Calcium hydroxide dressings using different preparation and application modes: density and dissolution by simulated tissue pressure.

AIM: To study the effect of different apical shapes in prepared simulated root canals on the application of a commercially prepared calcium hydroxide paste by a syringe or lentulo spiral. METHODOLOGY: Three different types of root canal preparation were performed in 90 simulated canals: group A to an apical size 20 and a 0.10 taper using hand and rotary instruments, group B to an apical size 30 and a 0.08 taper using GT rotary instruments and group C to an apical size 40 and a 0.04 taper using ProFile 0.04 instruments. The insertion of calcium hydroxide [Ca(OH)2] paste was accomplished using either a lentulo spiral or a syringe. After 1 week of simulated fluid pressure applied to the apical end of the canal using physiological saline solution, the solution was evaluated for released Ca(OH)2. The specimens were weighed initially, after preparation, after insertion of Ca(OH)2 paste, after temporization with Cavit and after 1 week of simulated fluid pressure. Digital radiographs of the filled canals were taken and canal areas in mm2, gray values of the Ca(OH)2 dressings, total area of voids in mm2, as well as location of voids in the apical, middle or coronal thirds of the root canals were measured. Analyses of variance, with Scheffe's post-hoc tests, as well as chi-square tests were performed. RESULTS: Canals in group C had significantly fewer (P < 0.01) radiographic voids than canals in groups A and B. Using a lentulo spiral resulted in significantly (P < 0.05) fewer voids compared with the injection technique. More voids were detected coronally compared with middle and apical root canal thirds (P < 0.05). CONCLUSIONS: Canal shape and method of application had an impact on the amount and radiodensity of calcium hydroxide dressings in simulated root canals. Canals prepared to an apical size 40 and a taper of 0.04 had the least number of voids; Ca(OH)2 was placed with significantly fewer voids using a lentulo spiral compared with the injection technique.

NCD activation of tubulin polymerization.

Tubulin dimer (tT) was purified from turkey erythrocytes. The motor domain of Drosophila non-claret disjunctional protein, NCD(335-700), was expressed in E. coli and purified. At 37 degrees C in the presence of GTP, the rate of polymerization of tT to microtubule (tMt) is accelerated over threefold by the presence of NCD(335-700). At 10 degrees C, the rate of tT polymerization is increased from zero, within experimental error, in the absence of NCD(335-700) to rates near those observed at 37 degrees C when NCD(335-700) is present. The NCD(335-700) concentration dependence of the rate indicated the reactive species was NCD(335-700)(n).tT, with n approximately 2. At 10 degrees C in the absence of GTP, polymerization does not occur, but tT activates NCD(335-700) MgATPase activity 10-fold. For the same conditions, using mians-NCD(335-700), which is modified with 2-(4'-maleimidylanilino) naphthalene-6-sulfonic acid, the apparent K(D) for binding to tT is 2.3 x 10(-5) M in the presence of MgADP. Replacing ADP with AMPPNP or ATP has a negligible effect on K(D). Mians-NCD(335-700) binding to tMt is 10-fold stronger than to tT. The above data indicate NCD(335-700) binds at a functional site on tT. The stoichiometry is consistent with the formation of NCD(335-700)(2).tT which in vitro accelerates self-assembly initiation and/or polymerization by binding a second tT in a position favorable for tubulin-tubulin interaction. The data suggest that in vivo functional NCD binding to microtubule involves one motor domain binding to alpha- and beta-subunits at the interface of two different tubulin dimers in a protofilament.

Myosin motor domain lever arm rotation is coupled to ATP hydrolysis.

