Actin Sites Research Articles

Intracellular effects of nitric oxide on force production and Ca2+ sensitivity of cardiac myofilaments.

The gaseous free radical nitric oxide (NO*) has been implicated in a wide range of physiological functions and also has a role in the pathogenesis of cellular injury. It has been suggested that NO* and its congeners may exert effects on actin-myosin crossbridge cycling by modulating critical thiols on the myosin head. To understand the mode and site of actin of NO* in myofibrils, the effects of the NO* donor 3-(2-hydroxy-1-methyl-2-nitrosohydrazine)-N-methyl-1-propanamine (NOC-7) have been studied in Triton X-100-treated rabbit cardiac fibers, in which isometric force was measured at controlled degrees of activation. Experiments were undertaken after previous exposure of the preparations to NOC-7 (for 30 min). We found that NO* induced several alterations of myofibrillar function, i.e., decrease in Ca2+ sensitivity and Hill coefficient and potentiation of rigor contracture. We attributed the effect on rigor contracture to strong inhibition of myofibrillar creatine kinase (CK) activity, because it could be prevented by exogeneously added CK; such CK inactivation afforded by NO* may result in the myofibrillar ATP-to-ADP ratio. In further experiments, concentration of NO* released from NOC-7 was determined by the electron spin resonance spin-trapping technique; N-(dithiocarboxy)sarcosine-Fe2+ complex was used as the spin-trap. NO* at cumulative concentration of 0.69 microM was effective in producing both enhancement of rigor contracture and decrease of myofibrillar-bound CK activity; however, Ca2+-sensitivity (pCa50) was significantly decreased at >5.6 microM of NO*, suggesting a result from different mechanisms. Thus, the observed decrease in Ca2+ sensitivity seems to be associated with direct modification of the regulatory proteins by a relatively higher concentration of NO*, and possibly not via inhibition of myofibrillar CK activity. The data reported here indicate that CK may be a pathophysiologically main target for increased NO* formation at low molecular range in the disease state in cardiac muscle.

Detection of nucleotide- and F-actin-induced movements in the switch II helix of the skeletal myosin using its differential oxidative cleavage mediated by an iron-EDTA complex disulfide-linked to the strong actin binding site.

We have synthesized the mixed disulfide, S-(2-nitro-5-thiobenzoic acid) cysteaminyl-EDTA, using a rapid procedure and water-soluble chemistry. Its disulfide-thiol exchange reaction with rabbit myosin subfragment-1 (S-1), analyzed by spectrophotometry, ATPase assays, and peptide mapping, led to the incorporation of the cysteaminyl-EDTA group into only Cys 540 on the heavy chain and into the unique cysteine on the alkali light chains. The former thiol, residing in the strong actin binding site, reacted at a much faster rate with a concomitant 3-fold decrease in the V(max) for acto-S-1 ATPase but without change in the essential enzymatic functions of S-1. Upon chelation of Fe(3+) ions to the Cys 540-bound EDTA and incubation of the S-1 derivative-Fe complex with ascorbic acid at pH 7.5, the 95 kDa heavy chain underwent a conformation-dependent, single-cut oxidative fragmentation within 5-15 A of Cys 540. Three pairs of fragments were formed which, after specific fluorescent labeling and SDS-PAGE, could be positioned along the heavy chain sequence as 68 kDa-26 kDa, 62 kDa-32 kDa, and 54 kDa-40 kDa. Densitometric measurements revealed that the yield of the 54 kDa-40 kDa pair of bands, but not that for the two other pairs, was very sensitive to S-1 binding to nucleotides or phosphate analogues as well as to F-actin. In binary complexes, all the former ligands specifically lowered the yield to 40% of S-1 alone, roughly in the following order: ADP = AMP-PNP > ATP = ADP.AlF(4) > ADP.BeF(x)() > PP(i). By contrast, rigor binding to F-actin increased the yield to 130%. In the ternary acto-S-1-ADP complex, the yield was again reduced to 80%, and it fell to 25% in acto-S-1-ADP.AlF(4), the putative transition state analogue complex of the acto-S-1 ATPase. These different quantitative changes reflect distinct ligand-induced conformations of the secondary structure element whose scission generates the 54 kDa-40 kDa species. According to the S-1 crystal structure, this element could be unambiguously assigned to the switch II helix (residues 475-507) whose N-terminus lies 14.2 A from Cys 540 and would include the ligand-responsive cleavage site. This motif is thought to be crucial for the transmission of sub-nanometer structural changes at the ATPase site to both the actin site and the lever arm domain during energy transduction. Our study illustrates this novel, actin site-specific chemical proteolysis of S-1 as a direct probe of the switch II helix conformational transitions in solution most likely associated with the skeletal cross-bridge cycle.

