MALEIMIDOBENZOYL-G-ACTIN - STRUCTURAL-PROPERTIES AND INTERACTION WITH SKELETAL MYOSIN SUBFRAGMENT-1

We have investigated various structural and interaction properties of maleimidobenzoyl-G-actin (MBS-actin), a new, internally cross-linked G-actin derivative that does not exhibit, at moderate protein concentration, the salt—and myosin subfragment 1 (S-1)—induced polymerizations of G-actin and reacts reversibly and covalently in solution with S-1 at or near the F-actin binding region of the heavy chain (Bettache, N., Bertrand, R., & Kassab, R. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6028-6032). The far-ultraviolet CD spectrum and α-helix content of the MBS-actin were identical with those displayed by native G-actin. 45Ca2+ measurements showed the same content of tightly bound Ca2+ in MBS-actin as in G-actin and the EDTA treatment of the modified protein promoted the same red shift of the intrinsic fluorescence spectrum as observed with native G-actin. Incubation of concentrated MBS-actin solutions with 100 mM KCl + 5 mM MgCl2led to the polymerization of the actin derivative when the critical monomer concentration reached 1.6 mg/mL, at 25 °C, pH 8.0. The MBS-F-actin formed activated the Mg2+-ATPase of S-1 to the same extent as native F-actin. The MBS-G-actin exhibited a DNase I inhibitor activity very close to that found with native G-actin and was not to be at all affected by its specific covalent conjugation to S-1. This finding led us to isolate, for the first time, by gel filtration, a ternary complex comprising DNase I tightly bound to MBS-actin cross-linked to the S-1 heavy chain, demonstrating that S-1 and DNase I bind at distinct sites on G-actin. Fluorescence measurements with pyrene-labeled MBS-G-actin were applied to monitor the solution interaction of the protein with the isolated S-1(A1) and S-1(A2) isoenzymes. The binding of either S-1 isoform did not cause polymerization of the fluorescent actin derivative and resulted in about 40% enhancement of the fluorescence emission of the pyrenyl probe. The dissociation constants (Kd) estimated at low ionic strength (2 mM) were 0.18 and 0.47 μM, for the complexes with S-1(A1) and S-1(A2), respectively. The addition of salt (100 mM) decreased the MBS-actin-S-1 affinity (Kd≥ 60 μM) and perturbed the structure of the actin derivative as assessed by limited proteolysis. Collectively, the data illustrate further the nativeness of the MBS-G-actin and its potential use in solution studies of the actin-myosin head interactions. © 1990, American Chemical Society. All rights reserved.