Systematic study of the structure and aggregation propensity of transthyretin under different pH conditions

Transthyretin (TTR) is a homo-tetrameric transport protein that carries thyroxine (T₄) and retinol-binding protein (RBP). Dissociation of native tetramer of TTR (due to mutations or age-related oxidative modifications) into misfolded monomeric subunits leads to the formation of amyloid fibrils which are deposited in the extra-cellular matrix and eventually cause myriad of human diseases including TTR amyloidosis, diabetes, preeclampsia, cognitive impairment, to name a few. Yet, the role of local micro-environmental factors such as pH in the modulation of tetramer stability is not well-understood. In this study, we performed a systematic analysis of the impact of pH on the structure-function paradigm and aggregation propensity of TTR, using a decreasing pH range of 8.0-2.2. The T₄-binding capacity was preserved at physiological pH, showed marked decrease below pH 6.6 and complete functional loss ≤ 3.3. Structural analyses revealed progressive β-sheet destabilization and increased exposure of aromatic residues. High thermodynamic stability was seen at neutral pH while thermal unfolding at low pH with pH 3.3 exhibiting lowest <i>T_m</i>_. Aggregation propensity of TTR was found to be lowest at physiological pH while it formed dense amorphous aggregates at pH 4.4 and distinct ThT-positive amyloid fibrils at pH 3.3. The study defines a pH-dependent threshold for TTR destabilization and aggregation, offering mechanistic insights into amyloid initiation and highlighting local pH as a determinant of disease-relevant aggregation pathways.