 2 mm 25 myosin V-HMM mm imidazole, pH 50 mm KCl, 2 mm MgCl2, mm EGTA, 2 mm 25 myosin V-HMM mm imidazole, pH 50 mm KCl, 2 mm MgCl2, 0.1 mm EGTA, 0.2 mm 2 mm 25 ± ± not ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± mant fluorescence (λex = 297 ± ± ± ± ± ± ± ± ± ± ± ± in 25 mm 50 mm KCl, 2 mm MgCl2, 1 mm EGTA, 1 mm dithiothreitol, pH 20 °C ± ± ± = stoichiometry of myosin heads actin ± ± ± mm = stoichiometry of myosin heads actin from extrapolated (Fig. ± ± 10 mm imidazole, pH 50 mm KCl, 2 mm MgCl2, mm EGTA, 2 mm 25 10 mm imidazole, pH 50 mm KCl, 2 mm MgCl2, 0.1 mm EGTA, 0.2 mm 2 mm 25 not mant fluorescence (λex = 297 in 25 mm 50 mm KCl, 2 mm MgCl2, 1 mm EGTA, 1 mm dithiothreitol, pH 20 °C Sweeney H.L. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google n = stoichiometry of myosin heads actin from extrapolated (Fig. 1B). in a The total pyrene quenching of myosin Va with bound ADP in the absence of calcium averaged ± of the in the presence of calcium over the actin We this to that when calcium is one head binds actin strongly and upon but the other remains weakly bound or detached and not quench fluorescence. In the total quenching in the presence and absence of calcium by consistent with actin binding ADP, presumably bound to the myosin motor domain. The equilibrium titrations (Fig. 1) suggest that only one head of myosin Va·ADP binds actin strongly in the absence of calcium. The that the quenching in the kinetic time courses (with actin ≫ do not change over a of actin is consistent with this mechanism. If two myosin Va in that bound actin strongly with both heads and the other weakly with both all myosin Va molecules would bind and quench pyrene actin fluorescence at saturating actin and this is not Time courses of actomyosin Va dissociation follow exponentials in the presence and absence of calcium 1 and 2) not Actomyosin Va·ADP dissociation double exponentials in the absence of calcium and is described by a in the presence of calcium 1 and 2) not dissociation is not observed in the presence of calcium, that calcium the population to a dissociating and that the actin filament binding affinity is in the presence of calcium, as from studies (22Tauhata S.B. dos Santos D.V. Taylor E.W. Mooseker M.S. Larson R.E. J. Biol. Chem. 2001; 276: 39812-39818Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). and Binding to Myosin binding to myosin Va in the absence of actin was measured from the fluorescence that occurs with binding La Cruz E.M. Wells A.L. Ostap E.M. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). Time courses of binding (Fig. are fitted by exponentials with observed rate constants that depend linearly on (Fig. Calcium not the association rate constant in the presence and absence of 1 and However, the of the fluorescence are ∼30 ± in the absence of calcium over the (Fig. not We can exclude the that nucleotide (i.e. ADP) the concentrations of heads of binding mantATP, the this would yield a biphasic time (i.e. binding to sites and binding to sites upon dissociation of bound which was not observed (Fig. In of myosin with effect not However, if bound nucleotide were and did not time courses of and binding would follow exponentials with as The that the of fluorescence change are in the absence and presence of calcium (Fig. is consistent with the interpretation that or nucleotide not account for the observed The of binding to myosin V-HMM are in the presence and absence of calcium (Fig. that the calcium-dependent observed with full-length myosin Va arise from with the motor Binding to Actomyosin binding to actomyosin Va was monitored from changes in pyrene actin fluorescence that arise with population of the weak binding actomyosin·ATP ensure that all reactions with dissociation of actomyosin from pyrene excess unlabeled was included in the ATP Time courses of pyrene fluorescence (Fig. after mixing pyrene actomyosin Va with ATP the absence or presence of free follow double exponentials with fast observed rate constants that depend on ATP (Fig. consistent with a two-step mechanism for ATP binding to actomyosin Va was also a phase that little dependence in the absence and presence of calcium with and 2 the rate of population of the weak actin binding state of myosin Va in the absence of calcium and in the presence of (Fig. 1, and 2) and the rate of ADP release from actomyosin (20De La Cruz E.M. Sweeney H.L. Ostap E.M. Biophys. J. 2000; 79: 1524-1529Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, La Cruz E.M. Wells A.L. Ostap E.M. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). We this phase to represent myosin Va dissociation from actin filaments J.E. Krementsova E.B. A. Trybus K.M. Proc. Natl. Acad.