Hydration (H₂O and D₂O) Dictates the Stabilities and Conformational Entropy of Transthyretin Tetramers

Transthyretin (TTR) is a 56 kDa tetrameric protein complex that plays a key role in transporting thyroxine and retinol. Despite its important biological role, TTR exhibits adverse effects, including disassembly into monomers, dimers, and trimers that then aggregate into disease-causing oligomers and fibrils. Hydration (referring to H₂O or D₂O) has been shown to have strong effects on TTR tetramer stabilities; however, probing how perturbation of hydration alters tetramer dynamics and stabilities is challenging. Here, we use variable-temperature (5-50 °C) electrospray ionization and ion mobility-mass spectrometry to better understand the influence of D₂O and H₂O hydration on the structures, stabilities, and dynamics of both wild-type TTR and several mutants (TTR^L55P, TTR^V30M, TTR^T119M, and TTR^V122I). Our findings include the following: (i) TTR tetramers in D₂O have lower average charge states compared to those in H₂O at temperatures between 5 and 50 °C; (ii) there are fewer disassembly products (monomers and dimers) in D₂O compared to H₂O at different temperatures; and (iii) the relative abundances of compact versus extended tetrameric forms are shifted in D₂O compared to H₂O. Results from hydrogen-deuterium exchange (HDX) combined with bottom-up proteomics of different TTR mutants are consistent with hydration in β-sheet and interface-forming regions providing tetramer stability. Additionally, HDX combined with bottom-up proteomics indicates that shifts in hydration at the interface are responsible for the observed differences between mutants. Overall, our results show that D₂O significantly alters the TTR stabilities and conformational entropy, highlighting the important role that hydration plays in protein dynamics.