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THz polarimetry on environmentally sensitive and microscopic samples can provide unique insight into underlying mechanisms of complex phenomena. For example, near-field THz anisotropic absorption s...

We examine temperature dependent picosecond dynamics as a function of structure and function for lysozyme and cytochrome c using temperature dependent terahertz permittivity measurements. A double Arrhenius temperature dependence with activation energies E1 ~ 0.1 kJ/mol and E2 ~10 kJ/mol fits the native 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 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. Critical to the function of proteins is both their three dimensional structure and the dynamics of this structure. On the picosecond timescale motions important for function include surface solvent, side chains and large scale correlated structural motions[1-3]. It has been suggested that the large scale correlated structural motions play a key role in the necessary structural reorganization that occurs during function [4, 5]. These correlated motions have recently been measured using coherent scattering techniques [6-8]. These exciting results raise the question of whether these correlated motions change with structure and function. Further, ref. 8 has suggested evidence of an optical mode at ~ 3 cm -1 , suggesting that optical techniques may be used to characterize these motions.

Terahertz near-field microspectroscopy is emerging as an essential tool for characterization of novel materials and biomolecules. It is important to ensure the near field geometry used does not introduce spectral artifacts. For example, many scanning techniques can be strongly influenced by the interaction between the scattering tip and the sample. Here we examine the spectroscopic effects of a $200\ \mu\mathrm{m}$ diameter aperture for THz near-field measurements. We use HFSS to model free-space transmission through samples with resonant absorbance as a function of sample thickness, lateral sample width, and aperture diameter. We examine the transmitted power and spectral fidelity for coupling of transmitted THz light onto a detector. These studies inform corrective post-measurement analysis algorithms and design of near-field detection systems.

We use massive averaging over the thermally populated energy landscape to calculate a realistic terahertz vibrational density of states (VDOS), isotropic absorption and anisotropic absorption for the protein chicken egg white lysozyme (CEWL). We find that the VDOS shows little variation with starting structure, however both the isotropic and anisotropic absorption vary considerably with starting structure. Averaging over the population leads to a smooth featureless average isotropic spectrum, in agreement with THz absorption measurements however resonant bands emerge for the anisotropic averaging. Further we find the vibrational ensemble in fact has frequency dependent features associated with large overlap with functional displacements.

We measure as a function of sample orientation, doping, temperature, and energy, the Faraday rotation of linearly polarized THz light by high temperature cuprate superconductors. We observe a linear dichroism signal which increases with decreasing frequency and may exhibit resonances in the THz (1–6 meV) range. Preliminary results also show strong temperature dependence to the Faraday rotation signal.

The dynamical transition (DT) corresponds to the rapid onset of atomic motions at ca. 200K seen for minimally hydrated biomolecules. The DT is present in solution phase terahertz protein absorption measurements suggesting a benchtop DT technique. To move towards this application, several outstanding questions must be addressed: 1) does the THz absorption of protein solution reflect the protein structural dynamics, or the biological water dynamics; and 2) are THz solution phase DT measurements consistent with direct root mean square displacement measurements of hydrated powders. We show that for T

Proteins are large complex biomolecules which have structural vibrations in the terahertz range. The vibrational spectrum is dependent on the protein structure, which fluctuates at biological temperatures. Recently it was shown that these fluctuations can be accounted for by massive sampling of the energy landscape to calculate the terahertz isotropic and anisotropic absorbance. The large number of vibrations contributing to the spectra however makes interpretation of resonant bands challenging. Here we examine how we can apply elastic network models to define fundamental vibrational displacements to analyze these massive data sets.

Terahertz time domain spectroscopy is used to measure the melting kinetics of fructose molecular crystals. Combining single crystal anisotropy measurements with density functional calculations we assign the phonon frequencies and interrogate how specific phonons behave with melting. While nearly all the low frequency phonons continuously red shift with heating and melting, the lowest energy phonon polarized along the c-axis blue shifts at the melting temperature, suggesting an initial structural change immediately before melting. We find that the kinetics follow a 3D growth model with large activation energies consistent with previous DSC measurements. The large activation energies indicate multiple H-bonds must break collectively for the transition. The results suggest THz TDS with sub picosecond resolution could be used to measure ultra-fast kinetics.

