Search for intermediate structures in transthyretin fibrillogenesis: Soluble tetrameric Tyr78Phe TTR expresses a specific epitope present only in amyloid fibrils

Familial Amyloidotic Polyneuropathy (FAP) is caused by the assembly of TTR into an insoluble beta -sheet. The TTR tetramer is thought to dissociate in to monomeric intermediates and subsequently polymerise into the pathogenic arnyloid form. The biochemical mechanism behind this transformation is unkn own. We characterised intermediate TTR structures in the in vitro amyloidog enesis pathway by destabilising the AB loop through substitution of residue 78. Changes at this residue, should destabilise the TTR tetrameric fold, b ased on the known crystallographic structure of a Leu55Pro transthyretin va riant. We generated a soluble tetrameric form of TTR that is recognised by a monoclonal antibody, previously reported to react only with highly amyloi dogenic mutant proteins lacking the tetrameric native fold and with amyloid fibrils. BIAcore system analysis showed that Tyr78Phe had similar binding properties as synthetic fibrils. The affinity of this interaction was 10(7) M-1 We suggest that the tetrameric structure of Tyr78Phe is altered due to the loosening of the AB loops of the tetramer, leading to a structure that might represent an early intermediate in the fibrillogenesis pathway. (C) 2000 Academic Press.