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.

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