Hydrogen Bond Exchange and Ca²⁺ Binding of Aqueous N-Methylacetamide Revealed by 2DIR Spectroscopy

Cation effects on proteins have been a challenge to understand. Herein, we present two-dimensional infrared (2DIR) spectroscopic measurements, coupled with molecular dynamics and spectroscopic calculations, of N-methylacetamide (NMA), a common model of the peptide backbone, in aqueous CaCl₂. The 2DIR spectra reveal that the dynamics of the amide carbonyl of NMA is dominated by exchange between two states of varying hydration, one possessing a structure similar to aqueous NMA and one that is dehydrated by one hydrogen bond. In addition, we demonstrate that at high (>5 M) CaCl₂ concentrations, direct binding of Ca²⁺ to the carbonyl of NMA occurs, stabilizing an iminium-type resonance structure of NMA, with a characteristic C═N⁺ stretch frequency at 1680 cm^-1. This species is only marginally populated and is only detectable in 2DIR spectra due to its larger transition strength.