Functional-State Dependence of Picosecond Protein Dynamics.

We examine temperature-dependent picosecond dynamics of two benchmarking proteins lysozyme and cytochrome c using temperature-dependent terahertz permittivity measurements. We find that a double Arrhenius temperature dependence with activation energies E1 ∼ 0.1 kJ/mol and E2 ∼ 10 kJ/mol fits the folded and ligand-free state response. The higher activation energy is consistent with the so-called protein dynamical transition associated with beta relaxations at the solvent-protein interface. The lower activation energy is consistent with correlated structural motions. When the structure is removed by denaturing, the lower-activation-energy process is no longer present. Additionally, the lower-activation-energy process is diminished with ligand binding but not for changes in the internal oxidation state. We suggest that the lower-energy activation process is associated with collective structural motions that are no longer accessible with denaturing or binding.

Tunable compact narrow band THz sources for frequency domain THz anisotropic spectroscopy.

We demonstrate frequency domain THz anisotropy signature detection for protein crystal models using newly developed compact tunable narrow band THz sources based on Orientation Patterned Gallium Phosphide for turn-key spectroscopic systems.

Near-field THz micropolarimetry.

We introduce a method for rapid determination of anisotropic terahertz absorption with sub micron resolution and high spectral integrity in the terahertz range. The method is ideal for microscopic and environmentally sensitive materials such as 2-D materials and protein crystals where the anisotropic absorption is critical to understanding underlying physics. We introduce the idea of using an iso-response relationship between the THz polarization and electro optic probe polarization to enable stationary sample polarization measurements covering a full 2π polarization dependence measurement.

Stationary Sample Anisotropic THz Spectroscopy using Discretely Tunable THz Sources.

We demonstrate anisotropic THz spectroscopy of sucrose using newly developed compact discretely tunable THz sources for turn-key spectroscopic systems.

THz Anisotropy Identification using Tunable Compact Narrow Band THz Sources.

We demonstrate THz anisotropy signature determination of a protein crystal model using newly developed compact tunable narrow band THz sources for turn-key spectroscopic systems for the bio molecular community.

Terahertz optical measurements of correlated motions with possible allosteric function.

A suggested mechanism for allosteric response is the distortion of the energy landscape with agonist binding changing the protein structure's access to functional configurations. Intramolecular vibrations are indicative of the energy landscape and may have trajectories that enable functional conformational change. Here, we discuss the development of an optical method to measure the intramolecular vibrations in proteins, namely, crystal anisotropy terahertz microscopy, and the various approaches which can be used to identify the spectral data with specific structural motions.

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.

Hydration and temperature interdependence of protein picosecond dynamics.

We investigate the nature of the solvent motions giving rise to the rapid temperature dependence of protein picoseconds motions at 220 K, often referred to as the protein dynamical transition. The interdependence of picoseconds dynamics on hydration and temperature is examined using terahertz time domain spectroscopy to measure the complex permittivity in the 0.2-2.0 THz range for myoglobin. Both the real and imaginary parts of the permittivity over the frequency range measured have a strong temperature dependence at >0.27 h (g water per g protein), however the permittivity change is strongest for frequencies <1 THz. The temperature dependence of the real part of the permittivity is not consistent with the relaxational response of the bound water, and may reflect the low frequency protein structural vibrations slaved to the solvent excitations. The hydration necessary to observe the dynamical transition is found to be frequency dependent, with a critical hydration of 0.19 h for frequencies >1 THz, and 0.27 h for frequencies <1 THz. The data are consistent with the dynamical transition solvent fluctuations requiring only clusters of ~5 water molecules, whereas the enhancement of lowest frequency motions requires a fully spanning water network.

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.

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.

Protein dynamical transition does not require protein structure.

Terahertz time domain spectroscopy shows that the protein dynamical transition, the rapid increase in protein dynamics occurring at approximately 200 K, needs neither tertiary nor secondary structure. Further, short chain alanine studies find a dynamical transition down to penta-alanine, with no transition observed for di-alanine or tri-alanine. These results reveal the temperature dependence arises strictly from the side-chain interaction with the solvent. The lack of a transition for shorter chain peptides may indicate a qualitative change in this interaction occurs at a specific peptide chain length.

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.

Large oxidation dependence observed in terahertz dielectric response for cytochrome c.

Far infrared dielectric response is used to characterize the collective mode density of states for cytochrome c as a function of oxidation state and hydration using terahertz time domain spectroscopy. A strong absorbance and refractive index increase was observed with the oxidation. A simple phenomenological fitting using a continuous distribution of oscillators reproduces the frequency dependence of the complex dielectric response as well as demonstrates quantitative agreement with a uniform increase in either mode density or polarizability with oxidation in the 5-80 cm(-1) frequency range. Hydration dependence measurements find that a difference in the equilibrium water content for ferri and ferro cytochrome c is not sufficient to account for the large change in terahertz response. The large dielectric increase at terahertz frequencies with oxidation suggests either a significant global softening of the potential and/or a significant increase in polarizability with oxidation.

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.