Equilibrium versus Nonequilibrium Peptide Dynamics: Insights into Transient 2D IR Spectroscopy.

Over the past two decades, two-dimensional infrared (2D IR) spectroscopy has evolved from the theoretical underpinnings of nonlinear spectroscopy as a means of investigating detailed molecular structure on an ultrafast time scale. The combined time and spectral resolution over which spectra can be collected on complex molecular systems has led to the precise structural resolution of dynamic species that have previously been impossible to directly observe through traditional methods. The adoption of 2D IR spectroscopy for the study of protein folding and peptide interactions has provided key details of how small changes in conformations can exert major influences on the activities of these complex molecular systems. Traditional 2D IR experiments are limited to molecules under equilibrium conditions, where small motions and fluctuations of these larger molecules often still lead to functionality. Utilizing techniques that allow the rapid initiation of chemical or structural changes in conjunction with 2D IR spectroscopy, i.e., transient 2D IR, a vast dynamic range becomes available to the spectroscopist uncovering structural content far from equilibrium. Furthermore, this allows the observation of reaction pathways of these macromolecules under quasi- and nonequilibrium conditions.

Tyrosine as a Non-perturbing Site-Specific Vibrational Reporter for Protein Dynamics.

The ability to detect changes in the local environment of proteins is pivotal to determining their dynamic nature during many biological processes. For this purpose, the utility of the tyrosine ring breathing vibration as a sensitive infrared reporter for measuring the local electric field in protein is investigated. Variations in the bandwidth of this vibrational transition in a variety of solvents indicate differences in microenvironment affect the inhomogeneous broadening and thus the frequency distribution. The ring mode is influenced by direct and indirect interactions associated with the charge distribution of the surrounding solvent molecules. Molecular dynamics simulations were implemented to obtain a correlation between the electric field induced by the solvent on the mode and the observed vibrational bandwidth. Moreover, the Trp-cage was synthesized as a model peptide system to access the efficacy of the correlation to predict the electric field strength within the hydrophobic core of the native and denatured states of the protein. The 2D IR spectra of tyrosine in dimethyl sulfoxide (DMSO) and water (D2O) show a two-fold difference in the time constant of the vibrational dynamics alluding to the dephasing mechanisms of the vibration and supporting the model put forth about the solvachromatic nature of the transition.

Two-Dimensional Infrared Study of Vibrational Coupling between Azide and Nitrile Reporters in a RNA Nucleoside.

The vibrations in the azide, N3, asymmetric stretching region and nitrile, CN, symmetric stretching region of 2'-azido-5-cyano-2'-deoxyuridine (N3CNdU) are examined by two-dimensional infrared (2D IR) spectroscopy. At earlier waiting times, the 2D IR spectrum shows the presence of both vibrational transitions along the diagonal and off-diagonal cross peaks indicating vibrational coupling. The coupling strength is determined from the off-diagonal anharmonicity to be 66 cm(-1) for the intramolecular distance of ∼7.9 Å, based on a structural map generated for this model system. In addition, the frequency-frequency correlation decay is detected, monitoring the solvent dynamics around each individual probe position. Overall, these vibrational reporters can be utilized in tandem to simultaneously track global structural information and fast structural fluctuations.