MOLECULAR CONTACTS BETWEEN NEBULIN AND ACTIN - CROSS-LINKING OF NEBULIN MODULES TO THE N-TERMINUS OF ACTIN

Nebulin, a giant actin binding protein, coextends with actin and is th ought to form a composite thin filament in the skeletal muscle sarcome re. To understand the molecular interactions between nebulin and actin , we have applied chemical cross-linking techniques to define molecula r contacts between actin and ND8, a two-module nebulin fragment that p romotes actin polymerization and inhibits depolymerization by binding to both G- and F-actin. The formation of a 1:1 complex with a dissocia tion constant of 4.9 mu M between ND8 and G-actin was demonstrated by fluorescence titration of dansyl-ND8 with G-actin. Treatment with a ze ro-length cross-linker, 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimi de (EDC), crosslinked the ND8-G-actin complex covalently without impai ring actin's ability to polymerize. End-labeling Western blot and sequ ence and mass analyses of purified conjugated peptides revealed the cr oss-linking between lysine 5 of ND8 and the two N-terminal acidic resi dues of G-actin. Similarly, we have shown by end-labeling that cross-l inking of ND8 to F-actin occurred at the N-terminus of actin protomer. The binding of nebulin to the N-terminus of actin is likely to be sig nificant in its ability to affect actin polymerization. Furthermore, t he association of nebulin modules with the actin N-terminus in subdoma in 1 supports the hypothesis that nebulin wraps around the outer edges of actin filaments where S1, tropomyosin, and several actin binding p roteins are known to interact.