The vibrational modes corresponding to protein tertiary structural motion lay in the far infrared or terahertz frequency range. These collective large scale motions depend on global structure and thus will necessarily be perturbed by ligand binding events. We discuss the use of terahertz dielectric spectroscopy to measure these vibrational modes and the sensitivity of the technique to changes in protein conformation, oxidation state and environment. A challenge of
applying this sensitivity as a spectroscopic assay for ligand binding is the sensitivity of the technique to both bulk water
and water bound to the protein. This sensitivity can entirely obscure the signal from the protein or protein-ligand complex itself, thus necessitating sophisticated sample preparation making the technique impractical for industrial applications. We discuss methods to overcome this background and demonstrate how terahertz spectroscopy can be used to quickly assay protein binding for proteomics and pharmaceutical research.
The terahertz dielectric response of partially thermally denatured, hen egg white lysozyme (HEWL) films is measured as a function of frequency and hydration using terahertz time domain spectroscopy (THz-TDS). Results are compared to similar measurements on native state samples. The frequency and hydration dependence of the absorbance for the two sample types are highly similar except for a notable suppression at ~ 0.4 THz (13 cm<sup>-1</sup>) in the partially denatured sample. In contrast to the native state sample which has a nearly frequency independent index of refraction, the index of the partially denatured sample decreases as a function of frequency. A transition is observed in both the absorbance and the index at a hydration level of ~ 0.25h (grams H<sub>2</sub>O per gram lysozyme). Below the transition, the response slowly increases while above 0.25h, the slope of both the absorbance and index sharply increases. Interestingly, we observed similar behavior in the native sample. The Cole-Cole plots exhibit a hydration dependence that is distinct from the native sample and indicative of neither pure resonance nor dielectric relaxation. We consider the implications of these results on THz biomolecular sensors.
We demonstrate the use of terahertz time domain spectroscopy for determination of ligand binding for biomolecules. Vibrational modes associated with tertiary structure conformational motions lay in the THz frequency range. We examine the THz dielectric response for hen egg white lysozyme (HEWL): free and bound with tri-N-acetyl-D-glucosamine. Transmission measurements on thin films show that there is a small change in the real part of the refractive index as a function of binding and a sizable decrease in the absorbance. A phenomenological model is used to determine the source of the absorbance change. A change in the vibrational mode density of states and net dipole moment changes will necessarily happen for all biomolecule-ligand binding, thus THz dielectric measurements may provide an universally applicable method to determine probe-target binding for biosensor applications.