Orange carotenoid protein (OCP) controls efficiency of the phycobilisome (PBS), the light harvesting antenna in cyanobacteria, to prevent oxidative damage. The OCP switches from resting state to photo protective state with intense blue light illumination. Questions persist as to why OCPR interaction increases with the PBS over that with the OCPO. Here we examine the role of long-range intramolecular vibrations within OCP. Using Stationary Sample Anisotropic Terahertz Microscopy (SSATM) we measure changes in the intramolecular vibrations with photo switching. We report the first observation of switching in the intramolecular vibrations with photoexcitation. Results suggest that there is a stiffening of the molecule in the photo protective state. This increase in structural stability may enhance the interaction with the PBS change in OCP interaction with PBS. In low light, carotenoid bound OCP appears orange (OCPo) and is inactive. Illumination by strong light converts OCP to the active, red (OCPR) state, which interacts with the PBS. A comparison of anisotropic THz microscopy measurements of dark adapted (OCPo) and illuminated OCP crystals indicate differences in their vibrational modes that may be important for OCP-PBS interactions.

Proteins are allosteric machines that couple motions at distinct, often distant, sites to control biological function. Low-frequency structural vibrations are a mechanism of this long-distance connection and are often used computationally to predict correlations, but experimentally identifying the vibrations associated with specific motions has proved challenging. Spectroscopy is an ideal tool to explore these excitations, but measurements have been largely unable to identify important frequency bands. The result is at odds with some previous calculations and raises the question what methods could successfully characterize protein structural vibrations. Here we show the lack of spectral structure arises in part from the variations in protein structure as the protein samples the energy landscape. However, by averaging over the energy landscape as sampled using an aggregate 18.5 μs of all-atom molecular dynamics simulation of hen egg white lysozyme and normal-mode analyses, we find vibrations with large overlap with functional displacements are surprisingly concentrated in narrow frequency bands. These bands are not apparent in either the ensemble averaged vibrational density of states or isotropic absorption. However, in the case of the ensemble averaged anisotropic absorption, there is persistent spectral structure and overlap between this structure and the functional displacement frequency bands. We systematically lay out heuristics for calculating the spectra robustly, including the need for statistical sampling of the protein and inclusion of adequate water in the spectral calculation. The results show the congested spectrum of these complex molecules obscures important frequency bands associated with function and reveal a method to overcome this congestion by combining structurally sensitive spectroscopy with robust normal mode ensemble analysis.

By measuring the polarization changes in THz, infrared, and visible radiation over an extended energy range (3-2330 meV), we observe symmetry-breaking in cuprate high temperature superconductors over wide energy, doping, and temperature ranges. We measure the polarization rotation and ellipticity of transmitted radiation though thin films as the sample is rotated. We observe a two-fold rotational symmetry in the Faraday angle, which is associated with linear dichroism (LD) and occurs when electromagnetic radiation polarized along one direction is absorbed more strongly than radiation polarized in the perpendicular direction. Such polarization anisotropies can be generally associated with symmetry breakings. We measure the amplitude of the LD signal and study its temperature, energy, and doping dependence. The LD signal shows a resonant behavior with a peak in the few hundred meV range, which is coincident with the mid-infrared optical feature that has been associated with the formation of the pseudogap state. The strongest LD signal is found in under-doped films, although it is also observed in optimally- and over-doped samples. The LD signal is consistent with nematic charge ordering as well as novel magnetoelectric effects., Comment: 10 pages, 4 figures

We have developed a stationary sample THz anisotropic dichroism microscope as a powerful tool for material characterization. The key innovation is the use of an iso-response curve defined by the terahertz and detection light polarizations. Here we show that following a continuous iso-response curve introduces angular distortion in the spectrum, and this distortion can be eliminated by using a discontinuous iso-response angle pairs avoiding large gradient regions. Employing this additional constraint on the relative polarizations provides a convenient technique for rapid anisotropic terahertz spectroscopy.

