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 disease-causing 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 alpha-helical protein in solution, as is evident from its alpha-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 (T(1)) and transverse (T(2)) relaxation experiments. Compared to WT, three mutants--L54R, A168D, and A171P--show a decreased alpha-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 alpha-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-beta 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.