Interaction of a spin-labeled analog of ATP with myosin

By means of spin labeled analogs of ATP we have shown that conformational changes in myosin molecule induced by variation of temperature take place in the region of the active centre. In case of Mg-ATP and unmodified myosin conformation of the active centre changes monotonously with the change in temperature but after the modification of S1 thiol groups by N-ethylmaleimide on the temperature dependence curve of rotational mobility of the spin label a discontinuous is observed at 14-16 degrees C. It is also observed in case of K+-EDTA-ATP, or Ca2+-ATP and unmodified myosin. It is shown that the chemical analogs of Mg2+-paramagnetic ion Mn2+ are directly connected with the myosin active centre in the presence of ATP(ADP), i. e. a triple complex enzyme-bivalent cation-substrate is formed.     

Interaction of spin-labeled methacyne analog with butyrylcholinesterase

Interaction between spin-labeled methacyne (I) and butyrylcholinesterase (BChE) was studied by ESR and enzyme kinetic methods. The compound (I) was shown to be a competitive reversible inhibitor, the value of Ki appeared to be 1.3 X 10(-5) M. Insertion of nitroxyl fragment in the methacyne molecule results in a two-fold increase of its inhibitory activity. The ESR spectrum of the enzyme-inhibitor complex was registered. This complex dissociates under the action of eserine, tetramethylammonium and hexamethonium. Scatchard plot reveals two different types of binding sites with Kdiss values 1.5 X 10(-5) M and 2.6 X 10(-4) M. One type of binding sites is identified as the enzyme active centre. The restricted motion of (I) in complex with BChE proves the assumption that the enzyme active centre is located in the split of macromolecule surface.     

Interaction of spin-labeled nucleotides with sarcoplasmic reticulum adenosinetriphosphatase

Spin-labeled derivatives of AMP-PCP, ATP, and 2'-deoxy-ATP, with a nitroxide moiety attached to the ribose ring [3'-O-(1-oxy-2,2,5,5-tetramethylpyrroline-3-carbonyl)nucleotide], are used to study the nucleotide binding site stoichiometry of sarcoplasmic reticulum (SR) ATPase. With all derivatives, a maximal binding of 4.5 nmol/mg of SR protein is found, a value close to the number of phosphorylation sites obtained with ATP. The spin-labeled nucleotides cannot be utilized by the enzyme as substrates. Binding of spin-labeled nucleotides is inhibited by labeling the ATPase with fluorescein 5'-isothiocyanate, indicating that all the labeled nucleotide is located at the catalytic site. Additions of spin-labeled ATP to vesicle suspensions during steady turnover demonstrate competitive inhibition of both catalysis and the regulatory effect normally exhibited by ATP. As secondary binding of spin-labeled ATP is not detected at pertinent concentrations, it is suggested that both functions of ATP may be effected through a single site.     

Interaction of C-protein with myosin

The effect of C-protein on the assembly reaction of myosin was studied by flow birefringence, electron microscopy, and ultracentrifugation. Myosin filaments were formed by dilution to a lower ionic strength. Thinner filaments of 70-110 A in diameter were formed in the presence of C-protein. When dilution was effected by moderately slow dilution (dilution time of 0.5-2 min) or by stepwise dilution, C-protein favored the formation of longer filaments. When dilution was effected by even slower dilution (dilution time above 2 min), C-protein favored the formation of shorter filaments. Longer filaments formed by slow dilution incorporated more C-protein than shorter ones formed by faster dilution. Addition of C-protein to a solution of myosin filaments caused association of the filaments into longer filaments. The elongation effect was slower and stronger for longer filaments.     

