ATP-linked monomer-polymer equilibrium of smooth muscle myosin: the free folded monomer traps ADP.Pi.

In vitro and at physiological ionic strength, unphosphorylated smooth muscle myosin filaments dissolve on addition of ATP, forming folded (10S) myosin monomers. By following the fate of ATP and the time course of filament disassembly we have established details of the mechanism of this process. Myosin filaments first bind and hydrolyse 2.0 mol/mol of ATP before significant filament dissolution occurs. Following dissolution, the hydrolysis products ADP.Pi are retained on the heads of the folded myosin monomers, and are released so slowly (half time approximately 100 min at 100 mM KCl) as to be effectively trapped. The straight (6S) conformation of myosin, stable at greater than 225 mM KCl, did not exhibit this product trapping, and neither did myosin filaments held under conditions which disfavour ATP-induced disassembly. The implications of these results for filament stability in vivo are discussed in terms of a simple, testable model for smooth muscle myosin self-assembly.     

Active site trapping of nucleotide by smooth and non-muscle myosins

The folded 10 S monomer conformation of smooth muscle myosin traps the hydrolysis products ADP and Pi in its active sites. To test the significance of this, we have searched for equivalent trapping in other conformational and assembly states of avian gizzard and brush border myosins, using formycin triphosphate (FTP) as an ATP analogue. When myosin monomers were in the straight-tail 6 S conformation, the hydrolysis products were released at about 0.03 s-1. Adoption of the folded 10 S monomer conformation reduced this rate by more than 100-fold, effectively trapping the products FDP and Pi in the active sites. This profound inhibition of product release occurred only on formation of the looped tail monomer conformation. In vitro-assembled myosin filaments released products at a comparable rate to free straight-tail 6 S monomers, and smooth muscle heavy meromyosin, which lacks the C-terminal two-thirds of the myosin tail, also did not trap the products in this way. Phosphorylation of the myosin regulatory light chain had no effect on the rate of product release from straight-tail 6 S myosin monomers or from myosin filaments. Rather, it allowed actin to accelerate product release. Phosphorylation acted also to destabilize the folded monomer conformation, causing the recruitment of molecules from the pool of folded monomers into the myosin filaments. The two processes of contraction and filament assembly are thus both controlled in vitro by light-chain phosphorylation. A similar linked control in vivo would allow the organization of myosin in the cell to adapt itself continuously to the pattern of contractile activity.     

Regulation in vitro of brush border myosin by light chain phosphorylation

Myosin was purified from chicken brush border cells to greater than 95% homogeneity and in a predominantly non-phosphorylated state. The effects of light chain phosphorylation by a Ca2+-calmodulin-dependent myosin light chain kinase on the conformational, enzymatic and filament assembly properties of this myosin were investigated. The actin-activated MgATPase activity of the non-phosphorylated myosin was low, and upon light chain phosphorylation an eight- to ninefold increase in this activity was observed, which was further potentiated by tropomyosin. Light chain phosphorylation was shown to control the assembly and disassembly of brush border myosin filaments. For example, turbidity measurements and electron microscopy demonstrated that MgATP disassembled non-phosphorylated myosin filaments; the disassembled myosin could reassemble when the light chains were phosphorylated, and could be disassembled again by dephosphorylating the light chains with phosphatase. In the electron microscope, the disassembled non-phosphorylated myosin molecules appeared in a folded conformation, and they were extended when phosphorylated. Proteolytic digestion was used to probe further the conformation of these folded and extended molecules, and their subunit organizations were characterized by a gel overlay technique. Quantitative analysis further demonstrated that light chain phosphorylation alters dramatically the monomer/polymer equilibrium of brush border myosin, shifting it towards filament formation. Comparison of analogous data for myosin from gizzard and thymus shows that each myosin has distinct solubility properties.     

