Yunfen He

Terahertz dielectric assay of solution phase protein binding

The authors demonstrate a method for rapid determination of protein-ligand binding on solution phase samples using terahertz dielectric spectroscopy. Measurements were performed using terahertz time domain spectroscopy on aqueous solutions below the liquid-solid transition for water. Small ligand binding sensitivity was demonstrated using triacetylglucosamine and hen egg white lysozyme with a decrease in dielectric response with binding. The magnitude of the change increases with frequency.

Photoactive Yellow Protein Terahertz Response: Hydration, Heating and Intermediate States

Photoactive yellow protein (PYP) is a model system for studies on functional protein dynamics and the role of protein flexibility during function. Functional conformational change is initiated by anharmonic collective vibrational modes that absorb in the far infrared (FIR) or terahertz (THz) region. We have used THz time-domain spectroscopy (THz-TDS) to investigate changes in the flexibility of PYP with functional state change (initial pG state to pB photo intermediate) induced by dehydration and photo excitation for both thin films and solutions. We find that the THz absorbance follows the dehydration induced capture of the pB state, indicating that the loss of photo cycling with the pB dehydration transition may be associated with the loss of picosecond flexibility. For hydrated films we find that previous reports of THz sensitivity to the pG to pB transition are likely in error and either arise from system drift or heating effects. We find no change in the dielectric response with photo induced occupation of the pB state. We compare these results with computational results and find that the THz dielectric response is dominated by relaxational motions of the solvent and surface side chains.

The role of structure in the protein dynamical transition

The protein dynamical transition is investigated as a function of protein structure using terahertz time domain spectroscopy (THz-TDS). Measurements performed for native state and denatured hen egg white lysozyme (HEWL) show that protein structure is not necessary for the dynamical transition. We find the temperature dependence follows activated behavior and there is no evidence of a fragile to strong transition. Measurements of short chain poly alanine show a dynamical transition down to penta-alanine, however no transition is observed for di-alanine or tri-alanine. These measurements demonstrate that the temperature dependence arises strictly from the interaction of the side chains with the solvent. The lack of a transition for shorter chain polypeptides may indicate the temperature dependence arises from a net ordering of the adjacent water which scales with the length of the polypeptide chain.

Protein Conformational Dynamics Measured With Terahertz Time Domain Spectroscopy

Tertiary structural vibrational modes of proteins are at far infrared or terahertz frequencies. These modes involve collective motion of many atoms and are indicative of the protein structure, hydration and binding with ligands and other proteins. The measurements of conformational change have focused on the hydration, denaturing effects, and photoactive proteins bacteriorhodopsin. In addition, a strong sensitivity of the terahertz response to heme protein oxidation state is shown. The impact of the results on biomolecular switching mechanisms as well as applications to biomolecular sensing is discussed.

The role of the protein surface on the local biological water dynamics

Protein function is reliant on structural flexibility and this flexibility is slaved to the surrounding solvent. Here we discuss how the exposed surface of the protein influences the solvent dynamics and thereby influences the proteins own structural dynamics. We discuss measurements of the THz absorption of water in the presence of hydrophilic and hydrophobic surfaces.

Dynamical Transition Observed in Lysozyme Solutions at THz Frequencies

Temperature-dependent THz dielectric response of hen egg-white lysozyme (HEWL) solution was measured using THz-TDS. We observe a dynamical transition at 200K, corresponding to greater protein flexibility as a function of increasing temperature.

Using terahertz spectroscopy as a protein binding assay

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.