We have investigated coupling of lever arm rotation to the ATP binding and hydrolysis steps for the myosin motor domain. In several current hypotheses of the mechanism of force production by muscle, the primary mechanical feature is the rotation of a lever arm that is a subdomain of the myosin motor domain. In these models, the lever arm rotates while the myosin motor domain is free, and then reverses the rotation to produce force while it is bound to actin. These mechanical steps are coupled to steps in the ATP hydrolysis cycle. Our hypothesis is that ATP hydrolysis induces lever arm rotation to produce a more compact motor domain that has stored mechanical energy. Our approach is to use transient electric birefringence techniques to measure changes in hydrodynamic size that result from lever arm rotation when various ligands are bound to isolated skeletal muscle myosin motor domain in solution. Results for ATP and CTP, which do support force production by muscle fibers, are compared to those of ATPgammaS and GTP, which do not. Measurements are also made of conformational changes when the motor domain is bound to NDP's and PP(i) in the absence and presence of the phosphate analogue orthovanadate, to determine the roles the nucleoside moieties of the nucleotides have on lever arm rotation. The results indicate that for the substrates investigated, rotation does not occur upon substrate binding, but is coupled to the NTP hydrolysis step. The data are consistent with a model in which only substrates that produce a motor domain-NDP-P(i) complex as the steady-state intermediate make the motor domain more compact, and only those substrates support force production.

CaATP as a substrate to investigate the myosin lever arm hypothesis of force generation.

In an effort to test the lever arm model of force generation, the effects of replacing magnesium with calcium as the ATP-chelated divalent cation were determined for several myosin and actomyosin reactions. The isometric force produced by glycerinated muscle fibers when CaATP is the substrate is 20% of the value obtained with MgATP. For myosin subfragment 1 (S1), the degree of lever arm rotation, determined using transient electric birefringence to measure rates of rotational Brownian motion in solution, is not significantly changed when calcium replaces magnesium in an S1-ADP-vanadate complex. Actin activates S1 CaATPase activity, although less than it does MgATPase activity. The increase in actin affinity when S1. CaADP. P(i) is converted to S1. CaADP is somewhat greater than it is for the magnesium case. The ionic strength dependence of actin binding indicates that the change in apparent electrostatic charge at the acto-S1 interface for the S1. CaADP. P(i) to S1. CaADP step is similar to the change when magnesium is bound. In general, CaATP is an inferior substrate compared to MgATP, but all the data are consistent with force production by a lever arm mechanism for both substrates. Possible reasons for the reduced magnitude of force when CaATP is the substrate are discussed.

Predicting conformational switches in proteins.

We describe a new computational technique to predict conformationally switching elements in proteins from their amino acid sequences. The method, called ASP (Ambivalent Structure Predictor), analyzes results from a secondary structure prediction algorithm to identify regions of conformational ambivalence. ASP identifies ambivalent regions in 16 test protein sequences for which function involves substantial backbone rearrangements. In the test set, all sites previously described as conformational switches are correctly predicted to be structurally ambivalent regions. No such regions are predicted in three negative control protein sequences. ASP may be useful as a guide for experimental studies on protein function and motion in the absence of detailed three-dimensional structural data.

Predicting allosteric switches in myosins.

The sequences of several members of the myosin family of molecular motors are evaluated using ASP (Ambivalent Structure Predictor), a new computational method. ASP predicts structurally ambivalent sequence elements by analyzing the output from a secondary structure prediction algorithm. These ambivalent sequence elements form secondary structures that are hypothesized to function as switches by undergoing conformational rearrangement. For chicken skeletal muscle myosin, 13 discrete structurally ambivalent sequence elements are identified. All 13 are located in the heavy chain motor domain. When these sequence elements are mapped into the myosin tertiary structure, they form two compact regions that connect the actin binding site to the adenosine 5'-triphosphate (ATP) site, and the ATP site to the fulcrum site for the force-producing bending of the motor domain. These regions, predicted by the new algorithm to undergo conformational rearrangements, include the published known and putative switches of the myosin motor domain, and they form plausible allosteric connections between the three main functional sites of myosin. The sequences of several other members of the myosin I and II families are also analyzed.

Reversible inactivation of myosin subfragment 1 activity by mechanical immobilization.

