An engineered transthyretin monomer that is nonamyloidogenic, unless it ispartially denatured

Transthyretin (TTR) is a soluble human plasma protein that can be converted into amyloid by acid-mediated dissociation of the homotetramer into monome rs. The pH required for disassembly also results in tertiary structural cha nges within the monomeric subunits. To understand whether these tertiary st ructural changes are required for amyloidogenicity, we created the Phe87Met /Leu110Met TTR variant (M-TTR) that is monomeric according to analytical ul tracentrifugation and gel filtration analyses and nonamyloidogenic at neutr al pH. Results from far- and near-UV circular dichroism spectroscopy, one-d imensional proton NMR spectroscopy, and X-ray crystallography, as well as t he ability of M-TTR to form a complex with retinol binding protein, indicat e that M-TTR forms a tertiary structure at pH 7 that is very similar if not identical to that found within the tetramer. Reducing the pH results in te rtiary structural changes within the M-TTR monomer, rendering it amyloidoge nic, demonstrating the requirement for partial denaturation. M-TTR exhibits stability toward acid and urea denaturation that is nearly identical to th at characterizing wild-type (WT) TTR at low concentrations (0.01-0.1 mg/mL) , where monomeric WT TTR is significantly populated at intermediate urea co ncentrations prior to the tertiary structural transition. However, the kine tics of denaturation and fibril formation are much faster for M-TTR than fo r tetrameric WT TTR, particularly at near-physiological concentrations, bec ause of the barrier associated with the tetramer to folded monomer preequil ibrium. These results demonstrate that the tetramer to folded monomer trans ition is insufficient for fibril formation; further tertiary structural cha nges within the monomer are required.