Capture of a dimeric intermediate during transthyretin amyloid formation

Point mutations in the human plasma protein transthyretin are associated wi th the neurological disorder familial amyloidosis with polyneuropathy type 1. The disease is characterized by amyloid fibril deposits causing damage a t the site of deposition. Substitution of two amino acids in the hydrophobi c core of transthyretin lead to a mutant that was very prone to form amyloi d. In addition, this mutant has also been shown to induce a toxic response on a neuroblastoma cell line. Renaturation of the transthyretin mutant at l ow temperature facilitated the isolation of an amyloid-forming intermediate state having the apparent size of a dimer. Increasing the temperature effe ctively enhanced the rate of interconversion from a partly denatured protei n to mature amyloid. Using circular dichroism the beta -sheet content of th e formed mature fibrils was significantly lower than that of the native fol d of transthyretin. Morphology studies using electron microscopy also indic ated a temperature-dependent transformation from amorphous aggregates towar d mature amyloid fibrils. In addition, 1-anilino-S-naphtalenesulfonate fluo rescence studies suggested the loss of the thyroxin-binding channel within both the isolated intermediate and the mature fibrils.