The Mg-ATPase activity of skeletal muscle myosin subfragment 1 (S1) is reversibly eliminated when it is aggregated by the force of osmotic pressure dehydration using polyethylene glycol (PEG). Several experiments indicate nucleotides bind aggregated S1, but the effects of binding are attenuated. Compared with S1 in solution, epsilonADP binds aggregated S1 with reduced affinity, and the bound epsilonADP fluorescence intensity is more effectively quenched by acrylamide. When ATP binds aggregated S1, the tryptophan intensity increases to only 50% of the solution level. Chemical cross-linking of cys-707 to cys-697 by p-phenylenedimaleimide is less efficient for aggregated S1 x MgADP. The data are consistent with aggregated S1 being able to bind nucleotide but not being able to complete the usual conformation change(s) in response to binding. If S1 is kept from aggregating by increasing the ionic strength at the same osmotic pressure, its Mg-ATPase activity and ATP-induced tryptophan fluorescence intensity increase are normal. The combined data are consistent with an ATP hydrolysis mechanism in which S1 segmental motion is coupled to its enzymatic activity. In this model, segmental motion is mechanically constrained by aggregation; the constrained S1 can bind ATP, but it cannot complete the hydrolysis mechanism.

Kinetic investigation of the ligand dependence of rabbit skeletal muscle myosin subfragment 1 Cys-697 and Cys-707 reactivities.

Rate constants for the reactions of Cys-697 and Cys-707 of skeletal muscle myosin subfragment 1 (S1) with N,N'-p-phenylenedimaleimide (pPDM) and its monofunctional analog phenylmaleimide (PM) were measured for S1 and S1 bound to nucleotides and/or actin. The [pPDM] and [PM] dependencies indicate that prereaction noncovalent complexes of S1 and the alkylating agents form. The rates of the pseudo-first-order reactions of the complexes depend on the nucleotide bound. For pPDM, only the rate constant ka (for Cys-707 modification) can be measured. The relative ka magnitudes are S1. MgATPgammaS > S1.MgADP > S1.MgPPi > S1.MgATP > actin.S1.MgADP > S1 > actin.S1 (for which ka approximately 0 s-1). For PM, only ka can be measured for S1.MgATPgammaS and S1.MgPPi. However, for S1, S1. MgADP, and S1.MgATP, ki (for the reaction of Cys-697) can also be measured, and it is also nucleotide sensitive. The data are consistent with a mechanism in which pPDM or PM binds S1 near Cys-707 to form a noncovalent complex that reacts at a rate determined by the relative orientation of the cysteine sulfhydryl and the bound reagent. The simplest mechanism for the cross-linking step that reconciles these data with earlier cross-linker length data and with S1-nucleotide atomic structures is one which has pPDM-S1 complexes exist part of the time in conformations having the helical Cys-697/Cys-707-pPDM region converted to a loop structure which cross-links. The fact that rigor actin.S1 is the slowest and the S1.MgATP analog S1.MgATPgammaS is the fastest to be cross-linked is discussed in terms of possible energetic roles for helix to loop transitions of the Cys-697/Cys-707 region during the ATP hydrolysis cycle.

Light chain-dependent myosin structural dynamics in solution investigated by transient electrical birefringence.

The technique of transient electrical birefringence was used to compare some of the electric and structural dynamic properties of myosin subfragment 1 (S1(elc, rlc)), which has both the essential and regulatory light chains bound, to S1(elc), which has only an essential light chain. The rates of rotational Brownian motion indicate that S1(elc, rlc) is larger, as expected. The permanent electric dipole moment of S1(elc, rlc) is also larger, indicating that the regulatory light chain portion of S1(elc, rlc) has a dipole moment and that it is aligned head-to-tail with the dipole moment of the S1(elc) portion. The permanent electric dipoles decrease with increasing ionic strength, apparently because of ion binding to surface charges. Both S1(elc, rlc) and S1(elc) have intrinsic segmental flexibility, as detected by the ability to selectively align segments with a brief weak electric field. However, unlike S1(elc), which can be structurally distorted by the action of a brief strong electric field, S1(elc, rlc) is stiffer and cannot be distorted by fields as high as 7800 V/cm applied to its approximately 8000 D permanent electric dipole moment. The S1 . MgADP . Pi analog S1 . MgADP . Vi is smaller than S1 . MgADP, for both S1(elc, rlc) and S1(elc). Interestingly, the smaller, stiffer S1(elc, rlc) . MgADP . Vi complex retains intrinsic segmental flexibility. These results are discussed within a framework of current hypotheses of force-producing mechanisms that involve S1 segmental motion and/or the loss of cross-bridge flexibility during force production.