Interactions of microsomal cytochrome P-450 with spin-labeled substrate analogs

The iodine-containing stable iminoxyl radicals with various distances between the N-O-group and the iodine atom are proposed to be used to study the structure of the active center of the microsomal cytochrome P-450. The radicals used induce changes in the optical spectra of the Fe3+ ion located in the active center of the enzyme, as in the case of type 1 substrates and inhibit essentially the microsomal oxidation of cytochrome P-450 substrates of type 1 and 2. This inhibition is neither due to suppression of the NADPH-cytochrome c reductase activity nor to cytochrome P-450 conversion to cytochrome P-420. Cytochrome P-450 substrates (aminopyrine) protect the enzyme against the radical-induced inactivation. The iodine-containing radicals are covalently bound to cytochrome P-450 in the vicinity of active center. The values of dissociation constants for the reversible enzyme-radical constants and the rate constants for the monomolecular transformation in the complex, k, were determined. The EPR method was used to detect the coupling between Fe3+ and the radical located in the active center of cytochrome P-450. The saturation curves of radical SPR spectra at 77 degrees K were employed to determine the contribution of Fe3+ to the relaxation time, T1, of the radicals covalently bound to cytochrome P-450 and to estimate the distances between the Fe3+ ion and the N-O-group of these radicals in the enzyme active center.     

Interaction of bacterial flagella with myosin

The interaction of isolated flagellar filaments of Bacillus brevis var. G.-B. P+ with skeletal muscle myosin has been investigated. Bacterial flagellar filaments co-precipitate with myosin at low ionic strength (at the conditions of myosin aggregation). Addition of bacterial flagellar filaments to myosin led to inhibition of its K+-EDTA- and Ca2+-ATPase activity, but had no influence on Mg2+-ATPase. Monomeric protein of bacterial flagella filaments (flagellin) did not co-precipitate with myosin and had no influence on its ATPase activity. The flagella filaments did not co-precipitate with myosin in the presence of F-actin if it was mixed with myosin before the filaments. If the flagella filaments were added to myosin solution before the addition of F-actin the amount of filaments and actin in myosin precipitate were comparable. In this case the presence of flagella filaments decreased activation of myosin Mg2+-ATPase by actin to 25-30%. Thus the bacterial flagellar filaments are able to interact with myosin and modify its ATPase activity. Probably, these properties of filaments are caused by resemblance of flagellin and actin. For instance, the unique origin of these proteins may be the reason of such resemblance.     

Orientation of spin-labeled myosin heads in glycerinated muscle fibers.

We have used electron paramagnetic resonance (EPR) spectra to study spin labels selectively and rigidly attached to myosin heads in glycerinated rabbit psoas muscle fibers. Because the angle between the magnetic field and the principal axis of the probe determines the position of the EPR absorption line, spectra from labeled fibers oriented parallel to the magnetic field yielded directly the distribution of spin label orientations relative to the fiber axis. Two spin labels, having reactivities resembling iodoacetamide (IASL) and maleimide (MSL), were used. In rigor fibers with complete filament overlap, both labels displayed a narrow angular distribution, full width at half maximum approximately 15 degrees, centered at angles of 68 degrees (IASL) and 82 degrees (MSL). Myosin subfragments (heavy meromyosin and subfragment-1) were labeled and allowed to diffuse into fibers. The resulting spectra showed the same sharp angular distribution that was found for the labeled fibers. Thus is appears that virtually all myosin heads in a rigor fiber have the same orientation relative to the fiber axis, and this orientation is determined by the actomyosin bond. Experiments with stretched fibers indicated that the spin labels on the fraction of heads not interacting with actin filaments had a broad angular distribution. Addition of ATP to unstretched fibers under relaxing conditions produced orientational disorder, resulting in a spectrum almost indistinguishable from that of an isotropic distribution of probes. Addition of either an ATP analog (AMPPNP) or pyrophosphate produced partial disorder. That is a fraction of the probes remained sharply oriented as in rigor while a second fraction was in a disordered distribution similar to that of relaxed fibers.     

Submillisecond rotational dynamics of spin-labeled myosin heads in myofibrils.