Monoclonal antibodies detect and stabilize conformational states of smooth muscle myosin

Antibodies with epitopes near the heavy meromyosin/light meromyosin junction distinguish the folded from the extended conformational states of smooth muscle myosin. Antibody 10S.1 has 100-fold higher avidity for folded than for extended myosin, while antibody S2.2 binds preferentially to the extended state. The properties of these antibodies provide direct evidence that the conformation of the rod is different in the folded than the extended monomeric state, and suggest that this perturbation may extend into the subfragment 2 region of the rod. Two antihead antibodies with epitopes on the heavy chain map at or near the head/rod junction. Magnesium greatly enhances the binding of these antibodies to myosin, showing that the conformation of the heavy chain in the neck region changes upon divalent cation binding to the regulatory light chain. Myosin assembly is also altered by antibody binding. Antibodies that bind to the central region of the rod block disassembly of filaments upon MgATP addition. Antibodies with epitopes near the COOH terminus of the rod, in contrast, promote filament depolymerization, suggesting that this region of the tail is important for assembly. The monoclonal antibodies described here are therefore useful both for detecting and altering conformational states of smooth muscle myosin.     

Control of filament length by the regulatory light chains in skeletal and cardiac myosins

The possible role of the regulatory light chains (LC2) in in vitro assembly of rabbit skeletal and dog cardiac myosins was examined by formation of minifilaments and synthetic thick filaments. After LC2 was removed, the resulting myosin preparations exhibited little aggregation in 0.5 M KCl and 0.05 M potassium phosphate (pH 6.5). Minifilaments migrated as a single, hypersharp peak during sedimentation velocity, but electron microscopic analysis revealed a more destabilized structure for LC2-deficient minifilaments. Thick filaments were formed in buffers containing 0.15 M KCl and the following: 20 mM imidazole; 20 mM imidazole, 5 mM ATP; or 20 mM imidazole, 5 mM ATP, and 5 mM MgCl2, all at pH 7.0. Skeletal and cardiac myosin filaments formed in imidazole buffer alone were bipolar, tapered at both ends, and about 1.6 micron long. Removal of LC2 resulted in the formation of shorter thick filaments (1.2 micron long). This effect could be reversed by reassociation with LC2. Inclusion of ATP in the buffer disrupted the filament structure, resulting in irregular, short filaments (less than 0.6 micron); addition of both ATP and MgCl2 largely reversed the effects of ATP alone. In cardiac myosin filaments, the bare zone diameter increased from 16 nm as measured in control and LC2-recombined samples to 20 nm in LC2-deficient myosin assemblies. These results implicate LC2 in an active role in controlling synthetic thick filament length in both skeletal and cardiac muscles.     

Assembly of smooth muscle myosin minifilaments: effects of phosphorylation and nucleotide binding

Small bipolar filaments, or "minifilaments," are formed when smooth muscle myosin is dialyzed against low ionic strength pyrophosphate or citrate/Tris buffers. Unlike synthetic filaments formed at approximately physiological ionic conditions, minifilaments are homogeneous as indicated by their hypersharp boundary during sedimentation velocity. Electron microscopy and hydrodynamic techniques were used to show that 20-22S smooth muscle myosin minifilaments are 380 nm long and composed of 12-14 molecules. By varying solvents, a continuum of different size polymers in the range of 15-30S could be obtained. Skeletal muscle myosin, in contrast, preferentially forms a stable 32S minifilament (Reisler, E., P. Cheung, and N. Borochov. 1986. Biophys. J. 49:335-342), suggesting underlying differences in the assembly properties of the two myosins. Addition of salt to the smooth muscle myosin minifilaments caused unidirectional growth into a longer "side-polar" type of filament, whereas bipolar filaments were consistently formed by skeletal muscle myosin. As with synthetic filaments, addition of 1 mM MgATP caused dephosphorylated minifilaments to dissociate to a mixture of folded monomers and dimers. Phosphorylation of the regulatory light chain prevented disassembly by nucleotide, even though it had no detectable effect on the structure of the minifilament. These results suggest that differences in filament stability as a result of phosphorylation are due largely to conformational changes occurring in the myosin head, and are not due to differences in filament packing.     

