Molecular contacts between nebulin and actin: Cross-linking of nebulin modules to the N-terminus of actin

Chaw Long Shih, Mu Jung G. Chen, Klaus Linse, Kuan Wang

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

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 μM between ND8 and G-actin was demonstrated by fluorescence titration of dansyl- ND8 with G-actin. Treatment with a zero-length cross-linker, 1-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 S1, tropomyosin, and several actin binding proteins are known to interact.

Original languageEnglish
Pages (from-to)1814-1825
Number of pages12
JournalBiochemistry
Volume36
Issue number7
DOIs
Publication statusPublished - Feb 18 1997
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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