A stationary sample anisotropic terahertz microscopy technique is used to characterize the intramolecular vibrations for lysozyme. Tetragonal and triclinic crystals are compared. We find excellent reproducibility within a single crystal symmetry group. Several resonant bands are present for both symmetry groups, indicating they originate with the intramolecular vibrations and not crystal lattice phonons. Bands become more pronounced and higher frequency resonant bands begin to emerge with slight dehydration.

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

We demonstrate tunable narrowband THz generation by optical rectification of a femtosecond pulse in Orientation Patterned Gallium Phosphide. Center frequencies of 0.9 - 3.8 THz with average power up to 15 μW were achieved using a 1.064 µm fiber laser for the pump laser. Biomolecular characterization for an early application of this system is also shown in this work by anisotropic spectroscopic signature detection of molecular crystals in the THz region.

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.

Protein structural vibrations impact biology by steering the structure to functional intermediate states; enhancing tunneling events; and optimizing energy transfer. Strong water absorption and a broad continuous vibrational density of states have prevented optical identification of these vibrations. Recently spectroscopic signatures that change with functional state were measured using anisotropic terahertz microscopy. The technique however has complex sample positioning requirements and long measurement times, limiting access for the biomolecular community. Here we demonstrate that a simplified system increases spectroscopic structure to dynamically fingerprint biomacromolecules with a factor of 6 reduction in data acquisition time. Using this technique, polarization varying anisotropy terahertz microscopy, we show sensitivity to inhibitor binding and unique vibrational spectra for several proteins and an RNA G-quadruplex. The technique’s sensitivity to anisotropic absorbance and birefringence provides rapid assessment of macromolecular dynamics that impact biology.

20 years ago Grischkowsky and coworkers began a revolution in the terahertz (THz) optical range [1]. Zhang and others extended this new field so that measurements unimagined previously became accessible in table top systems [2]. An immediate effort began to determine intramolecular dynamics of biomacromolecules such as RNA's and proteins. This talk will discuss how THz polarization techniques can be used to both fingerprint specific biomolecules and reveal their biologically important changes. I will also discuss challenges that must be addressed to fully realize THz light's impact on the biomedical community.

The so called protein dynamical transition has long been associated with thermally activated excitations of the solvent adjacent to the polypeptide surface. Recently we reported the somewhat surprising result that the low temperature (

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.

Nearly all protein functions require structural change, such as enzymes clamping onto substrates, and ion channels opening and closing. These motions are a target for possible new therapies; however, the control mechanisms are under debate. Calculations have indicated protein vibrations enable structural change. However, previous measurements found these vibrations only weakly depend on the functional state. By using the novel technique of anisotropic terahertz microscopy, we find that there is a dramatic change to the vibrational directionality with inhibitor binding to lysozyme, whereas the vibrational energy distribution, as measured by neutron inelastic scattering, is only slightly altered. The anisotropic terahertz measurements provide unique access to the directionality of the intramolecular vibrations, and immediately resolve the inconsistency between calculations and previous measurements, which were only sensitive to the energy distribution. The biological importance of the vibrational directions versus the energy distribution is revealed by our calculations comparing wild-type lysozyme with a higher catalytic rate double deletion mutant. The vibrational energy distribution is identical, but the more efficient mutant shows an obvious reorientation of motions. These results show that it is essential to characterize the directionality of motion to understand and control protein dynamics to optimize or inhibit function.

