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.

Terahertz near-field spectroscopy of filled subwavelength sized apertures in thin metal films.

We have measured terahertz near-field spectra of cesium iodide crystals as small as ~10 µm in diameter, which were deposited on single, sub-wavelength-sized apertures created in thin gold films on a substrate. The advantage of using small apertures for terahertz microspectroscopy is that only terahertz light that has interacted with the cesium iodide is observed. We find that around the transverse optical phonon frequency of cesium iodide, the amplitude transmission is as much influenced by the refractive index as by the absorption. We show that the ability to measure in the near-field of the apertures, where signals are relatively strong, allows us to measure on sample volumes as small as ~5×10(-16) m(3).

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.

Influence of the dielectric substrate on the terahertz electric near-field of a hole in a metal.

We have studied theoretically and experimentally the influence of a dielectric substrate on the frequency-dependent terahertz electric near-field of a small hole in a metal layer. We find that the near-field transmission spectrum and the two-dimensional field distribution of an empty hole in a thin metal layer on a substrate are almost identical to that of a hole which is also filled with the same dielectric material as the substrate. For thicker metal layers, however, the near-field spectra of filled and unfilled holes become very different. In addition, for thick metal layers, the two-dimensional field distributions are more strongly affected by the substrate, especially if we allow for an air gap between the metal and the substrate. Our results validate the -somewhat unusual- two-dimensional field distribution measured beneath a hole in a thick metal foil and highlight the effect that a substrate can have on the measurement of the near-field of an object.

Terahertz near-field vectorial imaging of subwavelength apertures and aperture arrays.

We present measurements of the complete terahertz (THz) electric near-field distribution, E(x), E(y) and E(z), in both the time- and frequency-domains, for subwavelength apertures and subsections of subwavelength aperture arrays. Measuring the individual components of the THz near-field with subwavelength spatial resolution, as they emerge from these structures, illustrates how the field interacts with the apertures. We observe the small but measurable y- and z-components of the electric field for both single apertures and arrays. Resonant contributions, attributed to Bloch modes, are detected and we observe the presence of a longitudinal field component, E(z), within the different array apertures, which can be attributed to a diffractive effect. These measurements illustrate in detail the individual THz field components emerging from subwavelength apertures and provide a direct measure of two important mechanisms that contribute to the net transmission of light through arrays.

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.

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.