Isotope-labeled aspartate sidechain as a non-perturbing infrared probe: Application to investigate the dynamics of a carboxylate buried inside a protein

[Display omitted] •We demonstrate a new protein vibrational probe.•This probe is useful to study electrostatic interactions with aspartate or glutamate.•Using this probe, we show that water may exist in the interior of a small protein. Because of their negatively charged carboxylates, aspartate and glutamate are frequently found at the active or binding site of proteins. However, studying a specific carboxylate in proteins that contain multiple aspartates and/or glutamates via infrared spectroscopy is difficult due to spectral overlap. We show, herein, that isotopic-labeling of the aspartate sidechain can overcome this limitation as the resultant 13COO− asymmetric stretching vibration resides in a transparent region of the protein IR spectrum. Applicability of this site-specific vibrational probe is demonstrated by using it to assess the dynamics of an aspartate ion buried inside a small protein via two-dimensional infrared spectroscopy.