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 thought to form a composite thin filament in the skeletal muscle sarcomere. To understand the molecular interactions between nebulin and actin, we have applied chemical cross-linking techniques to define molecular contacts between actin and ND8, a two-module nebulin fragment that promotes actin polymerization and inhibits depolymerization by binding to both G- and F-actin. The formation of a 1:1 complex with a dissociation constant of 4.9 microM between ND8 and G-actin was demonstrated by fluorescence titration of dansyl-ND8 with G-actin. Treatment with a zero-length cross-linker, l-ethyl-3-[3-(dimethylamino) propyl]carbodiimide (EDC), cross-linked the ND8-G-actin complex covalently without impairing actin's ability to polymerize. End-labeling Western blot and sequence and mass analyses of purified conjugated peptides revealed the cross-linking between lysine 5 of ND8 and the two N-terminal acidic residues of G-actin. Similarly, we have shown by end-labeling that cross-linking 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 significant in its ability to affect actin polymerization. Furthermore, the association of nebulin modules with the actin N-terminus in subdomain 1 supports the hypothesis that nebulin wraps around the outer edges of actin filaments where Sl, tropomyosin, and several actin binding proteins are known to interact.
Shih
C L
CL
Department of Chemistry and Biochemistry, University of Texas at Austin 78712, USA.
Chen
M J
MJ
Linse
K
K
Wang
K
K
D000199:Actins / Q000737:chemistry*; D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents; D005022:Ethyldimethylaminopropyl Carbodiimide; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009124:Muscle Proteins / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation
D000199:Actins; D003432:Cross-Linking Reagents; D009124:Muscle Proteins; C035620:nebulin; D008239:Lysine; D005022:Ethyldimethylaminopropyl Carbodiimide