Nebulin, a helical actin binding protein.

Abstract

Nebulin, a giant protein (molecular mass 800 kDa) specific for the skeletal muscle of vertebrates, has been suggested to be involved in the length regulation of the thin filament as a ‘molecular ruler’. Despite its size, nebulin appears to be composed mainly of small repeats of approximately 35 amino acids. We have characterized in this study the conformational and functional properties of single repeats. Complete repeats were found to bind to F‐actin while a truncated one did not. One repeat is therefore the smallest unit for nebulin‐‐actin interaction. Circular dichroism and nuclear magnetic resonance spectra measured for the peptides in water indicated a transient helical conformation. The folded region is located for them all around the conserved sequence SDxxYK. The helical conformation is strongly stabilized by anionic detergents and trifluoroethanol while uncharged or positively charged detergents have no effect. Since the surface of the actin filament is known to contain clusters of negative charges, anionic detergents may mimic the effect of an actin environment. 3D structures were calculated for three representative peptides in SDS. In vivo, the nebulin helices should form a complex with the actin filament. Based on the assumed importance of charge interactions between nebulin and actin, we propose a model for the structure of the F‐actin‐nebulin complex in vivo. According to that, two nebulin molecules occupy symmetrical positions along the central cleft of the actin filament bridging the two strands of the actin two‐start helix. The consistency of this model with experimental data is discussed.

Nebulin: A Study of Protein Repeat Evolution

“…19 However, the conformation of nebulin in its unbound form is not known. Nebulin units are transient helices in solution, 10 while long constructs of nebulin repeats aggregate at natural pH. 20 Attempts to model the structure of nebulin domains were not successful, since it lacks sufficiently close homologs in structure databases.…”

“…10 It contains a conserved SDxxYK sequence pattern that is responsible for binding to actin. 11 In some contexts, nebulin domains form an SR consisting of seven domains.…”

Proteolysis of specific muscle structural proteins by mu-calpain at low pH and temperature is similar to degradation in postmortem bovine muscle.

“…Nebulin, which is thought to be intimately associated with the thin filament and may even constitute part of a composite nebulin/thin filament (Pfuhl et al, 1994;Robson, 1995), may aid in anchoring the thin filament to the Z-line (Wang and Wright, 1988;Komiyama et al, 1992). Degradation of nebulin PM could weaken the thin filament linkages at the Z-line, and(or) of the thin filaments in the nearby I-band regions (Taylor et al, 1995), and thereby weaken the structure of the muscle cell.…”

Nebulin Interacts with CapZ and Regulates Thin Filament Architecture within the Z-Disc

“…Each module has a conserved motif thought to interact with a single actin monomer. In addition, modules 9-162 are organized into sets of seven-module super-repeats, which contain a second conserved motif that matches the periodicity of, and is thought to organize, the tropomyosin/troponin complexes (Jin and Wang, 1991;Pfuhl et al, 1994;Labeit and Kolmerer, 1995;Wang et al, 1996).…”