Two operational modes of atomic force microscopy reveal similar mechanical properties for homologous regions of dystrophin and utrophin

Abstract

Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by the absence of the protein dystrophin. Dystrophin is hypothesized to work as a molecular shock absorber that limits myofiber membrane damage when undergoing reversible unfolding upon muscle stretching and contraction. Utrophin is a dystrophin homologue that is under investigation as a protein replacement therapy for DMD. However, it remains uncertain whether utrophin can mechanically substitute for dystrophin. Here, we compared the mechanical properties of homologous utrophin and dystrophin fragments encoding the N terminus through spectrin repeat 3 (UtrN-R3, DysN-R3) using two operational modes of atomic force microscopy (AFM), constant speed and constant force. Our comprehensive data, including the statistics of force magnitude at which the folded domains unfold in constant speed mode and the time of unfolding statistics in constant force mode, show consistent results. We recover parameters of the energy landscape of the domains and conducted Monte Carlo simulations which corroborate the conclusions drawn from experimental data. Our results confirm that UtrN-R3 expressed in bacteria exhibits significantly lower mechanical stiffness compared to insect UtrN-R3, while the mechanical stiffness of the homologous region of dystrophin (DysN-R3) is intermediate between bacterial and insect UtrN-R3, showing greater similarity to bacterial UtrN-R3. SignificanceDuchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by mutations in DMD gene encoding dystrophin. Utrophin, a fetal homologue of dystrophin, is under active investigation as a dystrophin replacement therapy for DMD. However, it is still unknown if it can substitute dystrophin mechanically. Here, we report mechanical properties of both utrophin and dystrophin fragments encoding the N terminus through spectrin repeat 3 (UtrN-R3, DysN-R3) using atomic force microscope (AFM) through two operational modes, constant speed and constant force. Our data, consistent across both modes, confirm that UtrN-R3 expressed in bacteria exhibits significantly lower mechanical stiffness than insect UtrN-R3. Additionally, bacterial DysN-R3 lies between bacterial and insect UtrN-R3 in terms of mechanical properties, leaning closer to bacterial UtrN-R3.