The rotational motion of crossbridges, formed when myosin heads bind to actin, is an essential element of most molecular models of muscle contraction. To obtain direct information about this molecular motion, we have performed saturation transfer EPR experiments in which spin labels were selectively and rigidly attached to myosin heads in purified myosin and in glycerinated myofibrils. In synthetic myosin filaments, in the absence of actin, the spectra indicated rapid rotational motion of heads characterized by an effective correlation time of 10 microseconds. By contrast, little or no submillisecond rotational motion was observed when isolated myosin heads (subfragment-1) were attached to glass beads or to F-actin, indicating that the bond between the myosin head and actin is quite rigid on this time scale. A similar immobilization of heads was observed in spin-labeled myofibrils in rigor. Therefore, we conclude that virtually all of the myosin heads in a rigor myofibril are immobilized, apparently owing to attachment of heads to actin. Addition of ATP to myofibrils, either in the presence or absence of 0.1 mM Ca2+, produced spectra similar to those observed for myosin filaments in the absence of actin, indicating rapid submillisecond rotational motion. These results indicate that either (a) most of the myosin heads are detached at any instant in relaxed or activated myofibrils or (b) attached heads bearing the products of ATP hydrolysis rotate as rapidly as detached heads.     

Interaction of nonpolymerizable actins with myosin.

Polymerization of G-actin in the presence of salt and phalloidin was blocked by treatment of G-actin with m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) (designated as m-actin). The actin dimer produced by chemical crosslinking of F-actin with N,N'-p-phenylenedimaleimide did not polymerize and was still dimeric or tetrameric after further treatment with MBS (designated as d-actin). The m- and d-actins retained the ability to bind to myosin heads with apparent dissociation constants of 3-8 x 10(-6) and 3-5 x 10(-7) M, respectively. d-Actin formed a 1:1 actin monomer-myosin head complex. However, m-actin formed a 2:1 m-actin-head complex, suggesting the presence of at least two latent actin-binding sites on a myosin head. ATP weakens only 2- to 6-fold the binding of these complexes. One of two m-actins on a myosin head was replaced by d-actin. Native F-actin blocked the binding of both m- and d-actins to myosin heads in the presence and absence of ATP, although the affinities of myosin head for MBS-treated actins and F-actin are similar in the presence of ATP. These results suggest that there are at least three actin binding sites on a myosin head: one is responsible for binding of F-, m-, and d-actins, the second for binding of F- and m-actins, and the third for binding of F-actin at least in the presence of ATP. F-Actin binding to the third site may in some way block the first and second binding sites.     

Interaction of myosin subfragments with F-actin.

The effect of ionic strength, temperature, and divalent cations on the association of myosin with actin was determined in the ultracentrifuge using scanning absorption optics. The association constant (Ka) for the binding of heavy meromyosin (HmM) to F-actin was 1 X 10(7) M-1 at 20 degrees C, in 0.10 M KCl, 0.01 M imidazole (pH 7.0), 5 MM potassium phosphate, 1 mM MgCl2, and 0.3 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. Ka was the same for HMM prepared by trypsin or chymotrypsin. The affinity of subfragment 1 (S1) for actin under the same ionic conditions was 3 X 10(6) M-1. Varying the preparative procedure for S1 had little effect on Ka. The small difference in binding energy between HMM and S1 suggests that either only one head can bind strongly to actin at a time or that free energy is lost during the sterically unfavorable attachment of the two heads to actin.     

Resolution of conformational states of spin-labeled myosin during steady-state ATP hydrolysis

We have measured the conventional electron paramagnetic resonance (EPR) spectrum of spin-labeled myosin filaments as a function of the nucleotide occupancy of the active site of the enzyme. The probe used was 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidine-1-oxyl (IASL), which reacts specifically with sulfhydryl 1 of the myosin head. In the absence of nucleotide, the probe remains strongly immobilized (rigidly attached to the myosin head) so that no nanosecond rotational motions are detectable. When MgADP is added to IASL-labeled myosin filaments (T = 20 degrees C), the probe mobility increases slightly. During steady-state MgADP hydrolysis (T = 20 degrees C), the probe undergoes large-amplitude nanosecond rotational motion. These results are consistent with previous studies of myosin monomers, heavy meromyosin, and myosin subfragment 1. Isoclinic points observed in overlays of sequential EPR spectra recorded during ATP hydrolysis strongly suggest that the probes fall into two motional classes, separated by approximately an order of magnitude in effective rotational correlation time. Both of the observed states are distinct from the conformation of myosin in the absence of nucleotides, and the spectrum of the less mobile population is indistinguishable from that observed in the presence of MgADP. The addition of ADP and vanadate to IASL-myosin gives rise to two motional classes virtually identical with those observed in the presence of ATP, but the relative concentrations of the spin populations are significantly different. We have quantitated the percentage of myosin in each motional state during ATP hydrolysis. The result agrees well with the predicted percentages in the two predominant chemical states in the myosin ATPase cycle. Spectra obtained in the presence of nucleotide analogues permit us to assign the conformational states to specific chemical states. We propose that the two motional classes represent two distinct local conformations of myosin that are in exchange with one another during the ATP hydrolysis reaction cycle.     

