Nebulin Elasticity Pre-loads Thin Filaments of Skeletal Muscle: Unfolding of Transient a-helices

Abstract

Nebulin, a giant modular protein (600 - 900 kDa), acts as a thin filament ruler and regulator of contraction. The bulk of the protein consists of ∼200 tandem repeats of ∼35 residue actin-binding modules arranged as two single-repeat regions and 22 sets of seven-module super-repeats. The nanomechanics of nebulin were investigated with atomic force microscopy by tethering and stretching full length molecules between a pair of site-specific antibodies to either N or C terminus of nebulin, with one attached covalently to a functionalized self-assembled monolayer and the other to a functionalized cantilever. Upon stretch, single nebulin molecules extend to well over 1 μm and yield force curves with variable numbers and heights of peaks. An underlying periodicity at ∼22 and ∼15 nm was observed by periodogram analysis. Major force peaks appear to result from the re-orientation and unfolding of short α-helical bundles of nebulin modules, as suggested by circular dichroism, steered molecular dynamics simulations of computed structures as well as experimental force-extension curves of nebulin single and super repeats.The nebulin molecule exerts a compressive force at the ends and alongside the 1 μm long thin filaments. In the sarcomere, nebulin's compressive stress may stiffen actin filaments and assist the re-annealing of severed actin filaments under undue stress. In the skeletal muscle sarcomere, the elastic nebulin associates helically around actin filaments, thus behaving as a length regulating ruler only under stress and upon binding to the actin filaments. It may also act as a reversible and elastic tether between the myosin heads and actin during contraction. We propose the elasticity of nebulin as an integral component of its ruler function and acto-myosin regulation in preloaded thin filaments.