Effects of Hydration on Transthyretin Conformational Dynamics and Oligomerization

Transthyretin (TTR) is a 56 kDa tetrameric protein complex that transports thyroxine and retinol but can misfold, causing amyloid diseases, such as senile systemic amyloidosis, familial amyloid cardiomyopathy, and familial amyloid polyneuropathy. Previous studies have found that TTR aggregation is initiated when tetramers disassemble into monomers, dimers, and trimers, which misfold and assemble into heterogeneous oligomers. These oligomers are thought to be cytotoxic, yet their formation and composition remain poorly understood. To investigate monomer misfolding, ion mobility-mass spectrometry (IM-MS) was applied to wild-type TTR (wtTTR) and the pathogenic L55P variant under varying pH conditions. IM-MS revealed that acidic pH promotes extended monomer conformations for both wtTTR and L55P. Additionally, L55P showed a higher abundance of extended conformations that are attributed to its increased amyloidogenicity. Orbitrap-based charge detection mass spectrometry is used via the direct mass technology (DMT) mode to evaluate oligomeric species, revealing that acidic pH and lower temperatures promote oligomerization and L55P formed oligomers more readily than wtTTR. Together, these results show that oligomerization and conformational changes depend on solution pH, temperature, and proteoform, supporting the role that changes in hydration play in TTR aggregation. More broadly, these findings demonstrate the complementary strengths of IM-MS and DMT for characterizing aggregation intermediates and provide new insights into TTR aggregation.