Localization of Actin in Moloney Murine Leukemia Virus by Immunoelectron Microscopy

Immunoelectron microscopy was used to detect actin in wild-type (wt) Moloney murine leukemia virus (MoMuLV) and in virus-like particles (VLP) produced by recombinant Semliki Forest virus expressing only the MoMuLV gag polyprotein. Gold immunolabeling revealed the presence of actin on the surface of delipidized VLP and delipidized wt virus particles. Statistical evaluation of the number of colloidal gold particles per VLP revealed a large range of values and a prevalence of VLP with small numbers of gold particles. Labeling for actin was lost after prolonged treatment of VLP with 1% Nonidet-P40, high-pH buffer, or gelsolin. Gold immunolabeling with antibodies to gag proteins p15 (MA) and p12 and p30 (CA) was abundant and was not affected by treatment of VLP or wt virus with 1% Nonidet or gelsolin. VLP treated with a mixture of detergent and aldehyde fixatives showed more uniform and consistent labeling for actin than without fixatives. Negative staining or heavy metal shadowing revealed a globular surface of delipidized VLP. Stereomicrographs of gold-immunolabeled VLP showed that p15gag and p12gag were associated with the globular projections. Delipidized VLP were also well labeled with antibody to p30gag, which indicated that the gag shell permitted access of antibodies to p30gag and was therefore not a closely packed structure. Labeling for actin-binding proteins moesin and ezrin was negative in both the wt virus and the VLP. The absence of Gaussian distribution of actin in the sample of VLP suggests that actin is not a structural protein and its presence in MuLV virus particles may be fortuitous. This, however, does not rule out any possible role of actin in transport, assembly, budding, or release of virus particles, events which take place in the cytoplasm or at the plasma membrane. The site of actin in VLP is discussed in relation to the present knowledge of the molecular organization of the MuLV gag shell.

Cross bridge-dependent activation of contraction in cardiac myofibrils at low pH.

Striated muscle contracts in the absence of calcium at low concentrations of MgATP ([MgATP]), and this has been termed rigor activation because rigor cross bridges attach and activate adjacent actin sites. This process is well characterized in skeletal muscle but not in cardiac muscle. Rigor cross bridges are also thought to increase calcium binding to troponin C and play a synergistic role in activation. We tested the hypothesis that cross bridge-dependent activation results in an increase in contractile activity at normal and low pH values. Myofibrillar ATPase activity was measured as a function of pCa and [MgATP] at pH 7.0, and the data showed that, at pCa values of >/=5.5, there was a biphasic relationship between activity and [MgATP]. Peak activity occurred at 10-50 microM MgATP, and [MgATP] for peak activity was lower with increased pCa. The ATPase activity of rat cardiac myofibrils as a function of [MgATP] at a pCa of 9.0 was measured at several pH levels (pH 5.4-7.0). The ATPase activity as a function of [MgATP] was biphasic with a maximum at 8-10 microM MgATP. Lower pH did not result in a substantial decrease in myofibrillar ATPase activity even at pH 5.4. The extent of shortening, as measured by Z-line spacing, was greatest at 8 microM MgATP and less at both lower and higher [MgATP], and this response was observed at all pH levels. These studies suggest that the peak ATPase activity associated with low [MgATP] was coupled to sarcomere shortening. These results support the hypothesis that cross bridge-dependent activation of contraction may be responsible for contracture in the ischemic heart.

