The effect of the replacement of ATP with ADP on the conformational and dynamic properties of the actin monomer was investigated, by means of electron paramagnetic resonance (EPR) and fluorescence spectroscopic methods. The measurement of the ATP concentration during these experiments provided the opportunity to estimate the time dependence of ADP-Mg-G-actin concentration in the samples. According to the results of the fluorescence resonance energy transfer experiments, the Gln-41 and Cys-374 residues are closer to each other in the ADP-Mg-G-actin than in the ATP-Mg-G-actin. The fluorescence resonance energy transfer efficiency increased simultaneously with the ADP-G-actin concentration and reached its maximum value within 30 min at 20 degrees C. The EPR data indicate the presence of an ADP-Mg-G-actin population that can be characterized by an increased rotational correlation time, which is similar to the one observed in actin filaments, and exists only transiently. We suggest that the conformational transitions, which were reflected by our EPR data, were coupled with the transient appearance of short actin oligomers during the nucleotide exchange. Besides these relatively fast conformational changes, there is a slower conformational transition that could be detected several hours after the initiation of the nucleotide exchange.
Conformational and dynamic properties of actin filaments polymerized from ATP-or ADP-actin monomers were compared by using fluorescence spectroscopic methods. The fluorescence intensity of IAEDANS attached to the Cys 374 residue of actin was smaller in filaments from ADP-actin than in filaments from ATPactin monomers, which reflected a nucleotide-induced conformational difference in subdomain 1 of the monomer. Radial coordinate calculations revealed that this conformational difference did not modify the distance of Cys 374 from the longitudinal filament axis. Temperaturedependent fluorescence resonance energy transfer measurements between donor and acceptor molecules on Cys 374 of neighboring actin protomers revealed that the inter-monomer flexibility of filaments assembled from ADP-actin monomers were substantially greater than the one of filaments from ATP-actin monomers. Flexibility was reduced by phalloidin in both types of filaments.Actin is one of the most abundant proteins in biological systems and responsible for a number of cell functions in vivo (1, 2). Two principal forms of actin exist in living cells, the monomeric and the filamentous. Actins biological function depends on the actual dynamic and conformational properties of the protein and on the dynamic equilibrium between the two principal forms (1, 2).The polymerization of actin monomers with bound ATP is accompanied by the parallel hydrolysis of the nucleotide. However, the biological relevance of ATP hydrolysis by actin is still not well understood. Interestingly, ADP-monomeric actin also forms filaments (3), therefore, the presence of ATP and its hydrolysis are not essential for filament assembly. Previously, the hydrolysis of actin-bound ATP was assumed to play a key role in the steady-state treadmilling of actin filaments (4). Alternatively, Carlier (5, 6) suggested that ATP hydrolysis facilitated the rapid de-polymerization of actin.Recently, Janmey and colleagues (7) provided evidence that actin filaments polymerized from ATP-actin monomers were significantly stiffer than the ones obtained from ADP-monomers. Considering that both types of filaments consist mainly of ADP-actin protomers, this observation was explained by the assumption that the ATP-actin monomers were conformationally trapped following the hydrolysis of ATP, and the energy released during the hydrolysis was stored as elastic energy (7). The authors proposed that this elastic energy could play an important role when the actin filament interacted with actinbinding proteins. Although this exciting observation was later supported by other laboratories, a number of experimental results were contradictory.The direct effect of ATP on actin filaments was confirmed and extended to phalloidin-labeled actin by fluorescence microscopy experiments (8). Three-dimensional reconstructions from electron micrographs revealed similar effects of the nucleotides on the flexibility of actin filament (9, 10). The results of phosphorescence spectroscopic experiments apparently also supported the c...
The scavenging effect of the antimetabolite dihydrofolate reductase inhibitor methotrexate (MTX) and the isomers of its photoswitchable derivate, cis- and trans-phototrexate (PHX), have been compared by ESR spectroscopy, with the application of a cyclic hydroxylamine spin probe. The results showed the most pronounced scavenging effect in the presence of trans-phototrexate (trans-PHX). At a low concentration (100 µM) cis-PHX also showed a greater scavenging effect than the parent molecule MTX. Direct antioxidant properties of the investigated molecules were measured by ABTS scavenging assay, which showed no significant difference between trans-PHX and cis-PHX, but both of the isomers of PHX showed a higher antioxidant capacity than MTX. These findings imply that trans-PHX may have more pronounced anti-inflammatory and tissue-protective effects than MTX, despite the lack of its cytotoxic, antineoplastic effect.
Conventional and saturation transfer electron paramagnetic resonance spectroscopy (EPR and ST EPR) was used to study the orientation of probe molecules in muscle fibers in different intermediate states of the ATP hydrolysis cycle. A separate procedure was used to obtain ST EPR spectra with precise phase settings even in the case of samples with low spectral intensity.Fibers prepared from rabbit psoas muscle were labeled with isothiocyanate spin labels at the reactive thiol sites of the catalytic domain of myosin. In comparison with rigor, a significant difference was detected in the orientationdependence of spin labels in the ADP and adenosine 5¢- [b,c-imido] It is generally accepted that domain movements in the myosin head play a decisive role in the energy-transduction process of muscle contraction [1][2][3][4][5]. It is a multistep process which can produce several conformational states of myosin [6][7][8][9]. Extensive studies using different techniques have indicated that the nucleotide-binding pocket does not experience large conformational changes during the hydrolytic cycle [10][11][12]. However, small conformational changes induced by nucleotides in the motor domain should be converted into larger movements. The data show that, whereas the structure of the motor domain remains similar to rigor, the regulatory domain swings around a point at the distal end of the motor domain [8,[13][14][15][16][17]. The changes in the 50-kDa domain affect the segment of the 20-kDa domain that contains the essential thiol groups, SH1 (Cys707) and SH2 (Cys697). This part may be involved in the transducing of small conformational changes [18,21,22].Spectroscopic probes are widely used in muscle research. Paramagnetic probes provide a direct method by which dynamic changes and the rotation and orientation of specifically labeled proteins can be followed. In muscle fiber studies, the probe molecules, particularly the maleimide-based nitroxides and iodoacetamide spin labels, are usually attached to the reactive thiol site Cys707 of the motor domain [21-23], or to the regulatory light chain [9,24,25]. The main problems that limit interpretation of spectroscopic measurements are the location of the probe molecules on the proteins, the relative orientation of the spin labels with respect to the magnetic field, and the perturbing effect of probes on structure and function. We have previously observed that an isothiocyanate-based spin label is more sensitive to the domain orientation in the myosin head than the widely used maleimide spin label [26]. Selective modification of Cys707 strongly affects MgATP hydrolysis, and the motor function of myosin heads in the in vitro motility assay is blocked [27,28]. The most pronounced effect was observed in the intermediate complex ADP-P i [19,20].Spin label molecules bound to myosin are able to detect nucleotide binding and conformational changes in the myosin head related to the hydrolytic cycle of ATP [29]:where M denotes myosin, A stands for actin, and the asterisk (*) distinguishes inte...
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