Molecular Extensibility of Mini-dystrophins and a Dystrophin Rod Construct

Muscular dystrophies arise with various mutations in dystrophin, implicating this protein in force transmission in normal muscle. With 24 three-helix, spectrin repeats interspersed with proline-rich hinges, dystrophinʹs large size is an impediment to gene therapy, prompting the construction of mini-dystrophins. Results thus far in dystrophic mice suggest that at least one hinge between repeats is necessary though not sufficient for palliative effect. One such mini-dystrophin is studied here in forced extension at the single molecule level. Δ2331 consists of repeats (R) and hinges (H) H1-R1-2∼H3∼R22-24-H4 linked by native (−) and non-native (∼) sequence. This is compared to its core fragment R2∼H3∼R22 as well as an eight-repeat rod fragment middle (RFM: R8-15). We show by atomic force microscopy that all repeats extend and unfold at forces comparable to those that a few myosin molecules can generate. The hinge regions most often extend and transmit force while limiting tandem repeat unfolding. From 23–42 °C, the dystrophin constructs also appear less temperature-sensitive in unfolding compared to a well-studied βI-spectrin construct. The results thus reveal new modes of dystrophin flexibility that may prove central to functions of both dystrophin and mini-dystrophins.