Assembly of thick filaments and myofibrils occurs in the absence of the myosin head

We investigated the importance of the myosin head in thick filament formation and myofibrillogenesis by generating transgenic Drosophila lines expressing either an embryonic or an adult isoform of the myosin rod in their indirect flight muscles. The headless myosin molecules retain the regulatory light-chain binding site, the alpha-helical rod and the C-terminal tailpiece. Both isoforms of headless myosin co-assemble with endogenous full-length myosin in wild-type muscle cells. However, rod polypeptides interfere with muscle function and cause a flightless phenotype. Electron microscopy demonstrates that this results from an antimorphic effect upon myofibril assembly. Thick filaments assemble when the myosin rod is expressed in mutant indirect flight muscles where no full-length myosin heavy chain is produced. These filaments show the characteristic hollow cross-section observed in wild type. The headless thick filaments can assemble with thin filaments into hexagonally packed arrays resembling normal myofibrils. However, thick filament length as well as sarcomere length and myofibril shape are abnormal. Therefore, thick filament assembly and many aspects of myofibrillogenesis are independent of the myosin head and these processes are regulated by the myosin rod and tailpiece. However, interaction of the myosin head with other myofibrillar components is necessary for defining filament length and myofibril dimensions.

Polymerization of actin induced by a molar excess of ATP in a low salt buffer.

The polymerization of actin induced by dilution has previously been reported, where a 1000-fold molar excess of ATP over actin resulted when actin was diluted to 4.0 micrograms/ml in low salt buffer A (0.1 mM ATP, 0.1 mM CaCl2, 2 mM Tris-HCl, pH 8.0, 5 mM 2-mercaptoethanol, 1 mM NaN3). Filaments formed by the addition of ATP to a 1000-fold molar excess over actin in buffer B (0.1 mM CaCl2, 2 mM Tris-HCl, pH 8.0, 1 mM NaN3) were then separated by gel-filtration. When ATP was removed from these filaments using Dowex-1, depolymerization occurred. Thus, the reversible polymerization induced by the dilution of actin or by addition of ATP can be ascribed to the binding of ATP at the low affinity site of actin.

Fluorescence resonance energy transfer between points on tropomyosin and actin in skeletal muscle thin filaments: does tropomyosin move?

Fluorescence resonance energy transfer (FRET) spectroscopy has been used to determine spatial relationships between residues on tropomyosin and actin in reconstituted muscle thin filament, and to detect a positional change of tropomyosin relative to actin on the thin filament in the presence and absence of Ca2+ ions. In addition to Cys-190 which is a single cysteine residue in rabbit skeletal muscle alpha-tropomyosin, a new site, Cys-87 which is a unique cysteine residue in a mutant alpha-tropomyosin, was labeled with a resonance energy donor molecule, 5-(2-iodoacetylaminoethyl)aminonaphthalene 1-sulfonic acid (IAEDANS). On the other hand, Gln-41, Lys-61, Cys-374, and the ATP-binding site of actin were selectively labeled with acceptor probes: fluorescein cadaverine, fluorescein 5-isothiocyanate, 4-dimethyl-aminophenylazophenyl 4'-maleimide, and TNP-ATP (or TNP-ADP), respectively. The distances between probes attached to position 87 of the mutant tropomyosin and Gln-41, Lys-61, Cys-374, or the nucleotide-binding site of actin on the reconstituted thin filament in the presence of Ca2+ ion were measured to be 43.2, 49.7, 45.4, and 35.2 A, respectively, and the distance between probes attached to position 190 of tropomyosin and Gln-41 or the nucleotide-binding site of actin were 51.6 and 43.1 A, respectively. The transfer efficiencies between these donor and acceptor molecules were large, so that the efficiency should be very sensitive to changes in distance between probes attached to tropomyosin and actin. However, the transfer efficiency did not change appreciably upon removal of Ca2+ ions, suggesting that tropomyosin does not change its position on the reconstituted thin filament in response to a change in Ca2+ ion concentration. The present results do not support the notion of tropomyosin movement on skeletal muscle thin filaments as proposed in the steric blocking theory.

Identification of the basolateral targeting determinant of a peripheral membrane protein, MacMARCKS, in polarized cells.

