Myosin Subfragment 1 and Structural Elements of G-Actin: Effects of S-1(A2) on Sequences 39-52 and 61-69 in Subdomain 2 of G-Actin

The effect of myosin on the structure of two sequences on G-actin, a loop between residues 39 and 52 and a segment between residues 61 and 69 from the NH₂-terminus, was probed by limited proteolytic digestions of G-actin in the presence of the myosin subfragment 1 isozyme S-1(A2). Under the experimental conditions of this work, no polymerization of actin was induced by S-1(A2) [Chen & Reisler (1991) Biochemistry 30, 4546-4552]. S-1(A2) did not change the rates of subtilisin and chymotryptic digestion of G-actin at loop 39-52. In contrast to this, the second protease-sensitive region on G-actin, segment 61-69, was protected strongly by S-1(A2) from tryptic cleavage. The minor if any involvement of loop 39-52 in S-l binding was confirmed by determining the binding constants of S-1(A2) for pyrene-labeled G-actin (1.2 X 10⁶ M^-1), subtilisin-cleaved pyrenyl G-actin (0.3 X 10⁶ M^-1), and DNase I-pyrenyl G-actin complexes (0.3 X 10⁶ M^-1). Consistent with this, the activity of DNase I, which binds to actin loop 39-52 [Kabsch et al. (1990) Nature 347, 37-44], was inhibited almost equally well by actin in the presence and absence of S-1(A2). These results confirm the observation that DNase I and S-1(A2) bind to distinct sites on actin [Bettache et al. (1990) Biochemistry 29, 9085-9091] and demonstrate myosin-induced changes in segment 61—69 of G-actin.