A deficiency of functional dystrophin protein in muscle cells causes muscular dystrophy (MD). More than 50% of missense mutations that trigger the disease occur in the N-terminal actin binding domain (N-ABD or ABD1). We examined the effect of four diseasecausing mutations-L54R, A168D, A171P, and Y231N-on the structural and biophysical properties of isolated N-ABD. Our results indicate that N-ABD is a monomeric, well-folded α-helical protein in solution, as is evident from its α-helical circular dichroism spectrum, blue shift of the native state tryptophan fluorescence, well-dispersed amide crosspeaks in 2D NMR 15 N-1 H HSQC fingerprint region, and rotational correlation time calculated from NMR longitudinal ðT 1 Þ and transverse ðT 2 Þ relaxation experiments. Compared to WT, three mutants-L54R, A168D, and A171P-show a decreased α-helicity and do not show a cooperative sigmoidal melt with temperature, indicating that these mutations exist in a wide range of conformations or in a "molten globule" state. In contrast, Y231N has an α-helical content similar to WT and shows a cooperative sigmoidal temperature melt but with a decreased stability. All four mutants experience serious misfolding and aggregation. FT-IR, circular dichroism, increase in thioflavin T fluorescence, and the congo red spectral shift and birefringence show that these aggregates contain intermolecular cross-β structure similar to that found in amyloid diseases. These results indicate that disease-causing mutants affect N-ABD structure by decreasing its thermodynamic stability and increasing its misfolding, thereby decreasing the net functional dystrophin concentration.actin binding domain | Becker muscular dystrophy | calponin homology domain | Duchenne muscular dystrophy | protein aggregation

The C-linked carbo-beta-peptides, oligomers of a new class of C-linked carbo-beta3-amino acids, have been shown to generate mixed 12/10 and 10/12 helices. The design involves use of "alternating chirality" of the epimeric (at the amine center) monomers to control the stability of these helices. The observation of stable 12/10 helix in a tripeptide and 10/12 helix in a tetrapeptide is unprecedented.

New classes of alpha/gamma- and beta/gamma-hybrid peptides have been synthesized with novel 12/10- and 11/13-mixed helical patterns, respectively. The alpha/gamma-peptides were derived from the dipeptide repeats with alternating arrays of l-Ala and gamma-Caa((l)) (C-linked carbo-gamma-amino acid from d-mannose), which generated a new 12/10-mixed helix, for the first time, without a beta-amino acid. The beta/gamma-peptides made from an alternating arrangement of beta-Caa((x)) (C-linked carbo-beta-amino acid) and gamma-Caa((x)) (C-linked carbo-gamma-amino acid from d-xylose), on the other hand, resulted in an unprecedented 11/13-helix. The secondary structures in these peptides have been ascertained from detailed NMR studies, and CD spectroscopy and molecular dynamics investigations provided additional support for the structures derived.

Muscular dystrophy (MD) is the most common genetic lethal disorder in children. Mutations in dystrophin trigger the most common form of MD, Duchenne and its allelic variant Becker MD. Utrophin is the closest homologue and has been shown to compensate for the loss of dystrophin in human disease animal models. However, the structural and functional similarities and differences between utrophin and dystrophin are less understood. Both proteins interact with actin through their N-terminal actin-binding domain (N-ABD). In this study, we examined the thermodynamic stability and aggregation of utrophin N-ABD and compared with that of dystrophin. Our results show that utrophin N-ABD has spectroscopic properties similar to dystrophin N-ABD. However, utrophin N-ABD has decreased denaturant and thermal stability, unfolds faster, and is correspondingly more susceptible to proteolysis, which might account for its decreased in-vivo half-life compared to dystrophin. In addition, utrophin N-ABD aggregates to a lesser extent compared with dystrophin N-ABD, contrary to the general behavior of proteins in which decreased stability enhances protein aggregation. Despite these differences in stability and aggregation, both proteins exhibit deleterious effects of mutations. When utrophin N-ABD mutations analogous in position to the dystrophin disease-causing mutations were generated, they behaved similarly to dystrophin mutants in terms of decreased stability and the formation of cross-β aggregates, indicating a possible role for utrophin mutations in disease mechanisms.

In a twist: Tethering of short peptides with robust 10/12‐mixed helices, derived from C‐linked carbo‐β‐amino acids, to the turn‐inducing motif, β‐hGly‐D‐Pro‐Gly‐β‐hGly, permitted a de novo design of the helix–turn–helix motif in the foldamer domain.