Molecular extensibility of mini-dystrophins and a dystrophin rod construct.
Muscular dystrophies arise with various mutations in dystrophin, implicating this protein in force transmission in normal muscle. With 24 three-helix, spectrin repeats interspersed with proline-rich hinges, dystrophin's large size is an impediment to gene therapy, prompting the construction of mini-dystrophins. Results thus far in dystrophic mice suggest that at least one hinge between repeats is necessary though not sufficient for palliative effect. One such mini-dystrophin is studied here in forced extension at the single molecule level. Delta2331 consists of repeats (R) and hinges (H) H1-R1-2 approximately H3 approximately R22-24-H4 linked by native (-) and non-native (approximately) sequence. This is compared to its core fragment R2 approximately H3 approximately R22 as well as an eight-repeat rod fragment middle (RFM: R8-15). We show by atomic force microscopy that all repeats extend and unfold at forces comparable to those that a few myosin molecules can generate. The hinge regions most often extend and transmit force while limiting tandem repeat unfolding. From 23-42 degrees C, the dystrophin constructs also appear less temperature-sensitive in unfolding compared to a well-studied betaI-spectrin construct. The results thus reveal new modes of dystrophin flexibility that may prove central to functions of both dystrophin and mini-dystrophins.
Bhasin
Nishant
N
Pennsylvania Muscle Institute and Graduate Groups in Physics and Cell & Molecular Biology, University of Pennsylvania, Philadelphia, PA 19104, USA.
Law
Richard
R
Liao
George
G
Safer
Daniel
D
Ellmer
Jennifer
J
Discher
Bohdana M
BM
Sweeney
H Lee
HL
Discher
Dennis E
DE
D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D016189:Dystrophin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation
D016189:Dystrophin