Skeletal muscle myosin subfragment 1 dimers.

The rate of translational diffusion of skeletal muscle myosin subfragment 1 (S1) was determined from polarized dynamic light scattering autocorrelation measurements. Diffusion rates were expressed in terms of the hydrodynamic radii Rh. At 20 degrees C, in low ionic strength pH 8 solutions, Rh increased from 4.3 nm to 5.7 nm as [S1] was increased from 1.6 to 72 microM. Including MgATP to maintain S1. MgADP. Pi gave equivalent results. When the light scattering data were analyzed, assuming a monomer-dimer equilibrium, a dissociation constant of 83 microM was obtained. Steady state MgATPase activity measurements were made as a function of [ATP] for S1 in the 0.4-7 microM range, and analyzed assuming Michaelis-Menten kinetics. VMAX did not change, but KM increased about tenfold as [S1] was increased over this range. The light scattering and kinetic data were consistent with S1 aggregation at high [S1].

Myosin regulatory light chain and nucleotide modulation of actin binding site electric charge.

The ionic strength dependence of skeletal muscle myosin subfragment 1 (S1) binding to actin in the presence of ADP and ATP was measured for S1 with either only an essential light chain [S1(elc)] or with both an essential and the regulatory light chains [S1(elc,rlc)] bound. The data were analyzed to determine the apparent association constant, K(A), for actin binding and the absolute value of the product of the net effective electric charges at the actin-myosin interface, /ZMZA/. When MgADP is bound at the myosin active site, K(A) values at 0 M ionic strength for S1(elc) and S1(elc,rlc) are 12 and 4.9 x 10(6) M(-1), respectively, and /ZMZA/ values are 3.9 +/- 0.3 and 3.6 +/- 0.2 esu2. In the presence of ATP, K(A) values at 0 M ionic strength for S1(elc) and S1(elc,rlc) are 81 and 7.3 x 10(4) M(-1), respectively, and /ZMZA/ values are 14.7 esu2 for S1(elc) but only 6.4 esu2 for S1(elc,rlc). The Michaelis constant, K(M), for the actin activation of S1 steady-state MgATPase activity was significantly smaller for S1(elc), consistent with its greater K(A) and /ZMZA/. These data indicate that the regulatory light chain can allosterically regulate the interactions of myosin and actin by modulating the electric charge at the actin binding site. K(A) and /ZMZA/ were also measured at 25 degrees C for S1(elc,rlc) binding to actin in the presence of the ATP analog ATPgammaS. At 0 M ionic strength, K(A) is 8.0 x 10(4) M(-1), and /ZMZA/ is 0, within experimental uncertainty, suggesting that for S1 x MgATP the electric charge at the actin binding site is abolished. The results are interpreted in terms of possible roles of electrostatic interactions in mechanisms for S1 x MgATP dissociating from one actin and S1 x MgADP x Pi being guided electrostatically to bind to another.

Osmotic pressure probe of actin-myosin hydration changes during ATP hydrolysis.

Osmotic stress in the 0.5-5 x 10(6) dyne/cm2 range was used to perturb the hydration of actin-myosin-ATP intermediates during steady-state hydrolysis. Polyethylene glycol (PEG) (1000 to 4000 Da), in the 1 to 10 wt% range, which does not cause protein precipitation, did not significantly affect the apparent KM or the Vmax for MgATP hydrolysis by myosin subfragment 1 (S1) alone, nor did it affect the value for the phosphate burst. Consistent with the kinetic data, osmotic stress did not affect nucleotide-induced changes in the fluorescence intensities of S1 tryptophans or of fluorescein attached to Cys-707. The accessibility of the fluorescent ATP analog, epsilon ADP, to acrylamide quenching was also unchanged. These data suggest that none of the steps in the ATP hydrolysis cycle involve substantial hydration changes, which might occur for the opening or closing of the ATP site or of other crevices in the S1 structure. In contrast, KM for the interaction of S1.MgADP.Pi with actin decreased tenfold in this range of osmotic pressure, suggesting that formation of actin.S1.MgADP.Pi involves net dehydration of the proteins. The dehydration volume increases as the size of the PEG is increased, as expected for a surface-excluded osmolyte. The measured dehydration volume for the formation of actin.S1.MgADP.Pi was used to estimate the surface area of the binding interface. This estimate was consistent with the area determined from the atomic structures of actin and myosin, indicating that osmotic stress is a reliable probe of actin.myosin.ATP interactions. The approach developed here should be useful for determining osmotic stress and excluded volume effects in situ, which are much larger than those of typical in vitro conditions.