Order-disorder structural transitions in synthetic filaments of fast and slow skeletal muscle myosins under relaxing and activating conditions

In the previous study (Podlubnaya et al., 1999, J. Struc. Biol. 127, 1-15) Ca2+-induced reversible structural transitions in synthetic filaments of pure fast skeletal and cardiac muscle myosins were observed under rigor conditions (-Ca2+/+Ca2+). In the present work these studies have been extended to new more order-producing conditions (presence of ATP in the absence of Ca2+) aimed at arresting the relaxed structure in synthetic filaments of both fast and slow skeletal muscle myosin. Filaments were formed from column-purified myosins (rabbit fast skeletal muscle and rabbit slow skeletal semimebranosusproprius muscle). In the presence of 0.1 mM free Ca2+, 3 mM Mg2+ and 2 mM ATP (activating conditions) these filaments had a spread structure with a random arrangement of myosin heads and subfragments 2 protruding from the filament backbone. Such a structure is indistinguishable from the filament structures observed previously for fast skeletal, cardiac (see reference cited above) and smooth (Podlubnaya et al., 1999, J. Muscle Res. Cell Motil. 20, 547-554) muscle myosins in the presence of 0.1 mM free Ca2+. In the absence of Ca2+ and in the presence of ATP (relaxing conditions) the filaments of both studied myosins revealed a compact ordered structure. The fast skeletal muscle myosin filaments exhibited an axial periodicity of about 14.5 nm and which was much more pronounced than under rigor conditions in the absence of Ca2+ (see the first reference cited). The slow skeletal muscle myosin filaments differ slightly in their appearance from those of fast muscle as they exhibit mainly an axial repeat of about 43 nm while the 14.5 nm repeat is visible only in some regions. This may be a result of a slightly different structural properties of slow skeletal muscle myosin. We conclude that, like other filaments of vertebrate myosins, slow skeletal muscle myosin filaments also undergo the Ca2+-induced structural order-disorder transitions. It is very likely that all vertebrate muscle myosins possess such a property.     

Quantitative analysis of the kinetics of end-dependent disassembly of RecA filaments from ssDNA.

On linear single-stranded DNA, RecA filaments assemble and disassemble in the 5' to 3' direction. Monomers (or other units) associate at one end and dissociate from the other. ATP hydrolysis occurs throughout the filament. Dissociation can result when ATP is hydrolyzed by the monomer at the disassembly end. We have developed a comprehensive model for the end-dependent filament disassembly process. The model accounts not only for disassembly, but also for the limited reassembly that occurs as DNA is vacated by disassembling filaments. The overall process can be monitored quantitatively by following the resulting decline in DNA-dependent ATP hydrolysis. The rate of disassembly is highly pH dependent, being negligible at pH 6 and reaching a maximum at pH values above 7. 5. The rate of disassembly is not significantly affected by the concentration of free RecA protein within the experimental uncertainty. For filaments on single-stranded DNA, the monomer kcat for ATP hydrolysis is 30 min-1, and disassembly proceeds at a maximum rate of 60-70 monomers per minute per filament end. The latter rate is that predicted if the ATP hydrolytic cycles of adjacent monomers are not coupled in any way.     

Regulation of contraction and thick filament assembly-disassembly in glycerinated vertebrate smooth muscle cells

Isolated smooth muscle cells and cell fragments prepared by glycerination and subsequent homogenization will contract to one-third their normal length, provided Ca++ and ATP are present. Ca++- independent contraction was obtained by preincubation in Ca++ and ATP gamma S, or by addition of trypsin-treated myosin light chain kinase (MLCK) that no longer requires Ca++ for activation. In the absence of Ca++, myosin was rapidly lost from the cells upon addition of ATP. Glycerol-urea-PAGE gels showed that none of this myosin is phosphorylated. The extent of myosin loss was ATP- and pH-dependent and occurred under conditions similar to those previously reported for the in vitro disassembly of gizzard myosin filaments. Ca++-dependent contraction was restored to extracted cells by addition of gizzard myosin under rigor conditions (i.e., no ATP), followed by addition of MLCK, calmodulin, Ca++, and ATP. Function could also be restored by adding all these proteins in relaxing conditions (i.e., in EGTA and ATP) and then initiating contraction by Ca++ addition. Incubation with skeletal myosin will restore contraction, but this was not Ca++- dependent unless the cells were first incubated in troponin and tropomyosin. These results strengthen the idea that contraction in glycerinated cells and presumably also in intact cells is primarily thick filament regulated via MLCK, that the myosin filaments are unstable in relaxing conditions, and that the spatial information required for cell length change is present in the thin filament- intermediate filament organization.     