Dynamics of the nucleotide pocket of myosin measured by spin-labeled nucleotides

We have used electron paramagnetic probes attached to the ribose of ATP (SL-ATP) to monitor conformational changes in the nucleotide pocket of myosin. Spectra for analogs bound to myosin in the absence of actin showed a high degree of immobilization, indicating a closed nucleotide pocket. In the Actin.Myosin.SL-AMPPNP, Actin.Myosin.SL-ADP.BeF(3), and Actin.Myosin.SL-ADP.AlF(4) complexes, which mimic weakly binding states near the beginning of the power stroke, the nucleotide pocket remained closed. The spectra of the strongly bound Actin.Myosin.SL-ADP complex consisted of two components, one similar to the closed pocket and one with increased probe mobility, indicating a more open pocket, The temperature dependence of the spectra showed that the two conformations of the nucleotide pocket were in equilibrium, with the open conformation more favorable at higher temperatures. These results, which show that opening of the pocket occurs only in the strongly bound states, appear reasonable, as this would tend to keep ADP bound until the end of the power stroke. This conclusion also suggests that force is initially generated by a myosin with a closed nucleotide pocket.     

Interaction of crotoxin and its isolated subunits with spin-labeled fatty acids

We have investigated the interaction of crotoxin (component A-component B complex) and of its isolated phospholipase subunit (component B) with hydrophobic compounds by ESR, using spin-labeled fatty acids as probes. The phospholipase subunit alone (component B) binds more than three labeled fatty acid molecules/molecule with different affinities, the highest corresponding to a Kd of 10 microM in the case of 5-doxyl palmitic acid. In contrast, the noncatalytic subunit (component A) and the crotoxin complex do not bind fatty acids. ESR studies of the component B-fatty acid complex reveal a strong immobilization of the whole length of the fatty acid chain, strong spin-spin interactions between bound fatty acids, and nonaccessibility of the bound paramagnetic probe to Ni2+ ions. This suggests that the phospholipase component B possesses a hydrophobic cleft which may contain one or two fatty acids. This hydrophobic cleft is not accessible to spin-labeled fatty acids in the crotoxin complex. An overall rotational correlation time of about 200 ns of the phospholipase component B was determined by saturation transfer ESR. This high value is incompatible with the diffusion of a polypeptide of 14,500 molecular weight. The hydrodynamic analysis of the fatty acid-component B complex led us to estimate an apparent molecular weight of 95,000 which reveals that fatty acids induce the formation of polymers (most probably octamers) of component B. We propose a model in which the phospholipase component B exists in two conformational states which differ by their hydrophobicity.     

Chicken-gizzard actin. Interaction with skeletal-muscle myosin

Interaction of actin from chicken gizzard and from rabbit skeletal muscle with rabbit skeletal muscle myosin was compared by measuring the rate of superprecipitation, the activation of the Mg-ATPase and inhibition of K-ATPase activity of myosin and heavy meromyosin, and determination of binding of heavy meromyosin in the absence of ATP. Both the rate of superprecipitation of the hybrid actomyosin and the activation of myosin ATPase by gizzard actin are lower than those obtained with skeletal muscle actin. The activation of myosin Mg-ATPase by the two actin species also shows different dependence on substrate concentration: with gizzard actin the substrate inhibition starts at lower ATP concentration. The double-reciprocal plots of the Mg-ATPase activity of heavy meromyosin versus actin concentration yield the same value of the extrapolated ATPase activity at infinite actin concentration (V) for the two actins and nearly double the actin concentration needed to produce half-maximal activation (Kapp) in the case of gizzard actin. A corresponding difference in the abilities of the two actin species to inhibit the K-ATPase activity of heavy meromyosin in the absence of divalent cations was also observed. The results are discussed in terms of the effect of substitutions in the amino acid sequence of gizzard and skeletal muscle actins on their interaction with myosin.     

