Anion-Facilitated Hydrogen-Deuterium Exchange as a Tool to Probe Weak Anion-Protein Interactions Responsible for...

Impeded by the complexity of proteinaceous structure and the very weak nature of the noncovalent interactions involved, the detailed mechanisms by which anions induce salting-in Hofmeister effects in proteins and peptides remain unclear. Here, using β-hairpin peptides as models, we examine two approaches to qualify (map) anion binding: ¹H NMR chemical shifts and hydronium-catalyzed hydrogen-deuterium exchange (HDX) rate changes. We demonstrate that each salt investigated─despite an affinity too weak to quantify accurately, caused denaturation to an extent that is both peptide and anion-specific, with more charge-diffuse anions inducing a greater degree of unfolding. Our studies reveal that the HDX mapping provides more detail than chemical shift data. Thus, HDX mapping reveals two slightly different mechanisms of denaturation, depending on the nature of the anion. Namely, assisted by a N-terminal Arg residue, charge-dense Cl^- is chelated by the terminal N-H groups of the hairpin and induces a small degree of denaturation, whereas charge-diffuse anions intercalate deeply into the cation-π-hydrophobic core of the peptide and induce more substantial unfolding. These findings provide a glimpse of the different mechanisms by which anions can induce the salting-in Hofmeister effect in peptides and proteins and suggest HDX as a useful tool to map weak anion binding.