Although the molecular determinants that specify the targeting of transmembrane proteins to the apical or basolateral membrane domains within polarized epithelial cells have been well characterized, very little is known about the targeting of peripheral membrane proteins within these cells. MacMARCKS is a member of the MARCKS family of protein kinase C (PKC) substrates. This myristoylated protein regulates actin structure at cell membranes and is essential for the morphogenic movement of neuroepithelial cells during the formation of the neural tube. MacMARCKS was specifically targeted to sites of cell-cell contact in the basolateral domain of polarized Madin-Darby canine kidney (MDCK) epithelial cells and was displaced from this location upon activation of PKC. We defined the basolateral targeting determinant of MacMARCKS to be the effector domain, a basic region spanning 24 amino acids and containing the PKC phosphorylation sites as well as binding sites for calmodulin and actin. This domain, in conjunction with a myristoyl moiety, was sufficient to target a non-membrane-associated protein--green fluorescent protein--specifically to the basolateral surface of polarized MDCK cells. This is the first description of a specific amino acid sequence that specifies targeting of a peripheral membrane protein to the basolateral membrane in polarized epithelial cells.

α1-Syntrophin has distinct binding sites for actin and calmodulin

Overlay and co-sedimentation assays using recombinant α1-syntrophin proteins revealed that two regions of α1-syntrophin, i.e. aa 274–315 and 449–505, contain high-affinity binding sites for F-actin ( K d 0.16–0.45 μM), although only a single high-affinity site ( K d 0.35 μM) was detected in the recombinant full-length syntrophin. We also found that actomyosin fractions prepared from both cardiac and skeletal muscle contain proteins recognized by anti-syntrophin antibody. These data suggest a novel role for syntrophin as an actin binding protein, which may be important for the function of the dystrophin-glycoprotein complex or for other cell functions. We also found that α1-syntrophin binds calmodulin at two distinct sites with high ( K d 15 nM) and low ( K d 0.3 μM) affinity.

Ca2+-induced distance change between points on actin and troponin in skeletal muscle thin filaments estimated by fluorescence energy transfer spectroscopy.

Fluorescence resonance energy transfer spectroscopy has been used to study the spatial relationships between probes attached to actin and troponin in the reconstituted skeletal muscle thin filament in the presence and absence of Ca2+ ions. Gln-41 and the nucleotide-binding site of actin were selectively labeled with the acceptor probe: fluorescein cadaverine and 2'(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP), respectively. Troponin was selectively labeled at positions 9 or 133 of troponin-I and 98 of troponin-C with a donor probe; 5-(2-iodoacetylaminoethyl)aminonaphthalene 1-sulfonic acid (IAEDANS). The distances between probes attached to position 133 of TnI and Gln-41 or the nucleotide site of actin were determined to be 51.6+/-1.2 and 42.7+/-0.9 A respectively in the presence of Ca2+, and these distances decreased by 11.5 and 9.3 A respectively in the absence of Ca2+ ions. The distances between the probes attached to position 9 of TnI and Gln-41 or the nucleotide site of actin were determined to be 59.1+/-2.0 or 49.3+/-1.5 A respectively in the presence of Ca2+, and the distances decreased by 5.3 or 3.7 A in the absence of Ca2+. The distances between probes attached to position 98 of TnC and Gln-41 or the nucleotide site of actin were determined to be 55.1+/-1.7 and 57+/-5 A in the presence of Ca2+ and the distances increased slightly by approximately 1 A in the absence of Ca2+. The results suggest that the C-terminal domain of troponin I moves to the outer domain of actin during inhibition, while the C-terminal domain of TnC does not move much.

A strain-dependent ratchet model for [phosphate]- and [ATP]-dependent muscle contraction.

