Unfolding Transitions and Interdomain Coupling in Human Dystrophin Spectrin Repeats

Abstract

Dystrophin is a 427 kDa rod-shaped protein encoded by one of the largest mammalian genes. The protein is formed by 27 domains, with 24 of them being single spectrin repeats interposed by two hinge regions. Single point and missing domain mutations within Dystrophin have been linked to Becker's Muscular Dystrophy (BMD), which is a degenerative muscle disease. This suggests that one role for Dystrophin is to reduce mechanical force between the sarcolemma membrane and the cytoskeleton. The mechanism for this function is unknown. The observation that single point mutations within Dystrophin are correlated with BMD suggests there is communication between each amino acid within each Dystrophin domain and that this communication extends to its neighboring domains. This led to the hypothesis that as force is transduced through the spectrin repeats, this results in the partial unfolding of the repeats which then dissipates force. If this partial unfolding of a given domain is accentuated when another repeat domain is in tandem, then this is consistent with negative coupling. Conversely, stabilization of one domain by another is positive coupling. Molecular dynamics (MD) forced unfolding experiments were consistent with negative coupling through non-additive force trajectories for tandem domains compared to individual domains. Therefore, we designed an experimental approach to test the computational results.Using monomeric spectrin 17, monomeric spectrin 18, and dimeric spectrin 17-18, we experimentally defined the thermodynamic and structural basis of energetic coupling between the spectrin domains through differential scanning calorimetry (DSC), fluorescence lifetime (FLT), and Overhauser dynamic nuclear polarization (ODNP). A combined approach utilizing these methods will provide insight into how coupling between spectrin repeats affects signal propagation and mechanical force dissipation.