Elasticity of Intrinsically Disordered Nebulin Modules

Abstract

The elasticity of native full length nebulin, demonstrated recently via atomic force microscopy with site-specific antibody pairs as force handles (Langmuir, 2009, 25, 7496), suggests that in the thin filaments, nebulin is stretched to cope with the actin length and imposes significant force and influences the functions of the underlying actins. This pre-stressed mechanical state of thin filaments may have important implications for the role of nebulin as a length ruler and as a regulator of actomyosin interaction. The structural basis of nebulin elasticity remains open. We report here the structural characterization of modules from the super-repeat and single repeat regions by a combination of circular dichroism (CD), NMR, SAXS, AFM, structural predictions and steered molecular dynamics simulations. In aqueous solutions of common buffers, these modules are intrinsically disordered, but are poised to form alpha-helices, especially in the presence of trifluoroethanol. SAXS analysis of a four-module construct indicates an elongated structure with a radius of gyration of 3.6 nm and, as modeled with DAMMIN, shows a contour length of ∼15 nm. Interestingly, this extended structure is also evident in a small population of the structural models as predicted by ROSETTA++. AFM images of the modules on an inert surface are predominantly compact with an average height of ∼ 2.5 nm, consistent with the bulk of the ROSETTA predictions. These structural ensembles of compact and extended structures are significantly shorter than what it would take for nebulin modules to wrap around the perimeter of actin filaments (∼6 nm per module). We propose that nebulin modules’ disorder-order transition of alpha helices, contributes to its elasticity and how nebulin juxtapositions itself onto the actin to form a pre-stressed thin filaments in the muscle sarcomere.