A minimal strain-dependent ratchet model of muscle cross-bridge action is proposed which is broadly compatible with structural and kinetic constraints. Its essential features are: (1) dynamic binding of the S1-products complex to actin through a disorder-order transition coupled to the release of inorganic phosphate; (2) the absence of a force-generating rotation of the myosin head between the two force-holding states A.M.ADP and A.M; (3) strain-control of ADP release and ATP binding, giving net isometric tension and directed motility by the selective dissociation of negatively strained bound states. With a disordered pre-force state, the binding rate to state A.M.ADP need not be symmetric in x, the actin site displacement. With faster binding at positive x, the model predicts many steady-state and transient properties of striated muscle observed experimentally, including phases 2-4 of tension recovery from length changes and their dependence on excess phosphate (which enhances and accelerates phase 3) and reduced ATP (which gives a bimodal phase 2 and slows one mode). The response to large perturbations is often sensitive to the number of actin sites used, and to the inclusion of a 1 nm displacement of the neck region on release of ADP. The latter stabilizes the periodic tension behaviour produced by repeated releases.

Probing the effects of calcium on gelsolin.

Gelsolin is a calcium-regulated actin severing and capping protein that binds two calcium ions and has three sites for actin; two recognize monomeric actin and one attaches to the sides of filaments. It contains six repeating sequence segments (G1-6). Here, we have analyzed the effects of calcium ions on (i) limited proteolysis of bacterially expressed human gelsolin by plasmin and (ii) dynamic light scattering and circular dichroism of gelsolin and various of its subdomains. Following cleavage of gelsolin in the absence of calcium between Lys150 and His151 (the junction between G1 and G2), the molecule does not fall apart, nor does it bind actin without added calcium. This same molecule can be reconstituted by mixing an excess of G1 with G2-6 in EGTA. The noncovalently linked form of gelsolin shows three actin binding sites in calcium and requires 3 microM calcium for 50% activation of actin binding. Measurements of light scattering and circular dichroism revealed structural changes in response to calcium for intact gelsolin and a number of its actin-binding subdomains. Many of these changes occurred at calcium concentrations below 100 nM. These results are discussed in relation to the calcium control of gelsolin function and its three-dimensional structure (Burtnick et al.(1997) Cell 90, 661-670). Nanomolar concentrations of calcium initiate the unlatching of structural constraints that maintain the inaccessibility of the actin binding sites, but actin binding occurs only after additional micromolar calcium sites in both the N-terminal and C-terminal halves of the molecule are occupied.

A correlative analysis of actin filament assembly, structure, and dynamics.

The effect of the type of metal ion (i.e., Ca2+, Mg2+, or none) bound to the high-affinity divalent cation binding site (HAS) of actin on filament assembly, structure, and dynamics was investigated in the absence and presence of the mushroom toxin phalloidin. In agreement with earlier reports, we found the polymerization reaction of G-actin into F-actin filaments to be tightly controlled by the type of divalent cation residing in its HAS. Moreover, novel polymerization data are presented indicating that LD, a dimer unproductive by itself, does incorporate into growing F-actin filaments. This observation suggests that during actin filament formation, in addition to the obligatory nucleation- condensation pathway involving UD, a productive filament dimer, a facultative, LD-based pathway is implicated whose abundance strongly depends on the exact polymerization conditions chosen. The "ragged" and "branched" filaments observed during the early stages of assembly represent a hallmark of LD incorporation and might be key to producing an actin meshwork capable of rapidly assembling and disassembling in highly motile cells. Hence, LD incorporation into growing actin filaments might provide an additional level of regulation of actin cytoskeleton dynamics. Regarding the structure and mechanical properties of the F-actin filament at steady state, no significant correlation with the divalent cation residing in its HAS was found. However, compared to native filaments, phalloidin-stabilized filaments were stiffer and yielded subtle but significant structural changes. Together, our data indicate that whereas the G-actin conformation is tightly controlled by the divalent cation in its HAS, the F-actin conformation appears more robust than this variation. Hence, we conclude that the structure and dynamics of the Mg-F-actin moiety within the thin filament are not significantly modulated by the cyclic Ca2+ release as it occurs in muscle contraction to regulate the actomyosin interaction via troponin.

