Joseph R. Knab

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.

Evidence of Protein Collective Motions on the Picosecond Timescale

We investigate the presence of structural collective motions on a picosecond timescale for the heme protein, cytochrome c, as a function of oxidation and hydration, using terahertz (THz) time domain spectroscopy and molecular dynamics simulations. The THz response dramatically increases with oxidation, with the largest increase for lowest hydrations, and highest frequencies. For both oxidation states the THz response rapidly increases with hydration saturating above ∼25% (g H(2)O/g protein). Quasiharmonic vibrational modes and dipole-dipole correlation functions were calculated from molecular dynamics trajectories. The collective mode density of states alone reproduces the measured hydration dependence, providing strong evidence of the existence of these motions. The large oxidation dependence is reproduced only by the dipole-dipole correlation function, indicating the contrast arises from diffusive motions consistent with structural changes occurring in the vicinity of buried internal water molecules. This source for the observed oxidation dependence is consistent with the lack of an oxidation dependence in nuclear resonant vibrational spectroscopy measurements.

Terahertz Transmission Characteristics of High-Mobility GaAs and InAs Two-Dimensional-Electron-Gas Systems

Frequency-dependent complex conductivity of high-mobility GaAs and InAs two-dimensional-electron-gas (2DEG) systems is studied by terahertz time domain spectroscopy. Determining the momentum relaxation time from a Drude model, the authors find a lower value than that from dc measurements, particularly at high frequencies/low temperatures. These deviations are consistent with the ratio τt∕τq, where τq is the full scattering time. This suggests that small-angle scattering leads to weaker heating of 2DEGs at low temperatures than expected from dc mobility.

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.

Critical hydration and temperature effects on terahertz biomolecular sensing

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-1) 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 H2O 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.

Tagless and universal biosensor for point detection of pathogens

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.

Direct measurements of optical phonons in SrTiO3 nanosystems

We use terahertz time domain spectroscopy to examine finite size effects on the optical phonon modes in SrTiO3 thin films. In temperature-dependent measurements we find a near absence of mode softening in the TO1 phonon frequency. Furthermore we see an increase in the soft mode frequency with reduced thickness. Both of these results correlate well with the reduced dielectric response observed for nanoscale ferroelectric systems.

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.

Measuring protein flexibility with terahertz spectroscopy: basic research and applications

We have used terahertz time domain spectroscopy (TTDS) to measure protein dielectric response and investigate conformational vibrational modes. The bacteriorhodopsin discuss our more recent work on cytochrome c. Bacteriorhodopsin is a photoactive membrane protein from halobacterium salinarum.

Terahertz Dielectric Response Sensitivity to Protein Oxidation State

Previously we have shown that the terahertz dielectric response is sensitive to the oxidation state for cytochrome c (CytC) films. Here we discuss the origin of this sensitivity through hydration dependent measurements on films, solution phase measurements as a function of temperature and normal mode calculations as a function of hydration. We find that the hydration dependence of the terahertz response rapidly approaches its fully solvated value for ferri CytC, whereas for ferro CytC the effect of added water is more gradual, with the system not reaching fully hydrated values up to 0.5 gm water/gm protein. For solution phase samples below 270 K we do not see a significant difference in the terahertz response suggesting that the fully hydrated ferro cytochrome c has the same picosecond dynamics as ferri cytochrome c. These results contradict X-ray B factor measurements that suggest that ferri cytochrome c is significantly more flexible than ferro suggesting that the B-factor determination from X-ray crystal measurements may not represent in vivo values as crystal water is less than 0.5 gm water/gm protein.