The ATP-induced myosin subfragment-1 fluorescence intensity increase is due to one tryptophan.

The irradiation of skeletal muscle myosin subfragment 1 (S1) in the presence of 2,2,2-trichloroethanol (TCE) reduces S1 fluorescence intensity in two phases. In the first phase, there is an increase in MgATPase activity, and no significant change in the fluorescence intensity increase upon ATP binding. In the second phase, the activity remains elevated, but there is a complete loss of the ATP-induced intensity increase. Measurements on denatured S1 indicate that fluorescence intensity reductions of one fifth of the total occur during each of the two phases, consistent with the fluorescence intensity increase upon forming S1.MgADP.P(i) being due to one of the five heavy-chain tryptophans.

Cross-linking myosin subfragment 1 Cys-697 and Cys-707 modifies ATP and actin binding site interactions.

Skeletal muscle myosin is an enzyme that interacts allosterically with MgATP and actin to transduce the chemical energy from ATP hydrolysis into work. By modifying myosin structure, one can change this allosteric interaction and gain insight into its mechanism. Chemical cross-linking with N,N'-p-phenylenedimaleimide (pPDM) of Cys-697 to Cys-707 of the myosin-ADP complex eliminates activity and produces a species that resembles myosin with ATP bound (Burke et al., 1976). Nucleotide-free pPDM-modified myosin subfragment 1 (S1) was prepared, and its structural and allosteric properties were investigated by comparing the nucleotide and actin interactions of S1 to those of pPDM-S1. The structural properties of the nucleotide-free pPDM-S1 are different from those of S1 in several respects. pPDM-S1 intrinsic tryptophan fluorescence intensity is reduced 28%, indicating a large increase of an internal quenching reaction (the fluorescence intensity of the related vanadate complex of S1, S1-MgADP-Vi, is reduced by a similar degree). Tryptophan fluorescence anisotropy increases from 0.168 for S1 to 0.192 for pPDM-S1, indicating that the unquenched tryptophan population in pPDM-S1 has reduced local freedom of motion. The actin affinity of pPDM-S1 is over 6,000-fold lower than that of S1, and the absolute value of the product of the net effective electric charges at the acto-S1 interface is reduced from 8.1 esu2 for S1 to 1.6 esu2 for pPDM-S1. In spite of these changes, the structural response of pPDM-S1 to nucleotide and the allosteric communication between its ATP and actin sites remain intact. Compared to pPDM-S1, the fluorescence intensity of pPDM-S1 *MgADP is increased 50%(compared to 8 and 31% increases, respectively, for MgADP and MgATP binding to S1). Compared to acto-pPDM-S1, the absolute value of the product of the net effective electric charge at the actin binding interface of acto-pPDM-S1 *MgADP increases 7.3 esu2 (compared to a 0.9 esu2 decrease and an 11.0 esu2 increase, respectively, for MgADP and MgATP binding to acto-Sl).The interaction free energy for the ligands MgADP and actin, is -2.0 kcal/mol for pPDM-S1, compared to -1.2 kcal/mol for unmodified S1.

[2-3H]ATP synthesis and 3H NMR spectroscopy of enzyme-nucleotide complexes: ADP and ADP.Vi bound to myosin subfragment 1.

