Andrea Markelz

THz time domain spectroscopy of biomolecular conformational modes

We discuss the use of terahertz time domain spectroscopy for studies of conformational flexibility and conformational change in biomolecules. Protein structural dynamics are vital to biological function with protein flexibility affecting enzymatic reaction rates and sensory transduction cycling times. Conformational mode dynamics occur on the picosecond timescale and with the collective vibrational modes associated with these large scale structural motions in the 1-100 cm(-1) range. We have performed THz time domain spectroscopy (TTDS) of several biomolecular systems to explore the sensitivity of TTDS to distinguish different molecular species, different mutations within a single species and different conformations of a given biomolecule. We compare the measured absorbances to normal mode calculations and find that the TTDS absorbance reflects the density of normal modes determined by molecular mechanics calculations, and is sensitive to both conformation and mutation. These early studies demonstrate some of the advantages and limitations of using TTDS for the study of biomolecules.

Terahertz Dielectric Sensitivity to Biomolecular Structure and Function

A review of recent terahertz (THz) dielectric response measurements on large biomolecules is presented. The origin of the dielectric response in this frequency range is discussed. We specifically address the questions of the relative contributions of collective vibrational modes and relaxational contributions. When sample uniformity is controlled and multiple reflection effects included in the analysis, the overall dielectric response of large biomolecules in the THz range is broad and featureless. The response, however, is highly sensitive to hydration, temperature, binding, and conformational change. The origin of these effects is discussed with emphasis on the contribution of surface side chain relaxational loss and how this might be affected by environment, structure, and function.

Protein Flexibility and Conformational State: A Comparison of Collective Vibrational Modes of Wild-Type and D96N Bacteriorhodopsin

Far infrared (FIR) spectral measurements of wild-type (WT) and D96N mutant bacteriorhodopsin thin films have been carried out using terahertz time domain spectroscopy as a function of hydration, temperature, and conformational state. The results are compared to calculated spectra generated via normal mode analyses using CHARMM. We find that the FIR absorbance is slowly increasing with frequency and without strong narrow features over the range of 2-60 cm(-1) and up to a resolution of 0.17 cm(-1). The broad absorption shifts in frequency with decreasing temperature as expected with a strongly anharmonic potential and in agreement with neutron inelastic scattering results. Decreasing hydration shifts the absorption to higher frequencies, possibly resulting from decreased coupling mediated by the interior water molecules. Ground-state FIR absorbances have nearly identical frequency dependence, with the mutant having less optical density than the WT. In the M state, the FIR absorbance of the WT increases whereas there is no change for D96N. These results represent the first measurement of FIR absorbance change as a function of conformational state.

Terahertz Response and Colossal Kerr Rotation from the Surface States of the Topological Insulator Bi2Se3

We report the THz response of thin films of the topological insulator Bi2Se3. At low frequencies, transport is essentially thickness independent showing the dominant contribution of the surface electrons. Despite their extended exposure to ambient conditions, these surfaces exhibit robust properties including narrow, almost thickness-independent Drude peaks, and an unprecedentedly large polarization rotation of linearly polarized light reflected in an applied magnetic field. This Kerr rotation can be as large as 65° and can be explained by a cyclotron resonance effect of the surface states.

Optical measurements of long-range protein vibrations

Protein biological function depends on structural flexibility and change. From cellular communication through membrane ion channels to oxygen uptake and delivery by haemoglobin, structural changes are critical. It has been suggested that vibrations that extend through the protein play a crucial role in controlling these structural changes. While nature may utilize such long-range vibrations for optimization of biological processes, bench-top characterization of these extended structural motions for engineered biochemistry has been elusive. Here we show the first optical observation of long-range protein vibrational modes. This is achieved by orientation-sensitive terahertz near-field microscopy measurements of chicken egg white lysozyme single crystals. Underdamped modes are found to exist for frequencies >10 cm(-1). The existence of these persisting motions indicates that damping and intermode coupling are weaker than previously assumed. The methodology developed permits protein engineering based on dynamical network optimization.

INTERBAND IMPACT IONIZATION BY TERAHERTZ ILLUMINATION OF INAS HETEROSTRUCTURES

Experimental studies of InAs heterostructures illuminated by far‐infrared (FIR) radiation reveal an abrupt increase in the charge density for FIR intensities above a threshold value that rises with increasing frequency. We attribute this charge density rise to interband impact ionization in a regime in which ωτm∼1, where τm is the momentum relaxation time, and f=ω/2π is the FIR frequency. The dependence of the density rise on the FIR field strength supports this interpretation, and gives threshold fields of 3.7–8.9 kV/cm for the frequency range 0.3–0.66 THz.

Terahertz spectroscopy of bacteriorhodopsin and rhodopsin: similarities and differences.

We studied the low-frequency terahertz spectroscopy of two photoactive protein systems, rhodopsin and bacteriorhodopsin, as a means to characterize collective low-frequency motions in helical transmembrane proteins. From this work, we found that the nature of the vibrational motions activated by terahertz radiation is surprisingly similar between these two structurally similar proteins. Specifically, at the lowest frequencies probed, the cytoplasmic loop regions of the proteins are highly active; and at the higher terahertz frequencies studied, the extracellular loop regions of the protein systems become vibrationally activated. In the case of bacteriorhodopsin, the calculated terahertz spectra are compared with the experimental terahertz signature. This work illustrates the importance of terahertz spectroscopy to identify vibrational degrees of freedom which correlate to known conformational changes in these proteins.

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.

Temperature of quasi‐two‐dimensional electron gases under steady‐state terahertz drive

We use photoluminescence to study the time‐average energy distribution of electrons in the presence of strong steady‐state drive at terahertz (THz) frequencies, in a modulation‐doped 125 A AlGaAs/GaAs square well that is held at low lattice temperature TL. We find that the energy distribution can be characterized by an effective electron temperature, Te(≳TL), that agrees well with values estimated from the THz‐illuminated, dc conductivity. This agreement indicates that under strong THz drive, LO phonon scattering dominates both energy and momentum relaxation; that the carrier distribution maintains a heated, thermal form; and that phonon drift effects are negligible.