Science and technologies based on terahertz frequency electromagnetic radiation (100 GHz-30 THz) have developed rapidly over the last 30 years. For most of the 20th Century, terahertz radiation, then referred to as sub-millimeter wave or far-infrared radiation, was mainly utilized by astronomers and some spectroscopists. Following the development of laser based terahertz time-domain spectroscopy in the 1980s and 1990s the field of THz science and technology expanded rapidly, to the extent that it now touches many areas from fundamental science to 'real world' applications. For example THz radiation is being used to optimize materials for new solar cells, and may also be a key technology for the next generation of airport security scanners. While the field was emerging it was possible to keep track of all new developments, however now the field has grown so much that it is increasingly difficult to follow the diverse range of new discoveries and applications that are appearing. At this point in time, when the field of THz science and technology is moving from an emerging to a more established and interdisciplinary field, it is apt to present a roadmap to help identify the breadth and future directions of the field. The aim of this roadmap is to present a snapshot of the present state of THz science and technology in 2017, and provide an opinion on the challenges and opportunities that the future holds. To be able to achieve this aim, we have invited a group of international experts to write 18 sections that cover most of the key areas of THz science and technology. We hope that The 2017 Roadmap on THz science and technology will prove to be a useful resource by providing a wide ranging introduction to the capabilities of THz radiation for those outside or just entering the field as well as providing perspective and breadth for those who are well established. We also feel that this review should serve as a useful guide for government and funding agencies. OA hybrid

Modeling has predicted that intramolecular structural vibrations enables proteins to access functionally important structural change. We show that the vibrational density of states and the isotropic absorption in the terahertz range are only weakly dependent on the protein functional state for several bench marking proteins. At the same time the direction of motions changes dramatically with functional state and with a resulting impact on the anisotropic absorption. Our anisotropic THz microscopy (ATM) measurements confirm this sensitivity. Here we apply the technique to the question of whether the protein dynamical transition (DT) is important to protein function. We find a strong anisotropic resonance at 70 cm−1 rapidly increases in strength at temperatures above the DT. As these intramolecular vibrations enable protein structure to change conformation, the results suggest function will cease below DT for those proteins that require large scale conformational change.

Protein structural motion is necessary for function. A typical measure of protein flexibility is the average mean squared atomic displacement (MSD). Both X-ray and neutron scattering have found a strong increase in the MSD at 200-220 K for a number of proteins. This increase is often referred to as the dynamical transition (DT) [1]. Function ceases for many proteins below the DT. It was suggested that the DT indicated the temperature at which the motions necessary for function are frozen out. A question has been raised as to how long-range motions are affected by the transition. Specifically, would the long-range intramolecular vibrations associated with conformational changes be present at low frequencies, and then at higher temperatures red shift due to anharmonicity, as is seen in conventional solids? The different DT measurement techniques do not distinguish between local particle excitations and coherent long-range motions. Direct optical measurement of the long-range intramolecular vibrations requires spectroscopy within the energy range of 1-100 cm−1 (ν = 0.03-3 THz). Our recently developed technique, CATM (Crystal Anisotropy Terahertz Microscopy) [2], uses polarization dependent absorption on aligned molecules to isolate the long-range intramolecular vibrations from the local excitations. Temperature dependent CATM from 180K to 295K is used to investigate how the DT effects long-range vibrations. We find that a sharp 70 cm-1 band grows in as the temperature increases. We suggest that this new band above DT demonstrates that in fact the surrounding solvent acts as a frozen cage preventing long-range correlated motions, and as the surrounding solvent becomes more mobile, the large-scale motions necessary for function can occur.1.Doster, W., et al. Physical Review Letters, 2010. 104(9): p. 098101.2.Acbas, G., et al. Nature Communications, 2014. 5, 3076 http://dx.doi.org/10.1038/ncomms4076.

We report the anisotropic terahertz response of oxalic acid and sucrose crystals in the 0.2-3.0 THz range using terahertz time domain spectroscopy on large, single crystals. We compare the observed anisotropic response with the response calculated using solid-state density functional theory and find good agreement in the orientation dependence and relative intensities of the crystal phonons. It was found that oxalic dihydrate can be reversibly converted to anhydrous by controlled relative humidity. In addition, oxalic acid was found to have a large birefringence with Δn = 0.3, suggesting the material may be useful for terahertz polarization manipulation. Sucrose has a smaller birefringence of Δn = 0.05, similar to that of x-cut quartz. The anisotropic measurements provide both mode separation and symmetry determination to more readily achieve mode assignment for the more complex sucrose spectrum.

