Desmoplakin (DSP) is a large (~260 kDa) protein found in the desmosome, a subcellular complex that links the cytoskeleton of one cell to its neighbor. A mutation ‘hot-spot’ within the NH2-terminal third of the DSP protein (specifically, residues 299–515) is associated with both cardiomyopathies and skin defects. In select DSP variants, disease is linked specifically to the uncovering of a previously-occluded calpain target site (residues 447–451). Here, we partially stabilize these calpain-sensitive DSP clinical variants through the addition of a secondary single point mutation—tyrosine for leucine at amino acid position 518 (L518Y). Molecular dynamic (MD) simulations and enzymatic assays reveal that this stabilizing mutation partially blocks access to the calpain target site, resulting in restored DSP protein levels. This ‘molecular band-aid’ provides a novel way to maintain DSP protein levels, which may lead to new strategies for treating this subset of DSP-related disorders.

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migration in Cygb, Ngb and GbX, we replaced the non-fluorescent Fe protoporphyrin IX (FePPIX) with the fluorescent analogue Zn protoporphyrin IX (ZnPPIX) and characterized the ligand migration by monitoring the quenching of the ZnPPIX fluorescent emission by methyl viologen and the quenching of the ZnPPIX triplet state by oxygen. We observed an increase in the Ksv value for methyl viologen quenching in protein variants that are missing the internal disulfide bond, suggesting that the presence of the intraprotein disulfide bond regulates the accessibility of the heme binding pocket. Interestingly the lifetime of the triplet state for ZnPPIX incorporated in hexacoordinate vertebrate globins is not sensitive to changes in the heme environment as the observed lifetime is comparable to that determined previously for myoglobin.

force clamp experiments. Further experimentation using optical tweezers allowed the determination of unfolding rates and contour length changes associated with the folding/unfolding pathway of SNase.