Characteristics of troponin C binding to the myofibrillar thin filament: extraction of troponin C is not random along the length of the thin filament

Troponin C (TnC) is the Ca(2+)-sensing subunit of troponin responsible for initiating the cascade of events resulting in contraction of striated muscle. This protein can be readily extracted from myofibrils with low-ionic-strength EDTA-containing buffers. The properties of TnC extraction have not been characterized at the structural level, nor have the interactions of TnC with the native myofibrillar thin filament been studied. To address these issues, fluorescein-labeled TnC, in conjunction with high-resolution digital fluorescence microscopy, was used to characterize TnC binding to myofibrils and to determine the randomness of TnC extraction. Fluorescein-5-maleimide TnC (F5M TnC) retained biological activity, as evidenced by reconstitution of Ca(2+)-dependent ATPase activity in extracted myofibrils and binding to TnI in a Ca(2+)-sensitive manner. The binding of F5M TnC to highly extracted myofibrils at low Ca2+ was restricted to the overlap region under rigor conditions, and the location of binding was not influenced by F5M TnC concentration. The addition of myosin subfragment 1 to occupy all actin sites resulted in F5M TnC being bound in both the overlap and nonoverlap regions. However, very little F5M TnC was bound to myofibrils under relaxing conditions. These results suggest that strong binding of myosin heads enhances TnC binding. At high Ca2+, the pattern of F5M TnC binding was concentration dependent: binding was restricted to the overlap region at low F5M TnC concentration, whereas the binding propagated into the nonoverlap region at higher levels. Analysis of fluorescence intensity showed the greatest binding of F5M TnC at high Ca2+ with S1, and these conditions were used to characterize partially TnC-extracted myofibrils. Comparison of partially extracted myofibrils showed that low levels of extraction were associated with greater F5M TnC being bound in the nonoverlap region than in the overlap region relative to higher levels of extraction. These results show that TnC extraction is not random along the length of the thin filament, but occurs more readily in the nonoverlap region. This observation, in conjunction with the influence of rigor heads on the pattern of F5M TnC binding, suggests that strong myosin binding to actin stabilizes TnC binding at low Ca2+.

Type II myosin heavy chain encoded by the myo2 gene composes the contractile ring during cytokinesis in Schizosaccharomyces pombe.

We cloned the myo2 gene of Schizosaccharomyces pombe, which encodes a type II myosin heavy chain, by virtue of its ability to promote diploidization in fission yeast cells. The myo2 gene encodes 1,526 amino acids in a single open reading frame. Myo2p shows homology to the head domains and the coiledcoil tail of the conventional type II myosin heavy chain and carries putative binding sites for ATP and actin. It also carries the IQ motif, which is a presumed binding site for the myosin light chain. However, Myo2p apparently carries only one IQ motif, while its counterparts in other species have two. There are nine proline residues, which should break alpha-helix, in the COOH-terminal coiled-coil region of Myo2p. Thus, Myo2p is rather unusual as a type II myosin heavy chain. Disruption of myo2 inhibited cell proliferation. myo2Delta cells showed normal punctate distribution of interphase actin, but they produced irregular actin rings and septa and were impaired in cell separation. Overproduction of Myo2p was also lethal, apparently blocking actin relocation. Nuclear division proceeded without actin ring formation and cytokinesis in cells overexpressing Myo2p, giving rise to multinucleated cells with dumbbell morphology. Analysis using tagged Myo2p revealed that Myo2p colocalizes with actin in the contractile ring, suggesting that Myo2p is a component of the ring and responsible for its contraction. Furthermore, genetic evidence suggested that the acto-myosin system may interact with the Ras pathway, which regulates mating and the maintenance of cell morphology in S. pombe.

Molecular characterization of myosin IB from the lower eukaryote Entamoeba histolytica, a human parasite

The complete amino acid sequence of the Entamoeba histolytica unconventional myosin IB (Eh-myosin IB) is reported. Sequencing of overlapping cDNA fragments reveals a single open reading frame which predicts a 130 kDa protein of 1049 aa. Eh-myosin IB presents the three characteristics domains of myosins I subclass 1. This protein presents homology with myosins IB from other amoebae, but striking homologies with vertebrate unconventional myosins were also observed. The predicted actin and ATP-binding sites are located in the head domain. The heavy chain phosphorylation region is homologous to metazoan myosins I with an acidic residue present at the phosphorylation site. In the neck domain, an IQ motif indicates potential binding of calmodulin to the myosin I heavy chain. In the tail of Eh-myosin IB the three characteristic regions of myosin I are found. A putative membrane binding domain a very short domain rich in alanine and proline we demonstrate to be functional for actin binding, and the src-homology 3 domain. The Entamoeba histolytica myosin IB is the first unconventional myosin so far described in a lower eukaryote.

