Alternative splicing within proteins is common but not well understood in its influence on protein structure and stability. Filamins are ubiquitous actin-crosslinking proteins with two dozen Immunolgobulin (Ig) repeats and one alternatively-spliced ‘hinge’ that has been hypothesized to add flexibility. The hinge is also predicted to perturb folding. The molecular mechanics of filamins are probed here by AFM-forced extension, with a particular focus on the ∼30 aa hinge between repeats R15 and R16. After re-examining full-length filamin to clarify the single molecule limit for AFM experiments on long chains, short concatemers of (R15-R16)m and (R15-hinge-R16)m were studied by both AFM and solution structural methods. AFM shows that the hinged isoform extends and unfolds at smaller forces (60 pN) than the hinge-less form (80 pN), implying that the alternative splicing introduces a random coil that softens both adjacent domains. Circular Dichroism confirms that the hinge is a random coil, and thermal unfolding in solution suggests a weak destabilization by the hinge. Together with the rate-dependence of forced extension in AFM, the results reveal added resilience as the unfolding transition shifts to longer lengths upon insertion of the alternatively spliced hinge.
Alternative Splicing for Mechanical Resilience: The Softening Effect of Filamin’s Hinge
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Discher, DE, & Johnson, C. "Alternative Splicing for Mechanical Resilience: The Softening Effect of Filamin’s Hinge." Proceedings of the ASME 2007 Summer Bioengineering Conference. ASME 2007 Summer Bioengineering Conference. Keystone, Colorado, USA. June 20–24, 2007. pp. 789-790. ASME. https://doi.org/10.1115/SBC2007-176751
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