# Anion-Facilitated Hydrogen–Deuterium Exchange as a Tool to Probe Weak Anion–Protein Interactions Responsible for Hofmeister Effects

## TL;DR

This study explores how anions affect protein structure using hydrogen-deuterium exchange, revealing different denaturation mechanisms based on anion type.

## Contribution

The novel use of HDX mapping to detect weak anion-protein interactions and distinguish Hofmeister effect mechanisms.

## Key findings

- Charge-dense Cl– induces minor denaturation via N-terminal interactions.
- Charge-diffuse anions cause more significant unfolding by intercalating into the peptide core.
- HDX mapping provides more detailed insights than NMR chemical shifts for anion binding.

## Abstract

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.