Orientation of spin-labeled light chain 2 of myosin heads in muscle fibers

Electron paramagnetic resonance (e.p.r.) spectroscopy has been used to monitor the orientation of spin labels attached rigidly to a reactive SH residue on the light chain 2 (LC2) of myosin heads in muscle fibers. e.p.r. spectra from spin-labeled myosin subfragment-1 (S1), allowed to diffuse into unlabeled rigor (ATP-free) fibers, were roughly approximated by a narrow angular distribution of spin labels centered at 66 degrees relative to the fiber axis, indicating a uniform orientation of S1 bound to actin. On the other hand, spectra from spin-labeled heavy meromyosin (HMM) were roughly approximated by two narrow angular distributions centered at 42 degrees and 66 degrees, suggesting that the LC2 domains of the two HMM heads have different orientations. In contrast to S1 or HMM, the spectra from rigor fibers, in which LC2 of endogenous myosin heads was labeled, showed a random orientation which may be due to distortion imposed by the structure of the filament lattice and the mismatch of the helical periodicities of the thick and thin filaments. However, spectra from the fibers in the presence of ATP analog 5'-adenylyl imidodiphosphate (AMPPNP) were approximated by two narrow angular distributions similar to those obtained with HMM. Thus, AMPPNP may cause the LC2 domain to be less flexible and/or the S2 portion to be more flexible, so as to release the distortion of the LC2 domain and make it return to its natural position. At high ionic strength, AMPPNP disoriented the spin labels as ATP did under relaxing conditions, suggesting that the myosin head is detached from and/or weakly (flexibly) attached to a thin filament.     

A spin-labeled abasic DNA substrate for AP endonuclease.

We report the first observation of a spin-labeled ds 23-mer oligonucleotide by high-field electron spin resonance (ESR) and demonstrate that it interacts with AP endonuclease, the key enzyme in DNA abasic site repair. The spin labeled 23-mer with a U at position 12 of the upper strand is processed by uracil DNA glycosylase to provide the abasic substrate. With a spin-label two nucleotides away from the abasic site, AP endo binds and cleaves when the label is 3' but not 5' to the abasic site. These results confirm that the disposition of the bases immediately upstream of the abasic site is particularly critical for cleavage by AP endo, and establish that DNA-protein interactions in this important enzyme can be examined using spin-labeled substrates.     

Interaction of tryptophanase with substrate analogs

Tryptophanase from Escherichia coli was studied with respect to its interactions with L-alanine, beta-chloro-L-alanine, L-phenylalanine, L-methionine, L-threonine, beta-phenyl-DL-serine (threo form) and also with a new tryptophan analog oxindolyl-L-alanine. Slow transamination of L-alanine in the active site of the enzyme was observed. Some evidence is presented which indicates that the side transamination reaction occurs during incubation of tryptophanase with an adequate substrate, beta-chloro-L-alanine. Absorption and circular dichroism (CD) spectra of the enzyme-quasisubstrate complexes have been recorded. Addition of beta-phenylserine and threonine to the enzyme induces a decrease of absorbance at 337 nm and an increase of absorbance at 420 nm. The spectral changes are associated with inversion of the CD sign, i.e. with disappearance of positive CD in the 420 nm band and appearance of negative CD in this band. It is inferred that beta-phenylserine and threonine form an external coenzyme-substrate aldimine which undergoes slow conversion to give a keto acid and the free enzyme. Addition of oxindolylalanine to tryptophanase results in the formation of an intense narrow absorption band at 504 nm with a shoulder at about 475 nm. This band belongs to a quinonoid intermediate. A positive CD is seen in the 504 nm band; the dissymmetry factor (delta A/A) in this band is much smaller than that in the absorption bands of the free enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)