Chemical dissection and reassembly of amyloid fibrils formed by a peptide fragment of transthyretin

We have examined the chemical dissection and subsequent reassembly of fibri ls formed by a ten-residue peptide to probe the forces that drive the forma tion of amyloid. The peptide, TTR10-19, encompasses the A strand of the inn er beta-sheet structure that lines the thyroid hormone binding site of the human plasma protein transthyretin. When dissolved in water under low pH co nditions the peptide readily forms amyloid fibrils. Electron microscopy of these fibrils indicates the presence of long (>1000 nm) rigid structures of uniform diameter (approximately 14 nm). Addition of urea (3 M) to preforme d fibrils disrupts these rigid structures. The partially disrupted fibrils form flexible ribbon-like arrays, which are composed of a number of clearly visible protofilaments (3-4 run diameter). These protofilaments are highly stable, and resist denaturation in 6 M. urea at 75 degrees C over a period of hours. High concentrations (>50%, v/v) of 2,2,2-trifluoroethanol also d issociate TTR10-19 fibrils to the constituent protofilaments, but these slo wly dissociate to monomeric, soluble peptides with extensive cl-helical str ucture. Dilution of the denaturant or co-solvent at the stage when dissocia tion to protofilaments has occurred results in the efficient reassembly of fibrils. These results indicate that assembly of fibrils from protofilament s involves relatively weak and predominantly hydrophobic interactions,. whe reas assembly of peptides into protofilaments involves both electrostatic a nd hydrophobic forces, resulting in a highly stable and compact structures. (C) 2000 Academic Press.