A biophysical map of the dystrophin rod

Abstract

We have conducted a biophysical scan of the rod region of dystrophin, targeting all 24 single spectrin type repeat, STR, motifs and 23 2-STR tandem motifs. Of these 47 targets, we were able to express and purify 39 and have characterized them with regard to various stability metrics: thermodynamic stability as assessed by thermal and solvent denaturation, as well as resistance to proteolysis. We find that while all measured parameters varied greatly throughout the rod, there was no general stabilization of the 2-STR motifs over single STR motifs. However, stabilization by thermodynamic interaction was seen in six regions: strongly in D16:17 and D21:22 and to a lesser extent in D2:3, D4:5, D6:7 and D20:21. This indicates that these STRs interact structurally. In the rest of the rod, no cooperativity was seen and STRs appear to be thermodynamically independent. Stability also varied widely along the rod, with some motifs that are barely stable, beginning to unfold at physiological temperatures; these are largely found in the central rod region from D7 to D15. Regions of high stability were found in the interacting motifs, as well as a general trend toward increasing stability at the C-terminus of the rod. Interestingly, the rod region nNOS binding site occurs at such an interacting, very stable site, D16:17. Overall this describes a highly heterogeneous rod region.