Water Plays Key Roles in Stabilities of Wild Type and Mutant Transthyretin Complexes.

Transthyretin (TTR), a 56 kDa homotetramer that is involved in the transport of thyroxine and retinol, has been linked to amyloidosis through disassembly of tetramers to form monomers, dimers, and trimers that then reassemble into higher order oligomers and/or fibrils. Hybrid TTR (hTTR) tetramers are found in heterozygous individuals that express both wild type TTR (wt-TTR) and mutant TTR (mTTR) forms of the protein, and these states display increased rates of amyloidosis. Here we monitor subunit exchange (SUE) reactions involving homomeric and mixed tetramers using high resolution native mass spectrometry (nMS). Our results show evidence that differences in TTR primary structure alter tetramer stabilities, and hTTR products can form spontaneously by SUE reactions. In addition, we find that solution temperature has strong effects on TTR tetramer stabilities and formation of SUE products. Lower temperatures promote formation of hTTR tetramers containing L55P and V30M subunits, whereas small effects on the formation of hTTR tetramers containing F87A and T119M subunits are observed. We hypothesize that the observed temperature dependent stabilities and subsequent SUE behavior are a result of perturbations to the network of "two kinds of water": hydrating and structure stabilizing water molecules (Spyrakis et al. J. Med. Chem. 2017, 60 (16), 6781-6827; Xu et al. Soft Matter 2012, 8, 324-336) that stabilize wt-TTR and mTTR tetramers. The results presented in this work illustrate the utility of high resolution nMS for studies of the structures, stabilities, and dynamics of protein complexes that directly influence SUE reactions.