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 (TTRL55P, TTRV30M, TTRT119M, and TTRV122I). 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.