Properties of porcine platelet myosin. II. Shape change of the molecule

Porcine platelet myosin molecules were examined by electron microscopy for changes in their shape. At high ionic strength, the molecules were morphologically indistinguishable from skeletal muscle myosin, except for a slight difference in the bent regions of their tails. At physiological ionic strength, however, the following important difference was observed between the two myosins. Unlike skeletal muscle myosin, the filaments of nonphosphorylated platelet myosin could be disassembled by stoichiometric ATP into a monomeric form with sharply bent or folded tail, and reassembled after ATP hydrolysis. Similar disassembly changes could be induced by various nucleotide triphosphates (CTP, GTP, ITP, and UTP) and to a lesser extent by ADP, AMP, and AMPPNP. These results suggest that ATP binds to the hydrolytic sites in platelet myosin molecule and induces the molecular shape change.     

Actin-facilitated assembly of smooth muscle myosin induces formation of actomyosin fibrils

To identify regulatory mechanisms potentially involved in formation of actomyosin structures in smooth muscle cells, the influence of F-actin on smooth muscle myosin assembly was examined. In physiologically relevant buffers, AMPPNP binding to myosin caused transition to the soluble 10S myosin conformation due to trapping of nucleotide at the active sites. The resulting 10S myosin-AMPPNP complex was highly stable and thick filament assembly was suppressed. However, upon addition to F-actin, myosin readily assembled to form thick filaments. Furthermore, myosin assembly caused rearrangement of actin filament networks into actomyosin fibers composed of coaligned F-actin and myosin thick filaments. Severin-induced fragmentation of actin in actomyosin fibers resulted in immediate disassembly of myosin thick filaments, demonstrating that actin filaments were indispensable for mediating myosin assembly in the presence of AMPPNP. Actomyosin fibers also formed after addition of F-actin to nonphosphorylated 10S myosin monomers containing the products of ATP hydrolysis trapped at the active site. The resulting fibers were rapidly disassembled after addition of millimolar MgATP and consequent transition of myosin to the soluble 10S state. However, reassembly of myosin filaments in the presence of MgATP and F-actin could be induced by phosphorylation of myosin P-light chains, causing regeneration of actomyosin fiber bundles. The results indicate that actomyosin fibers can be spontaneously formed by F-actin-mediated assembly of smooth muscle myosin. Moreover, induction of actomyosin fibers by myosin light chain phosphorylation in the presence of actin filament networks provides a plausible hypothesis for contractile fiber assembly in situ.     

Stepping between Membrane Microdomains

In isolated thick filaments from many types of muscle, the two head domains of each myosin molecule are folded back against the filament backbone in a conformation called the interacting heads motif (IHM) in which actin interaction is inhibited. This conformation is present in resting skeletal muscle, but it is not known how exit from the IHM state is achieved during muscle activation. Here, we investigated this by measuring the in situ conformation of the light chain domain of the myosin heads in relaxed demembranated fibers from rabbit psoas muscle using fluorescence polarization from bifunctional rhodamine probes at four sites on the C-terminal lobe of the myosin regulatory light chain (RLC). The order parameter 〈P₂〉 describing probe orientation with respect to the filament axis had a roughly sigmoidal dependence on temperature in relaxing conditions, with a half-maximal change at ∼19°C. Either lattice compression by 5% dextran T500 or addition of 25 μM blebbistatin decreased the transition temperature to ∼14°C. Maximum entropy analysis revealed three preferred orientations of the myosin RLC region at 25°C and above, two with its long axis roughly parallel to the filament axis and one roughly perpendicular. The parallel orientations are similar to those of the so-called blocked and free heads in the IHM and are stabilized by either lattice compression or blebbistatin. In relaxed skeletal muscle at near-physiological temperature and myofilament lattice spacing, the majority of the myosin heads have their light chain domains in IHM-like conformations, with a minority in a distinct conformation with their RLC regions roughly perpendicular to the filament axis. None of these three orientation populations were present during active contraction. These results are consistent with a regulatory transition of the thick filament in skeletal muscle associated with a conformational equilibrium of the myosin heads.     