Spectroscopic analysis using the Terahertz frequencies between 0.1-15 THz (3–500 cm−1) has been underutilised by the biochemistry community but is starting to yield some scientifically interesting information. Analysis of structures from simple molecules like N-methylacetamide, to polyamides, peptides and relatively complex proteins provides different types of information dependant on the molecular size. The absorbance spectrum of small molecules is dominated by individual modes and specific hydrogen bonds, peptide spectra have peaks associated with secondary structure, while protein spectra are dominated by ensembles of hydrogen bonds and/or collective modes. Protein dynamics has been studied using Terahertz spectroscopy using proteins like bacteriorhodopsin, illustrating a potential application where this approach can provide complementary global dynamics information to the current nuclear magnetic resonance and fluorescence-based techniques. Analysis of higher-order protein structures like polyomavirus virus-like particles generate quite different spectra compared to their constituent parts. The presence of an extended hydration layer around proteins, first postulated to explain data generated using p-germanium spectroscopy may present a particularly interesting opportunity to better understand protein’s complex interaction with water and small solutes in an aqueous environment. The practical aspects of Terahertz spectroscopy including sample handling, the use of molecular dynamics simulation and orthogonal experiment design are also discussed.

Protein hydration plays a critical role in protein dynamics and biological processes. Pump-probe transmission measurement has been applied to investigate the hydration effects on the energy relaxation of a heme protein ferric Cytochrome c (Cyt c) film after soret-band photoexcitation. Transient dynamics study indicates that the energy internal conversion time of ~300 fs is independent of hydration. The vibrationally excited electronic ground-state recovery rates show two transitions at the hydration level of h = 12.4-16.5% and 21.7-23.5%. The first transition occurs at the hydration level for the onset of an increasing ferric Cyt c flexibility while the second transition occurs at the saturated hydration level. The hydration dependence of steady-state electronic absorption spectrum results shows that the Q-band peak is nearly constant in center wavelength, but the line width surprisingly narrows with increasing hydration. For the ~695 nm absorbance associated with the MET80-Fe bond, the intensity increases with increasing hydration and slightly blue shifts. The 695 nm peak grows rapidly at h = 12.4% and then plateaus at h = 21.7%. This research shows that ~695 nm absorbance and ground-state recovery rates are sensitive to the hydration of the protein. This study will aid in understanding how hydration modulates the activity of the protein dynamics at a local level.

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.

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.

Collective structural vibrational modes of proteins lay in the terahertz frequency range. We discuss several measurements exploring picosecond dynamics in hen egg white lysozyme suggesting that terahertz time domain spectroscopy can provide unique insight to critical protein dynamics. We find a rapid increase in terahertz absorbance and index at both a critical hydration point and separately a critical temperature. Specifically, we discuss the hydration and temperature transitions and suggest that these are possibly linked. We discuss the possibility that the terahertz response is mainly determined by relaxational response of side chains within the protein and the energy barriers for these motions are hydration dependent. Finally we show that simple normal mode calculations do not reproduce the strong hydration dependence, further suggesting that resonant vibrational response is not sufficient to describe the terahertz dielectric response.

THz time domain spectroscopy of biomolecules was performed to determine applicability of the technique for chemical and conformational identification of biomolecules. Measurements were performed on samples of DNA, bovine serum albumin, collagen, hen egg white lysozyme, myoglobin, and bacteriorhodopsin as a function of temperature, hydration and photoexcitation. The results are compared to normal mode calculations. We demonstrate a clear resemblance of the observed broad and near featureless THz absorbance spectrum to the calculated density of normal modes. While the magnitude of the absorbance and the center of the broad response depends on biomolecular species, unique chemical identification would appear challenging. The observed dependence on hydration is in agreement with mass and dielectric loading, and the dependence on temperature is in agreement with decreasing conformational flexibility with reduced temperature. Finally THz absorbance dependence on the biomolecular conformation and mutation is demonstrated for bacteriorhodopsin.

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.

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.

