LONG-RANGE CONFORMATIONAL EFFECTS OF PROTEOLYTIC REMOVAL OF THE LAST 3 RESIDUES OF ACTIN

Truncated derivatives of actin devoid of either the last two (actin(-2 C)) or three residues (actin(-3C)) were used to study the role of the C-terminal segment in the polymerization of actin. The monomer critica l concentration and polymerization rate increased in the order: intact actin < actin(-2C) < actin(-3C). Conversely, the rate of hydrolysis o f actin-bound ATP during spontaneous polymerization of Mg-actin decrea sed in the same order, so that, for actin(-3C), the ATP hydrolysis sig nificantly lagged behind the polymer growth. Probing the conformation of the nucleotide site in the monomer form by measuring the rates of t he 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 subtilisi n cleavage of monomeric actin within the DNAse-I binding loop, whereas in F-actin subunits the susceptibility of this and of another site wi thin 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 ob servations made in this work it has been concluded that perturbation o f the C-terminal structure in monomeric actin is transmitted to the cl eft, where nucleotide and bivalent cation are bound, and to the DNAse- I binding loop on the top of subdomain 2. Further changes at these sit es, 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 asso ciated with the polymerization process.