Alteration of tropomyosin function and folding by a nemaline myopathy-causing mutation

Mutations in the human TPM3 gene encoding gamma -tropomyosin (ac-tropomyosi n-slow) expressed in slow skeletal muscle fibers cause nemaline myopathy. N emaline myopathy is a rare, clinically heterogeneous congenital skeletal mu scle disease with associated muscle weakness, characterized by the presence of nemaline rods in muscle fibers. In one missense mutation the codon corr esponding to Met-8, a highly conserved residue, is changed to Arg. Here, a rat fast alpha -tropomyosin cDNA with the Met8Arg mutation was expressed in Escherichia coli to investigate the effect of the mutation on in vitro fun ction. The Met8Arg mutation reduces tropomyosin affinity for regulated acti n 30- to 100-fold. Ca2+-sensitive regulatory function is retained, although activation of the actomyosin S1 ATPase in the presence of Ca2+ is reduced. The poor activation may reflect weakened actin affinity or reduced effecti veness in switching the thin filament to the open, force-producing state. T he presence of the Met8Arg mutation severely, but locally, destabilizes the tropomyosin coiled coil in a model peptide, and would be expected to impai r end-to-end association between TMs on the thin filament. In muscle, the m utation may alter thin filament assembly consequent to lower actin affinity and altered binding of the N-terminus to tropomodulin at the pointed end o f the filament. The mutation may also reduce force generation during activa tion.