Subunit exchange between smooth muscle myosin filaments

Filaments formed from phosphorylated smooth muscle myosin are stable in the presence of MgATP, whereas dephosphorylated filaments are disassembled to a mixture of folded monomers and dimers. The stability of copolymers of phosphorylated and dephosphorylated myosin was, however, unknown. Gel filtration, sedimentation velocity, and pelleting assays were used to show that MgATP could dissociate dephosphorylated myosin from copolymers containing either rod and myosin or dephosphorylated and phosphorylated myosin. Copolymers were typically formed by dialyzing monomeric mixtures into filament-forming buffer but, unexpectedly, could also be formed within minutes of mixing preformed rod and myosin minifilaments. This result suggested that molecules can rapidly and extensively exchange between filaments, presumably via the monomeric pool of myosin in equilibrium with polymer. An exchange of molecules between filaments was demonstrated directly by electron microscopy using gold-labeled streptavidin or antibody to detect the exchanged species. By this approach it was shown that smooth muscle myosin filaments, like other macromolecular assemblies, are dynamic structures that can readily alter their composition in response to changing solvent conditions. Moreover, because folded monomeric myosin is unable to polymerize, these experiments suggest a mechanism for the disassembly of the filament by MgATP.     

Correlation of conformation and phosphorylation and dephosphorylation of smooth muscle myosin

The rate of phosphorylation and dephosphorylation of smooth muscle myosin by myosin light chain kinase and by two myosin light chain phosphatases (gizzard phosphatase IV and aorta phosphatase) are measured in various conditions; the relationship between the rate of phosphorylation and dephosphorylation of myosin and the myosin conformation is also studied. The rate of dephosphorylation of myosin was completely inhibited in the presence of 1 mM MgCl2 and ATP at low ionic strength where phosphorylated myosin forms a folded conformation. The inhibition was released when myosin formed either an extended monomer or filaments. The rate of phosphorylation of myosin was also affected by the conformation of myosin. The rate for a folded myosin was slower than those for an extended monomer and filamentous myosin. The phosphorylation and dephosphorylation of heavy meromyosin, subfragment-1, and the isolated 20,000-dalton light chain are not inhibited at low ionic strength, and the rate of phosphorylation and dephosphorylation was decreased with increasing ionic strength. KCl dependence of the rate of phosphorylation and dephosphorylation of myosin was normalized by using KCl dependence of subfragment-1, and it was found that the marked inhibition of the rate of phosphorylation and dephosphorylation of myosin is closely related to the change from an extended to a folded conformation of myosin.     

Tropomyosin prevents depolymerization of actin filaments from the pointed end

Regulation of the pointed, or slow-growing, end of actin filaments is essential to the regulation of filament length. The purpose of this study is to investigate the role of skeletal muscle tropomyosin (TM) in regulating pointed end assembly and disassembly in vitro. The effects of TM upon assembly and disassembly of actin monomers from the pointed filament end were measured using pyrenyl-actin fluorescence assays in which the barbed ends were capped by villin. Tropomyosin did not affect pointed end elongation; however, filament disassembly from the pointed end stopped in the presence of TM under conditions where control filaments disassembled within minutes. The degree of protection against depolymerization was dependent upon free TM concentration and upon filament length. When filaments were diluted to a subcritical actin concentration in TM, up to 95% of the filamentous actin remained after 24 h and did not depolymerize further. Longer actin filaments (150 monomers average length) were more effectively protected from depolymerization than short filaments (50 monomers average length). Although filaments stopped depolymerizing in the presence of TM, they were not capped as shown by elongation assays. This study demonstrates that a protein, such as TM, which binds to the side of the actin filament can prevent dissociation of monomers from the end without capping the end to elongation. In skeletal muscle, tropomyosin could prevent thin filament disassembly from the pointed end and constitute a mechanism for regulating filament length.     

