Using proteolytic susceptibility as a probe, we have identified four regions of the actin polypeptide chain where structural rearrangements, dependent on the nature of the tightly bound metal ion and/or nucleotide, take place. Replacement of the tightly bound Ca²⁺ by Mg²⁺ in ATP‐actin strongly affected the regions around Arg26 and Lys68, as judged from nearly complete inhibition of tryptic cleavages of the polypeptide chain at these residues. It also significantly diminished the rates of splitting by trypsin of the peptide bonds involving carbonyl groups of Arg372 and of Lys373 in the C‐terminal segment. Conversion of ATP‐actin to ADP‐actin to (with Mg²⁺ as the tightly bound cation) abolished the protective effect of Mg²⁺ on specific tryptic cleavage and, in contrast, largely inhibited proteolysis at specific sites for subtilisin and for a novel protease from Escherichia coli A2 strain within a surface loop of residues 39–51. We also examined the effect of proteolytic cleavage or chemical modification at certain sites on the kinetics of proteolysis at other sites of the molecule. These experiments demonstrated structural relationships between loop 39–51 and regions involving Lys61 and Lys68. It is suggested that the conformational transitions reflected in the observed changes in proteolytic susceptibility may underlie the known influence of the nature of the tightly bound cation and nucleotide on the kinetics of actin polymerization and stability of the polymer.

Actin can be specifically cleaved between residues 42 and 43 with a novel protease from Escherichia coli A2 strain (ECP) [Khaitlina, S. Y., Collins, J. H., Kuznetsova, I. M., Pershina, V. P., Synakevich, I. G., Turoverov, K. K. & Usmanova, A. M. (1991) FEBS Lett. 279, 49–51]. The resulting C‐terminal and N‐terminal fragments remained associated to one another in the presence of either Ca²⁺ or Mg²⁺. The protease‐treated actin was, however, neither able to spontaneously assemble into filaments nor to copolymerize with intact actin unless its tightly bound Ca²⁺ was replaced with Mg²⁺. Substitution of Mg²⁺ for the bound Ca²⁺ was also necessary to partially restore the ability of the protease‐treated actin to inhibit the DNase I activity.The critical concentration for KCl‐induced polymerization of ECP‐treated ATP–Mg–G‐actin, determined by measuring the fluorescence of pyrenyl label, was approximately threefold higher than that for actin cleaved between residues 47 and 48 using subtilisn, and 36‐fold higher than the critical concentration for polymerization of intact actin under the same conditions. Morphologically, the filaments of ECP‐treated actin were indistinguishable from those of intact actin. Comparison of the fluorescence spectra of pyrenyl‐labelled actins and chemical cross‐linking with N,N′‐1,2‐phenylenebismaleimide have, however, revealed structural differences between the filaments assembled from ECP‐treated actin and those of intact as well as subtilisin‐treated actin. Moreover, the filaments of ECP‐treated actin were easily disrupted by centrifugal forces or shearing stress unless they were stabilized by phalloidin. The results are consistent with the direct participation of the region around residues 42 and 43 in the monomer/monomer interactions as predicted from the atomic model of F‐actin [Holmes, K. C., Popp, D., Gebhard, W. & Kabsch, W. (1990) Nature 347, 44–49] and suggest that the interactions involving this region are of primary importance for stabilization of the actin filament. The mechanism of the regulation of actin polymerization by the tightly bound divalent cation is also discussed.

Cofilins are evolutionary conserved proteins present in all Eukaryotic cells. Their primary function is dynamic reorganization of actin cytoskeleton. Two cofilin isoforms are known: cofilin 1, present in all studied non-muscle cells and in embryonic muscle cells, and cofilin 2, which dominates in mature skeletal and cardiac muscles. Polypeptide chains of both isoforms fold into a structure homological to a conservative ADF (actin depolymerizing factor) domain, which is characteristic of actin depolymerizing factor. In cofilin molecule two actin-binding sites were found. One site binds monomeric and filamentous actin, the second one interacts only with the filament. Binding of cofilin to actin filament causes a change in the orientation of subunits, which results in filament severing. This increases number of ends which can either elongate or shorten the filament, depending on the conditions. Cofilin interactions with monomeric actin decreases availability of polymerization-competent actin subunits. Cofilin activity is controlled by phosphorylation, binding membrane phospholipids, local pH and oxidative stress. Under conditions of oxidative stress oxidation of cysteine residues leads to formation of dimers, which are able to cross-link actin filaments. Stable actin-cofilin rods save cellular ATP, which is not used during active polymerization process. This facilitates faster cell recovery from the stress. The final cellular reaction on the environmental stimuli is a resultant of cofilin activity and activities of other actin-binding proteins, which function either synergistically or antagonistically. Due to the central role in the regulation of actinfilaments dynamics, cofilin is involved in development of cancer, neurodegenerative diseases, congenital myopathies and cardiomyopathies.

Truncated derivatives of actin devoid of either the last two (actin-2C) or three residues (actin-3C) were used to study the role of the C-terminal segment in the polymerization of actin. The monomer critical concentration and polymerization rate increased in the order: intact actin < actin-2C < actin-3C. Conversely, the rate of hydrolysis of actin-bound ATP during spontaneous polymerization of Mg-actin decreased in the same order, so that, for actin-3C, the ATP hydrolysis significantly lagged behind the polymer growth. Probing the conformation of the nucleotide site in the monomer form by measuring the rates of the bound nucleotide exchange revealed a similar change upon removal of either the two or three residues from the C-terminus. The C-terminal truncation also resulted in a slight decrease in the rate of subtilisin cleavage of monomeric actin within the DNAse-I binding loop, whereas in F-actin subunits the susceptibility of this and of another site within this loop, specifically cleaved by a proteinase from Escherichia coli A2 strain, gradually increased upon sequential removal of the two and of the third residue from the C-terminus. From these and other observations made in this work it has been concluded that perturbation of the C-terminal structure in monomeric actin is transmitted to the cleft, where nucleotide and bivalent cation are bound, and to the DNAse-I binding loop on the top of subdomain 2. Further changes at these sites, observed on the polymer level, seem to result from elimination of the intersubunit contact between the C-terminal residues and the DNAse-I binding loop. It is suggested that formation of this contact plays an essential role in regulating the hydrolysis of actin-bound ATP associated with the polymerization process.