Global protein vibrations have long been associated with protein functionality. Long-range intramolecular vibrations have been measured in proteins using anisotropic absorption in the terahertz frequency range [1,2]. These measurements directly correspond to the directionality of vibrations and show large changes in the directionality of the vibrational displacements for free chicken egg white lysozyme (CEWL) and inhibitor bound CEWL. Normal mode ensemble analysis (NMEA) and quasiharmonic analysis (QHA) calculations of the free and tri-acetylglucosamine (3NAG) bound CEWL dynamics were performed and also show large changes in vibration directionality with binding. This in spite of the calculated energy distribution showing very little change, in agreement with neutron scattering measurements. We investigate the importance of the protein directionality by extending the calculations to a double deletion mutant (DD CEWL) with an activity rate 1.4 times that of WT [3]. The deletions are far from the active site, indicating that the increased activity arises from changes in the dynamics. The calculated anisotropic spectra show large changes with mutation, whereas the energy distribution show practically no change from the WT. Similarities in the bound WT, free DD, and bound DD CEWL NMEA spectra indicate the mutation may be changing the directionality of the vibrations toward more efficient motions. Projections of the low frequency vibrations, from QHA, on the functional displacement show higher overlap of residues around Glu35 and Asp52 in the mutant system. These residues are known to be critical in CEWL functionality. The results reveal that the mutation may be steering the protein toward more functional dynamics without significantly affecting the energy distribution. [1] K.A. Niessen, et al. (2015) DOI: 10.1007/s12551-015-0168-4[2] G. Acbas, et al. (2014) DOI: 10.1038/ncomms4076[3] S. Mine, et al. (1999) DOI: 10.1006/jmbi.1999.2572. Supported by NSF (DBI 1556359, MCB 1616529) and DOE (DE-SC0016317).

Orange carotenoid protein (OCP) is the only photosensory protein described to date having a carotenoid as the active chromophore. Upon strong green-blue (or white) illumination, OCP goes from the orange inactive state (OCPO) to the red active state (OCPR) [1]. OCPR interacts with the phycobilisome (PB), assisting in thermal energy dissipation. This dissipation diminishes the formation of reactive oxygen which damages the photosynthetic apparatus and leads to cell death [2]. OCP has a large conformational change as it goes from OCPO to OCPR, and this structural change is thought to be essential to the energy quenching interaction with the PB. However, the conversion from inactive to active state has been recently shown to also occur with high chaotrope concentrations. Specifically high concentration NaSCN produces a long lived red state in the absence of photoexcitation [3]. We suggest that both the susceptibility of OCP to large conformational change and its interaction with PB are associated with changes in the long range picosecond structural flexibility. We measure OCP protein flexibility changes with photoactivation and chemical activation using terahertz absorption spectroscopy. Temperature dependent terahertz time domain spectroscopy is measured in the 100 - 290 K range on OCP solutions as a function of illumination and NaSCN concentration. A rapid increase in the THz absorbance is observed in the 180-220 K range. We quantify the picosecond structural flexibility by both the net THz absorption and the dynamical transition temperature, which scales with structural stability. R.E.B acknowledges DOE award DE-FG02-07ER15902 and A.G.M acknowleges NSF awards DBI 1556359 and MCB 1616529, and DOE award DE-SC0016317 for support of the work.1. Wilson, A. et al. Proc. Natl. Acad. Sci. U. S. A. 105:12075-12080(2008).2. Wilson, A. et al. Plant Cell, 18:992-1007(2006).3. King, J.D. et al. FEBS Lett, 588:4561-4565(2014).