Organized unidirectional waves of ATP hydrolysis within a RecA filament

The RecA protein forms nucleoprotein filaments on DNA, and individual monomers within the filaments hydrolyze ATP. Assembly and disassembly of filaments are both unidirectional, occurring on opposite filament ends, with disassembly requiring ATP hydrolysis. When filaments form on duplex DNA, RecA protein exhibits a functional state comparable to the state observed during active DNA strand exchange. RecA filament state was monitored with a coupled spectrophotometric assay for ATP hydrolysis, with changes fit to a mathematical model for filament disassembly. At 37 °C, monomers within the RecA-double-stranded DNA (dsDNA) filaments hydrolyze ATP with an observed k_cat of 20.8 ± 1.5 min⁻¹. Under the same conditions, the rate of end-dependent filament disassembly (k_off) is 123 ± 16 monomers per minute per filament end. This rate of disassembly requires a tight coupling of the ATP hydrolytic cycles of adjacent RecA monomers. The relationship of k_cat to k_off infers a filament state in which waves of ATP hydrolysis move unidirectionally through RecA filaments on dsDNA, with successive waves occurring at intervals of approximately six monomers. The waves move nearly synchronously, each one transiting from one monomer to the next every 0.5 s. The results reflect an organization of the ATPase activity that is unique in filamentous systems, and could be linked to a RecA motor function.     

MgATP specifically controls in vitro self-assembly of vertebrate skeletal myosin in the physiological pH range

The appearances in the electron microscope of rat and rabbit skeletal muscle myosin filaments and rod aggregates, formed in the presence of variable amounts of MgATP, were compared at different pH values. It is shown that small amounts of MgATP, similar to those sufficient to trigger the dissociation of the actomyosin complex, were able to modify the geometry of myosin filaments profoundly in the physiological pH range, whereas the conformation of rod aggregates remained unchanged even in the presence of high concentrations of MgATP. Myosin filaments formed in the absence of MgATP displayed the classical spindle-shaped conformation and variable diameters at all pH values, whereas myosin filaments formed in the presence of MgATP in the physiological pH range had constant diameters, similar to those of natural thick filaments. These filaments of constant diameter frayed, rapidly and reversibly, into two types of subfilaments with respective diameters of 4 to 5 nm and 9 to 10 nm, when the pH of the medium was raised above 7.2. Spindle-shaped myosin filaments and rod aggregates remained unchanged by such small changes in pH. It was possible to change the conformation of preformed spindle-shaped filaments by simply adding MgATP to the medium, but this reaction was slow and took several hours to be completed. Relatively high concentrations of MgATP, similar to those in the living cell, increased the solubility of both myosin filaments and rod aggregates in the alkaline pH range (pH greater than or equal to 7.0). Low pH values (less than or equal to 6.5) and excess free Mg2+ (greater than or equal to 6 to 7 mM) abolished both the specific effect of MgATP on myosin filament conformation and its solubilizing effect on both myosin filaments and rod aggregates. The degree of purity of the myosin preparations and the level of phosphorylation of the LC-2 light chains did not influence filament behaviour noticeably and rat and rabbit myosins behaved similarly.     

