Nanomechanics of Full-Length Nebulin: An Elastic Strain Gauge in the Skeletal Muscle Sarcomere

Muscle Proteomics and Nanotechnology Section, Laboratory of Muscle Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Langmuir (Impact Factor: 4.46). 06/2009; 25(13):7496-505. DOI: 10.1021/la9009898
Source: PubMed


Nebulin, a family of giant modular proteins (MW 700-800 kDa), acts as a F-actin thin filament ruler and calcium-linked regulator of actomyosin interaction. The nanomechanics of full length, native rabbit nebulin was investigated with an atomic force microscope by tethering, bracketing, and stretching full-length molecules via pairs of site-specific antibodies that were attached covalently, one to a protein resistant self-assembled monolayer of oligoethylene glycol and the other to the cantilever. Using this new nanomechanics platform that enables the identification of single molecule events via an unbiased analysis of detachment force and distance of all force curves, we showed that nebulin is elastic and extends to approximately 1 microm by external force up to an antibody detachment force of approximately 300-400 pN. Upon stretching, nebulin unravels and yields force spectra with craggy mountain range profiles with variable numbers and heights of force peaks. The peak spacings, analyzed by the model-independent, empirical Hilbert-Huang transform method, displayed underlying periodicities at approximately 15 and approximately 22 nm that may result from the unfolding of one or more nebulin modules between force peaks. Nebulin may act as an elastic strain gauge that interacts optimally with actin only under appropriate strain and stress. This stretch to match protein ruler may also exert a compressive force that stabilizes thin filaments against stress during contraction. We propose that the elasticity of nebulin is integral and essential in the muscle sarcomere.

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Available from: Jeffrey Forbes, May 22, 2014
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    • "Another possibility is that thin filaments could be stabilized by nebulin via compressive forces. Atomic force microscopy revealed that nebulin is moderately compliant and, when extended, could provide substantive restorative forces to the thin filament (Yadavalli et al., 2009). This force could mechanically protect the filament from contractile-induced strain, preventing actin depolymerization. "

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    • "In 2009, Yadavalli et al. reported that they extracted full-length nebulin from rabbit longissimus dorsi muscle using 0.45% sodium deoxycholate (DOC), and then purified it with an NaCl gradient in the presence of 0.45% DOC. However, the detailed preparation method has not been published yet [13]. "
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    ABSTRACT: Nebulin is about 800 kDa filamentous protein that binds the entire thin filament of vertebrate skeletal muscle sarcomeres. Nebulin cannot be isolated from muscle except in a completely denatured form by direct solubilization of myofibrils with SDS because nebulin is hardly soluble under salt conditions. In the present study, nebulin was solubilized by a salt solution containing 1 M urea and purified by DEAE-Toyopearl column chromatography via 4 M urea elution. Rotary-shadowed images of nebulin showed entangled knit-like particles, about 20 nm in diameter. The purified nebulin bound to actin filaments to form loose bundles. Nebulin was confirmed to bind actin, alpha-actinin, beta-actinin, and tropomodulin, but not troponin or tropomyosin. The data shows that full-length nebulin can be also obtained in a functional and presumably native form, verified by data from experiments using recombinant subfragments.
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