The synthesis of [2-3H]ATP with specific activity high enough to use for 3H NMR spectroscopy at micromolar concentrations was accomplished by tritiodehalogenation of 2-Br-ATP. ATP with greater than 80% substitution at the 2-position and negligible tritium levels at other positions had a single 3H NMR peak at 8.20 ppm in 1D spectra obtained at 533 MHz. This result enables the application of tritium NMR spectroscopy to ATP utilizing enzymes. The proteolytic fragment of skeletal muscle myosin, called S1, consists of a heavy chain (95 kDa) and one alkali light chain (16 or 21 kDa) complex that retains myosin ATPase activity. In the presence of Mg2+, S1 converts [2-3H]ATP to [2-3H]ADP and the complex S1.Mg[2-3H]ADP has ADP bound in the active site. At 0 degrees C, 1D 3H NMR spectra of S1.Mg[2-3H]ADP have two broadened peaks shifted 0.55 and 0.90 ppm upfield from the peak due to free [2-3H]ADP. Spectra with good signal-to-noise for 0.10 mM S1.Mg[2-3H]ADP were obtained in 180 min. The magnitude of the chemical shift caused by binding is consistent with the presence of an aromatic side chain being in the active site. Spectra were the same for S1 with either of the alkali light chains present, suggesting that the alkali light chains do not interact differently with the active site. The two broad peaks appear to be due to the two conformations of S1 that have been observed previously by other techniques. Raising the temperature to 20 degrees C causes small changes in the chemical shifts, narrows the peak widths from 150 to 80 Hz, and increases the relative area under the more upfield peak. Addition of orthovanadate (Vi) to produce S1.Mg[2-2H]ADP.Vi shifts both peaks slightly more upfield without changing their widths or relative areas.

Myosin-ATP chemomechanics.

The hydrodynamic size of rabbit skeletal muscle myosin subfragment 1 (S1) is decreased when S1 and MgATP form the steady-state intermediate S1-MgADP,P(i). The rotational decay time, tau, determined by transient electrical birefringence techniques was 259 ns for S1-MgADP,P(i) and 271 ns for S1-MgADP at 3 degrees C in low ionic strength solutions. The data were interpreted using a hydrodynamic model consisting of a rigid linear four-bead structure that had a point at the center of one of the inner beads about which the structure can bend. The structure of S1-MgADP was approximated by adjusting the bend angle to 20 degrees. The best fit to the S1-MgADP,P(i) decay time was then obtained when the angle was increased to 38 degrees. The results obtained using this simple model suggest that MgATP binding and hydrolysis changes the structure of S1 so that one end of it moves by at least 3.9 nm. The reverse of this process, during product release, would provide a displacement large enough to account for most of the ATP-driven filament sliding that occurs in muscle or in in vitro motility assays.

Nucleotide- and temperature-induced changes in myosin subfragment-1 structure.

The effects of nucleotide binding and temperature on the internal structural dynamics of myosin subfragment 1 (S1) were monitored by intrinsic tryptophan phosphorescence lifetime and fluorescence anisotropy measurements. Changes in the global conformation of S1 were monitored by measuring its rate of rotational diffusion using transient electric birefringence techniques. At 5 degrees C, the binding of MgADP, MgADP,P and MgADP,V (vanadate) progressively reduce the rotational freedom of S1 tryptophans, producing what appear to be increasingly more rigidified S1-nucleotide structures. The changes in the luminescence properties of the tryptophans suggest that at least one is located at the interface of two S1 subdomains. Increasing the temperature from 0 to 25 degrees C increases the apparent internal mobility of S1 tryptophans in all cases and, in addition, a reversible temperature-dependent transition centered near 15 degrees C was observed for S1, S1-MgADP and S1-MgADP,P, but not for S1-MgADP,V. The rotational diffusion constants of S1 and S1-MgADP were measured at temperatures between 0 and 25 degrees C. After adjusting for the temperature and viscosity of the solvent, the data indicate that the thermally induced transition at 15 degrees C comprises local conformational changes, but no global conformational change. Structural features of S1-MgADP,P, which may relate to its role in force generation while bound to actin, are presented.