INTERMOLECULAR COUPLING BETWEEN LOOP-38-52 AND THE C-TERMINUS IN ACTIN-FILAMENTS

The recently reported structural connectivity in F-actin between the D Nase I binding loop on actin (residues 38-52) and the C-terminus regio n was investigated by fluorescence and proteolytic digestion methods. The binding of copper to Cys-374 on F- but not G-actin quenched the fl uorescence of dansyl ethylenediamine (DED) attached to Gln-41 by more than 50%. The blocking of copper binding to DED-actin by N-ethylmaleim ide labeling of Cys-374 on actin abolished the fluorescence quenching. The quenching of DED-actin fluorescence was restored in copolymers (1 :9) of N-ethylmaleimide-DED-actin with unlabeled actin. The quenching of DED-actin fluorescence by copper was also abolished in copolymers ( 1:4) of DED-actin and N-ethylmaleimide-actin. These results show inter molecular coupling between loop 38-52 and the C-terminus in F-actin. C onsistent with this, the rate of subtilisin cleavage of actin at loop 38-52 was increased by the bound copper by more than 10-fold in F-acti n but not in G-actin. Neither acto-myosin subfragment-1 (S1) ATPase ac tivity nor the tryptic digestion of G-actin and F-actin at the Lys-61 and Lys-69 sites were affected by the bound copper. These observations suggest that copper binding to Cys-374 does not induce extensive chan ges in actin structure and that the perturbation of loop 38-52 environ ment results from changes in the intermolecular contacts in F-actin.