DYNAMIC PROPERTIES OF ACTIN - STRUCTURAL-CHANGES INDUCED BY BERYLLIUMFLUORIDE

Beryllium fluoride (BeFx) has been widely used as a phosphate analogue in nucleotide-binding proteins. It was found to bind tightly to F- bu t not G-actin (Combeau C., and Carlier M. F. (1988) J. Biol. Chem. 263 , 17429-17436) and to affect the three-dimensional structure of filame nts by stabilizing the subdomain 2 region of the actin protomer (Orlov a, A., and Egelman, E. H. (1992) J. Mol. Biol. 227, 1043-1053). In thi s work we examined the BeFx-induced structural and functional changes in G- and F-actin by using proteolysis, chemical modifications, ATPase , and in vitro motility assays. The results of proteolysis studies sho w that BeFx binds also to MgADP-G-actin and renders its subdomain 2 re gion more similar to that in MgATP-G-actin. This is manifested in enha nced subtilisin and decreased tryptic digestions in subdomain 2 of G-a ctin. BeFx had a strong effect on the proteolysis of MgADP-F-actin: bo th the tryptic and sub digestions in subdomain 2 were completely inhib ited. Significant protection against proteolysis in this region was ob served even at 1:14 molar ratios of BeFx to actin indicating cooperati ve effects on the structure of the actin filament. A similar although milder effect of phosphate on the proteolysis of F-actin suggests that BeFx acts as a phosphate analogue in this system. BeFx also induces c hanges in the subdomain 1 region of F-actin. This is revealed via redu ced rates of Cys-374 alkylation with thylamino-3-(4'-maleimidylphenyl) -4-methylcoumarin and an increased subtilisin cleavage near the C term inus of actin in the presence of BeFx. The BeFx-induced structural cha nges in actin have little effect on its interactions with myosin. BeFx inhibits only slightly the actin-activated ATPase activity of S1 by d ecreasing V-max without affecting K-M. Additionally, the binding of Be Fx to actin does not change the sliding velocity of actin filaments in the in vitro motility assays. The BeFx-induced specific and distinct changes in G- and F-actin point to the dynamic nature of actin structu re and the local differences between monomeric and polymeric forms of actin.