The Two-Dimensional Vibrational Echo of a Nitrile Probe of the Villin HP35 Protein

Two-dimensional (2D) IR spectroscopy was used to probe the hydrophobic core structure of the 35-residue villin headpiece subdomain, HP35, by monitoring the C N vibrational stretching band of a cyano-substituted phenylalanine (Phe). The presence of two humps in the vibrational frequency distribution in the folded equilibrium state is revealed. They represent two states that exchange more slowly than ca. -10 ps. The two CN stretch mode peak frequencies (and their equilibrium populations ) are 2228.7 (44%) and 2234.5 cm(-1) (56%). The two CN modes have different frequency-frequency correlation times of 7.4 and 1.6 ps, respectively. These results suggest that the population with the higher frequency CN group is partly exposed, whereas the other CN mode experiences a hydrophobic-like environment.