The protein dynamical transition is the remarkable increase in the average atomic root mean squared displacement (RMSD) in the 180-220 K range. The effect is associated with the onset of anharmonic motions critical to biological function. However evidence of the actual biological relevance of the dynamical transition is inconsistent. While for some proteins, function ceases below the dynamical transition, for other proteins the dynamical transition appears to have no effect. Here we report measurements that suggest the difference arises from the dependence of function on large scale conformational change, and specifically the reliance on long range vibrations to access these structural changes. The dynamical transition has been extensively observed using X-ray, neutron scattering, NMR and terahertz absorption spectroscopy [1,2], with the results indicating it arises from thermally activated solvent motions. Those techniques measure all motions contributing to the RMSD including both localized motions and intramolecular vibrations. To isolate the vibrations and examine how the dynamical transition impacts them, we use a new technique, anisotropy terahertz microscopy (ATM) [3]. This unique method suppresses the background from the localized motions giving unprecedented access to the long range motions that enable large scale conformational changes. ATM measurements of lysozyme anisotropic optical absorbance in the 150-300 K temperature range show that the resonant vibrational bands rapidly increase in intensity at the dynamical transition, and surprisingly blue shift with increasing temperature, in contrast to the expected anharmonicity. The measurements demonstrate that the surrounding solvent below the dynamical transition acts as a frozen cage preventing the vibrations necessary for functional conformational change. This solvent slaving of the long range vibrations potentially impacts protein structural stability and vulnerability to structural disorder. This work was supported by NSF (DBI 1556359 and MCB 1616529), and DOE DE-SC0016317. 1. Doster,W., et al. Phys.Rev.Lett., 2010.104(9):098101. 2. Niessen,K., et al. Biophys.Rev., 2015.7,201. 3. Acbas,G., et al. Nat.Commun., 2014.5,3076.

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.

The higher transmission through tissues of long wavelength light motivates the development of fluorescent proteins with excitation shifted to the red. However red fluorescent proteins (RFPs) are more susceptible to photobleaching than their shorter wavelength counterparts. In particular RFPs are more susceptible to photobleaching [1]. A possible reason for this is a decrease in the structural stability of the beta barrel. Measurements of structural stability include atomic root mean squared displacement measured by the X-ray B-factor and neutron quasi elastic scattering. To date, X-ray measurements of RFP's do not indicate a structural stability change and systematic scattering studies have not been performed. Using THz dielectric response we examine if the picosecond structural flexibility decreases with increasing FP stability.

One can achieve a more uniform native-state structure and remove scattering concerns by using solution phase samples. To avoid strong background from bulk solvent, one can form thin (~100 μm) lms of solution and dry these in a controlled way, using, for example, a closed cell with humidity control. e hydration can be set high enough to achieve protein structural homogeneity in the lm, but suciently low so that bulk water is not present and cannot contribute to absorption. Terahertz measurement of such samples, however, still had glasslike absorption. While closer examination of the complex permittivity of the hydrated lms revealed that the response could not be entirely accounted for with just relaxational response, but also required at least one damped harmonic oscillator (Knab et al. 2006), it is clear that for fully hydrated proteins, we must consider multiple contributions to the dielectric response. By proper decomposition of the data, enhanced by systematic variation of temperature, solvent content, or protein functional state, one can isolate the component arising from the protein structural vibrations. We can write down an approximate decomposition for solution phase proteins using the following formula:e e e e e= + + +W bW p relax p vib, , , (11.1)where the subscripts W, bW, p, relax, and p, vib refer to permittivity contributions from the bulk water, bound water, protein relaxational response, and protein long-range structural vibrational response, respectively. is decomposition is a vast simplication, ignoring any interaction between dierent components, but it expresses the various sources of dielectric response for the complex sample. e rst three components are modeled very well using dielectric relaxation, and the last component is a sum of damped harmonic oscillators. In this chapter, we will discuss the dierent relaxational contributions to the terahertz response for hydrated proteins and methods to remove these so that one can extract the contribution from protein structural motions. We will discuss general aspects of relaxational response, the permittivity of water, in all its various forms, present in biological samples, and the relaxational response from amino acid side chains. Finally, we will discuss, in light of all the various models for relaxational eects, how the current measurements for protein samples suggest that the long-range vibrations of proteins do contribute to the terahertz response.

We report the first use of pulsed terahertz spectroscopy to examine low-frequency collective vibrational modes of biomolecules. Broadband absorption increasing with frequency was observed for lyophilized powder samples of calf thymus DNA, bovine serum albumin and collagen in the 0.06–2.00 THz (2–67 cm−1) frequency range, suggesting that a large number of the low-frequency collective modes for these systems are IR active. Transmission measurements at room temperature showed increasing FIR absorption with hydration and denaturing.