Anion shielding of electrostatic repulsions in transthyretin modulates stability and amyloidosis: Insight into the chaotrope unfolding dichotomy

The balance between stabilizing forces and the localized electrostatic repu lsions destabilizing the transthyretin (TTR) tetramer is tunable via anion shielding. The two symmetrical anion interaction sites in TTR are comprised of residues Lys15 and Lys15 from opposing subunits on the periphery of the two thyroxine binding sites. These is an element of -ammonium groups repel one another and destabilize the tetramer, unless an appropriate anion is p resent, which stabilizes the tetramer. Chaotrope denaturation of TTR exhibi ts unusual behavior in that urea appears to be a stronger denaturant than G dmCl (guanidinium chloride), even though GdmCl is typically twice as powerf ul as a denaturant. The shift in the midpoint of the urea denaturation curv e to higher concentrations as well as the increase in the mole fraction of tetramer that is highly resistant to denaturation with increasing KCI conce ntration provides strong evidence that anion shielding stabilizes the TTR t etramer. A consequence of tetramer stabilization is folding hysteresis, bec ause the high GdmCl concentrations required to denature the anion-stabilize d tetramer do not allow refolding of the unfolded monomers. The formation o f amyloid fibrils by TTR requires that its normal tetrameric structure diss ociate to alternatively folded monomers, a process mediated by acidificatio n (pH 5-4). This process is inhibited by Cl- ions in a concentration-depend ent fashion. Chloride ion may not be the relevant physiological TTR stabili ty modulator, but it is the main focus of these studies explaining the hyst eresis observed in the denaturation and refolding studies with GdmCl.