Polymerization of vertebrate non-muscle and smooth muscle myosins

We investigated how light chain phosphorylation controls the stability of filaments of vertebrate non-muscle myosins (from bovine thymocytes and chicken intestine epithelial brush border cells) and smooth muscle myosin (from chicken gizzard) in vitro. Using a sedimentation assay, the solubilities of the myosins were determined by measuring the amounts of myosin monomers (Cm) and filaments (Cp) present under a given set of conditions as a function of the total myosin concentration (Ct). Below 200 mM-NaCl, each myosin displayed distinct "critical monomer concentrations" (Cc) for polymerization, which were dependent on the salt concentration, the state of light chain phosphorylation and the presence of MgATP. At 150 mM-NaCl, MgATP increased the Cc of non-phosphorylated brush border myosin approximately five to tenfold, thymus myosin approximately 10 to 15-fold, and gizzard myosin approximately 25 to 50-fold. When these myosins were phosphorylated, MgATP had little effect on their solubilities, and their Cc values remained low. Analytical ultracentrifugation and electron microscopy demonstrated that the myosins were present in three different conformational states under the conditions used in the sedimentation assays, i.e. filaments, extended monomer (6 S) and folded monomer (10 S). Since at equilibrium only filaments and monomers were observed, we suggest that the polymerization pathway for these myosins can be analysed in terms of a dynamic monomer-polymer equilibrium (polymer in equilibrium 6 S monomer in equilibrium 10 S monomer). At roughly physiological ionic strength, light chain dephosphorylation (in the presence of MgATP) promotes the folded state (10 S), whereas phosphorylation promotes the extended state (6 S), and thereby favours filament assembly. The relevance of the monomer-polymer equilibrium to the state of organization of the myosin in vivo is discussed.     

Orientation of the N- and C-Terminal Lobes of the Myosin Regulatory Light Chain in Cardiac Muscle

The orientations of the N- and C-terminal lobes of the cardiac isoform of the myosin regulatory light chain (cRLC) in the fully dephosphorylated state in ventricular trabeculae from rat heart were determined using polarized fluorescence from bifunctional sulforhodamine probes. cRLC mutants with one of eight pairs of surface-accessible cysteines were expressed, labeled with bifunctional sulforhodamine, and exchanged into demembranated trabeculae to replace some of the native cRLC. Polarized fluorescence data from the probes in each lobe were combined with RLC crystal structures to calculate the lobe orientation distribution with respect to the filament axis. The orientation distribution of the N-lobe had three distinct peaks (N1–N3) at similar angles in relaxation, isometric contraction, and rigor. The orientation distribution of the C-lobe had four peaks (C1–C4) in relaxation and isometric contraction, but only two of these (C2 and C4) remained in rigor. The N3 and C4 orientations are close to those of the corresponding RLC lobes in myosin head fragments bound to isolated actin filaments in the absence of ATP (in rigor), but also close to those of the pair of heads folded back against the filament surface in isolated thick filaments in the so-called J-motif conformation. The N1 and C1 orientations are close to those expected for actin-bound myosin heads with their light chain domains in a pre-powerstroke conformation. The N2 and C3 orientations have not been observed previously. The results show that the average change in orientation of the RLC region of the myosin heads on activation of cardiac muscle is small; the RLC regions of most heads remain in the same conformation as in relaxation. This suggests that the orientation of the dephosphorylated RLC region of myosin heads in cardiac muscle is primarily determined by an interaction with the thick filament surface.     

Disassembly of Escherichia coli RecA E38K/��C17 Nucleoprotein Filaments Is Required to Complete DNA Strand Exchange

Disassembly of RecA protein subunits from a RecA filament has long been known to occur during DNA strand exchange, although its importance to this process has been controversial. An Escherichia coli RecA E38K/ΔC17 double mutant protein displays a unique and pH-dependent mutational separation of DNA pairing and extended DNA strand exchange. Single strand DNA-dependent ATP hydrolysis is catalyzed by this mutant protein nearly normally from pH 6 to 8.5. It will also form filaments on DNA and promote DNA pairing. However, below pH 7.3, ATP hydrolysis is completely uncoupled from extended DNA strand exchange. The products of extended DNA strand exchange do not form. At the lower pH values, disassembly of RecA E38K/ΔC17 filaments is strongly suppressed, even when homologous DNAs are paired and available for extended DNA strand exchange. Disassembly of RecA E38K/ΔC17 filaments improves at pH 8.5, whereas complete DNA strand exchange is also restored. Under these sets of conditions, a tight correlation between filament disassembly and completion of DNA strand exchange is observed. This correlation provides evidence that RecA filament disassembly plays a major role in, and may be required for, DNA strand exchange. A requirement for RecA filament disassembly in DNA strand exchange has a variety of ramifications for the current models linking ATP hydrolysis to DNA strand exchange.