No abstract

Spectrin repeat domains are a highly conserved biological motif found in many human structural proteins. Dystrophin contains 24 tandem spectrin repeats which provide structural support through mediating interactions between intracellular actin filaments and the extracellular matrix. However, the molecular mechanism by which dystrophin provides this support is unknown. Understanding this underlying structure/function relationship is important because mutations in dystrophin directly cause muscular dystrophy. Thus far, the following constructs have been expressed in E. coli, purified using chromatography, and thermodynamically characterized: S 17 , S 17-18, S 17-19. Parameters were determined through globally fitting thermal denaturation signals from Fluorescence Spectroscopy (FS), Circular Dichroism (CD), and Fluorescence Lifetime Spectroscopy (FLT) to a two-state model of unfolding. Fits were constrained using DC p values determined using Differential Scanning Calorimetry (DSC). This parameterization then allowed for determination of the free energy of stability (DG unfolding) of each construct. Results indicate that the DG unfolding of S 17 is nearly double that of S 17-19. This pronounced non-additivity indicates that tandem spectrin repeats mutually destabilize each other, termed negative coupling. Additionally, the comparison of Electron Paramagnetic Resonance (EPR) spectra of S 17 and S 17-19 indicate that the trimer exhibits greater local confirmational flexibility, consistent with decreased stability. To further test this negative coupling hypothesis, we are purifying S 19 for thermodynamic characterization. This will allow for the comparison of the sum of S 19 and S 17-18 's DG unfolding values with that of the trimer, helping further reveal the energetic and structural basis of dystrophin's mechanism.