The binding of protein to metal ions is essential to the functioning of thousands of enzymes. Although the action of many proteins is well known, there is less concrete knowledge about the mechanisms behind them because traditional methods such as NMR are orders of magnitude too slow to observe the dynamics. Two-dimensional infrared spectroscopy is able to probe molecular structure and dynamics on the sub-picosecond time scale. The fundamentals of 2D-IR spectroscopy is developed to enable the understanding of the process. The binding sites of many proteins such as those containing the widespread EF-hand motif rely on carbonyl ligands, both backbone amides and side-chain carboxylates. Butyramide and EDTA are analyzed with linear Fourier-transform and two-dimensional infrared spectroscopy. The data are consistent with published literature. Butyramide shows a binding time scale with water of 600 fs, which is consistent with the known weak binding of amides to metal in aqueous solutions. Vibrational frequencies are calculated using density functional theory. The carboxylate normal modes of calcium-bound EDTA show delocalization and energy splitting consistent with vibrational excitons