Effect of metal cations on the conformation of myosin subfragment-1-ADP-phosphate analog complexes: a near-UV circular dichroism study.

The interaction of myosin with actin, coupled with hydrolysis of ATP, is the molecular basis of muscle contraction. The head segment of myosin, called S1, contains the distinct binding sites for ATP and actin and is responsible for the ATPase activity. The myosin-catalyzed ATP hydrolysis consists of several intermediate steps and each step is accompanied by conformational changes in the S1 segment. The rate-limiting step of the ATP hydrolysis is the dissociation of the S1 x ADP x Pi complex which is accelerated by actin. The substitution of Pi with phosphate analogs (PA), such as vanadate, beryllium fluoride (BeFx) or aluminum fluoride (AlF4-), yields stable complexes which mimic the intermediates of the ATP hydrolysis. In this work, tertiary structure changes in S1 in the vicinity of aromatic residues was studied by comparing near-UV circular dichroism (CD) spectra from S1-nucleotide-phosphate analog complexes in the presence of Mg2+ and other cations. A significant difference between the MgATP and MgADP spectra indicated notable tertiary structural changes accompanying the M**ADP x Pi --> M*ADP transition. The spectra of the S1 x MgADP x BeFx and S1 x MgADP x AlF4- complexes resemble to those obtained upon addition of MgATPgammaS and MgATP to S1, and correspond to the M* x ATP and M** x ADP x Pi intermediates, respectively. We have found recently that the presence of divalent metal cations (Me2+) is essential for the formation of stable S1 x MeADP x PA complexes. Moreover, the nature of the metal cations strongly influences the stability of these complexes [Peyser, Y. M., et al. (1996) Biochemistry 35, 4409-4416]. In the present work we studied the effect of Mg2+, Mn2+, Ca2+, Ni2+, Co2+, and Fe2+ on the near-UV CD spectrum of the ATP, ADP, ADP x BeFx, and ADP x AlF4- containing S complexes. The CD spectra obtained with ADP, ATP ADP x BeFx and ADP x AlF4- were essentially identical in the presence of Co2+ and rather similar in the case of Ca2+, while they were partially different in other cases. An interesting correlation was found between actin activation and ATP versus ADP difference spectra in the presence of various metal ions. The distribution of the fractional concentration of the intermediates of ATP hydrolysis was estimated in the presence of each cation from the CD spectra with phosphate analogs. In the presence of Mg2+ the predominant intermediate is the M** x ADP x Pi state, which is in accordance with the kinetic studies. On the other hand with non-native cations the predominant intermediate is the M* x ADP state and the release of ADP is the rate limiting step in the myosin-catalyzed ATP hydrolysis. According to the results, the near-UV CD spectrum originating from aromatic residues in S1 not only can distinguish identifiable states in the ATP hydrolysis cycle but can also pinpoint to changes in the tertiary structure caused by complex formation with nucleotide or nucleotide analog and various divalent metal cations. These findings, that are correlative with actin activation, and thus with the power stroke, suggest new strategies for perturbing S1 structure in the continuous efforts directed toward the elucidation of the mechanism of muscle contraction.

Purification of a 47-kDa calmodulin-binding polypeptide as an actin-binding protein from Neurospora crassa

We have enriched a 47-kDa polypeptide (p47) from Neurospora crassa on the basis of its affinity to calmodulin. The p47 was purified to homogeneity by chromatography on a Mono S cation exchange column and evidence is presented that the polypeptide co-sediments specifically with F-actin. The intracellular distribution of p47 and actin was also examined using indirect double immunofluorescence staining of cells at different stages of development. Our results suggest that by altering the conformation binding site of actin to p47, calmodulin could play a regulatory role in the polarized hyphal growth of N. crassa.

