The Acid-Mediated Denaturation Pathway of Transthyretin Yields a Conformational Intermediate That Can Self-Assemble into Amyloid

Abstract

Transthyretin (TTR) amyloid fibril formation is observed during partial acid denaturation and while refolding acid-denatured TTR, implying that amyloid fibril formation results from the self-assembly of a conformational intermediate. The acid denaturation pathway of TTR has been studied in detail herein employing a variety of biophysical methods to characterize the intermediate(s) capable of amyloid fibril formation. At physiological concentrations, tetrameric TTR remains associated from pH 7 to pH 5 and is incapable of amyloid fibril formation. Tetrameric TTR dissociates to a monomer in a process that is dependent on both pH and protein concentration below pH 5. The extent of amyloid fibril formation correlates with the concentration of the TTR monomer having an altered, but defined, tertiary structure over the pH range of 5.0-3.9. The inherent Trp fluorescence-monitored denaturation curve of TTR exhibits a plateau over the pH range where amyloid fibril formation is observed (albeit at a higher concentration), implying that a steady-state concentration of the amyloidogenic intermediate with an altered tertiary structure is being detected. Interestingly, 1-anilino-8-naphthalenesulfonate fluorescence is at a minimum at the pH associated with maximal amyloid fibril formation (pH 4.4), implying that the amyloidogenic intermediate does not have a high extent of hydrophobic surface area exposed, consistent with a defined tertiary structure. Transthyretin has two Trp residues in its primary structure, Trp-41 and Trp-79, which are conveniently located far apart in the tertiary structure of TTR. Replacement of each Trp with Phe affords two single Trp containing variants which were used to probe local pH-dependent tertiary structural changes proximal to these chromophores. The pH-dependent fluorescence behavior of the Trp-79-Phe mutant strongly suggests that Trp-41 is located near the site of the tertiary structural rearrangement that occurs in the formation of the monomeric amyloidogenic intermediate, likely involving the C-strand-loop-D-strand region. Upon further acidification of TTR (below pH 4.4), the structurally defined monomeric amyloidogenic intermediate begins to adopt alternative conformations that are not amyloidogenic, ultimately forming an A-state conformation below pH 3 which is also not amyloidogenic. In summary, analytical equilibrium ultracentrifugation, SDS-PAGE, far- and near-UV CD, fluorescence, and light scattering studies suggest that the amyloidogenic intermediate is a monomeric predominantly beta-sheet structure having a well-defined tertiary structure.

Protofilaments, Filaments, Ribbons, and Fibrils from Peptidomimetic Self-Assembly: Implications for Amyloid Fibril Formation and Materials Science

“…A Congo red stock solution was prepared by dissolving recrystallized Congo red as described previously. 89 The concentration of the Congo red stock was determined by measuring the absorbance of a diluted aliquot at 477 nm ( 477 nm ) 34 722 cm -1 M -1 ). 94 Buffered Congo red solutions at 2.5 µm were prepared by dilution of an aliquot of the stock solution in 50 mM acetate, phosphate, or carbonate-bicarbonate buffer solutions at the desired pH.…”

Potentially amyloidogenic conformational intermediates populate the unfolding landscape of transthyretin: Insights from molecular dynamics simulations

“…7 Additionally, amyloidogenic TTR variants were shown to produce, in solution and even at neutral pH, higher amounts of partially unfolded monomeric species with enhanced propensity for ordered aggregation into amyloid fibrils. 10,11 Therefore, the conformational stability of the TTR monomers undoubtedly plays a critical role in the process of amyloid formation.…”

The Sequence-Dependent Unfolding Pathway Plays a Critical Role in the Amyloidogenicity of Transthyretin

“…Such intermediates, once formed, undergo a downhill self-assembly process (15). The monomeric intermediate was first characterized as adopting an altered tertiary structure, possibly with the C strandloop-D strand motif dislocated from the core of the protein (16). This partial misfolding mechanism was supported later by a search for structural determinants of the amyloidogenic fold (17), in which two monoclonal antibodies bind specifically to regions comprising residues 39-44 (in the C strand) and 56-61 (after the D strand) only when the C and D strands adopt a non-native conformation.…”