Infrared probes based on nitrile-derivatized prolines: Thermal insulation effect and enhanced dynamic range

Vibrational chromophores that are sensitive to local electrostatic environment are useful probes of structural variations of proteins on subnanosecond time scales, but their short vibrational lifetimes often limit their applicability. Here we explore a possibility to increase the lifetime of nitrile probes by introducing heavy atoms between the probe and protein side chains. Stereoisomers of thiocyanato- and selenocyanato-derivatized prolines, Pro-SCN and Pro-SeCN, are synthesized, and their CN stretch lifetimes in D ₂O and chloroform are measured with polarization-controlled IR pump-probe spectroscopy. The measured lifetimes of 170-330 ps for Pro-SeCN are three to four times longer than those for Pro-SCN, indicating that selenium atom is more effective than sulfur atom in blocking the intramolecular vibrational relaxation pathways of the CN stretch mode. This is further confirmed by carrying out nonequilibrium molecular dynamics simulations of the vibrational relaxation processes. Given the crucial role of the proline residue in determining protein structures, we anticipate that the Pro-SeCN probe can be an excellent site-specific probe of changes in protein local environment.