Thermodynamically Coupled Unfolding Transitions in Dystrophin ABD1

Abstract

Dystrophin, a 427 kDa protein located on one of the largest genes in the human genome, has been implicated in myocytes’ ability to dissipate mechanical forces transduced between cytoskeletal and membrane features. Single point mutations in Dystrophin can result in myocyte membrane shearing under normal muscular flexion and is diagnosed as Becker's Muscular Dystrophy. Given point mutations outside of direct binding sites can render Dystrophin at a loss of function, the amino acids within each domain are likely stabilized or destabilized allosterically. Our research aims to determine how secondary and tertiary structures of Dystrophin can effectively couple during unfolding transitions within and between protein domains. Here, we study the actin binding domain (ABD1) of Dystrophin which contains the highest frequency of point mutations resulting in Becker's muscular dystrophy. In order to gain insight as to how ABD1 may couple within itself, we designed a series of thermodynamic analyses to describe ABD1 unfolding semi-mechanistically. We use differential scanning calorimetry as well as additional spectroscopic techniques (CD and FLT) to monitor ABD1 unfolding energetics and determine how energy may be stored in ABD1's secondary structure or in its buried hydrophobic residues. Relating these approaches provides a comparative approach as to how different structural features may affect ABD1's ability to dissipate mechanical stress through folding and unfolding thermodynamically favorable folding features.