Assessing Mechanical Properties of a Utrophin Fragment with Two Operational Modes

Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by the absence of the protein dystrophin. Utrophin, a dystrophin homologue, is being explored as a protein replacement therapy for DMD. Here, we study mechanical properties of a utrophin fragment encoding the N terminus through spectrin repeat 3 (UtrN-R3) using atomic force microscopy (AFM). We perform single molecule force spectroscopy using two operational modes of AFM, constant speed and constant force. In constant speed mode, the molecule is stretched continually while constant force mode maintains a steady force on the molecule. Under both modes, the bound domains unbound where the force at which the domains unravel and the time of unbinding are recorded under the constant speed and the constant force modes respectively. The Dudko-Hummer-Szabo method [Dudko et. al 2008] relates the statistics of force magnitude data from constant speed mode to the time to unravel statistics of the constant force mode. Our data shows consistent results using constant speed and constant force modes. We also recover parameters of the energy landscape of the domains and perform a homogeneity analysis for UtrN-R3. To further validate our results, Monte Carlo simulations are conducted which corroborate the conclusions drawn from experimental data. We include a Python-based toolbox including a simulation engine and different modeling methods for protein analysis.