Energy-filtered electron microscopy reveals that talin is a highly flexible protein composed of a series of globular domains.

Talin is a multidomain cytoskeletal protein containing discrete binding sites for acidic phospholipids, beta-integrin, actin and vinculin. Hence, it is thought to link microfilaments to the cytoplasmic membrane in cell-matrix adhesion sites, and this should critically depend on talin structure. To obtain more information on the latter, we used energy-filtered transmission electron microscopy of negatively stained talin purified from chicken smooth muscle. We show that in buffers of physiological ionic strength, talin adopts an elongated shape (56 +/- 7 nm in length), consisting of a series of globular masses. While these compact elements, arranged like beads on a string, were of rather uniform dimensions (3.8 nm in diameter), their center-to-center spacings varied, indicating the flexibility of the connecting strands. The ends of the elongated molecules frequently formed loops. The images obtained are consistent with the assumption that, under the conditions used, the majority of the talin molecules are monomeric. A minor fraction appeared as dimers, composed of two chains only partially intertwined, thus giving rise to Y-shaped particles. Electron micrographs revealed that the biochemically defined 50-kDa N-terminal talin head domain is composed of two globular subunits, while chemical cross-linking provided evidence that the C-terminal 220-kDa fragment is solely responsible for dimerization. These results imply that in the dimeric molecules, the polypeptide chains are arranged in parallel, in contrast to what has been described for human-platelet talin. In buffers of low ionic strength (0.02 M instead of 0.15 M KCl), the molecules collapsed into a compact shape. By showing the high flexibility and versatility of its morphology, our data favour the concept of talin as an important resilient link in microfilament-plasma-membrane attachment.

Three-dimensional Structure of Nucleotide-bearing Crossbridges in Situ: Oblique Section Reconstruction of Insect Flight Muscle in AMPPNP at 23°C

We have explored the three-dimensional structure of myosin crossbridges in situin order to define the structural changes that occur when nucleotide binds to the myosin motor. When AMPPNP binds to rigor insect flight muscle, each half sarcomere lengthens by ∼2.0 nm and tension is reduced by ∼70% without a reduction in stiffness, suggesting partial reversal of the power stroke. We have obtained averaged oblique section three-dimen sional reconstructions of mechanically monitored insect flight muscle in AMPPNP that permit simultaneous examination of all myosin crossbridges within the unit cell and direct comparison of calculated transforms with X-ray diagrams of the native fibers. Transforms calculated from the oblique section reconstruction of AMPPNP insect flight muscle at 23 °C show good agreement with native X-ray diagrams, suggesting that the average crossbridge forms in the reconstruction reflect the native structure. In contrast to the rigor lead and rear crossbridges in the double chevrons, the averaged reconstruction of AMPPNP fibers show only one crossbridge class, in the position of the rigor lead bridge. The portion of the crossbridge close to the thick filament appears broader than in rigor, and shows a small 0.5 to 1.0 nm M-ward shift of the regulatory domain region of myosin. In transverse view, AMPPNP “lead” crossbridges are less azimuthally bent than rigor. Fitting the atomic model of actomyosin subfragment 1 to the averaged crossbridges shows that the detectable differences between rigor bridges and between rigor and AMPPNP bridges occur in the alignment and angles of the regulatory domains and suggests that rear bridges are more strained than lead crossbridges. The apparent absence of rear bridges in AMPPNP in averaged reconstructions indicates detachment of a number of force-bearing bridges, which conflicts with the maintained stiffness of the fibers used for the reconstruction. This conflict may be explained if rigor rear bridges become distributed irregularly over more actin sites in AMPPNP, so that their average density is too low to appear in the averaged reconstructions. The reconstructions indicate that in insect flight muscle the response of in siturigor crossbridges to AMPPNP binding is not uniform. Lead bridges persist but have altered structure in the light chain domain, whereas rear bridges detach and possibly redistribute. Shape changes in attached myosin heads within the myofibrillar lattice are in the appropriate direction and of the appropriate magnitude needed to explain the sarcomere lengthening. This could be a direct response to nucleotide binding, a passive response to rear bridge detachment, or a combination of both.