Force-bearing linkages between the cytoskeleton and extracellular matrix are clearly important to normal cell viability-as is evident in a disease such as Duchenne muscular dystrophy (DMD) which arises in the absence of the linkage protein dystrophin. Therapeutic approaches to DMD include antisense-mediated skipping of exons to delete nonsense mutations while maintaining reading frame, but the structure and stability of the resulting proteins are generally unclear. Here we use mass spectrometry to detect most dystrophin exons, and we express and physically characterize dystrophin "nano"-constructs based on multiexon deletions that might find use in a large percentage of DMD patients. The primary structure challenge is addressed first with liquid chromatography tandem mass spectrometry (LC-MS/MS) which can detect tryptic peptides from 53 of dystrophin's 79 exons; equivalent information from immunodetection would require 53 different high-specificity antibodies. Folding predictions for the nano-constructs reveal novel helical bundle domains that arise out of exon-deleted "linkers," while secondary structure studies confirm high helicity and also melting temperatures well above physiological. Extensional forces with an atomic force microscope nonetheless unfold the constructs, and the ensemble of unfolding trajectories reveal the number of folded domains, proving consistent with structure predictions. A mechanical cooperativity parameter for unfolding of tandem domains is also introduced as the best predictor of a multiexon deletion that is asymptomatic in humans. The results thereby provide insight and confidence in exon-skipped designs.

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2996834PMC
http://dx.doi.org/10.1002/cm.20489DOI Listing

Publication Analysis

Top Keywords

mass spectrometry
12
duchenne muscular
8
muscular dystrophy
8
exon-skipped dystrophins
4
dystrophins treatment
4
treatment duchenne
4
dystrophy mass
4
spectrometry mapping
4
exons
4
mapping exons
4

Similar Publications

Want AI Summaries of new PubMed Abstracts delivered to your In-box?

Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!