Experimental and computational investigations of the

Experimental and computational investigations of the THz spectra of dipeptide nanotubes K. M. Siegrista, C. Pfefferkornb, A. Schwarzkopfb, V. B. Podobedovb, D. F. Plusquellicb a University of Maryland, 1000 Hilltop Circle, Baltimore, MD, USA 21250; b National Institute of Standards and Technology, Gaithersburg, MD, USA 20899. ABSTRACT Continuous wave THz spectroscopy has been used to obtain spectra for four isostructural dipeptide nanotubes at 4.2K from 2 cm-1 to 100 cm-1 (0.05 to 3 THz). Line-narrowing of spectral features by a factor of 2 to 4 is observed for the crystalline dipeptide films investigated by absorption spectroscopy using a plane parallel waveguide, compared to spectra from pressed disks of polyethylene-diluted samples. The x-ray determined crystal structures of these peptides formed the basis for a parallel computational investigation. Spectral predictions from the ab initio level computational package DMOL3 and the empirical force field model CHARMM22 are compared to the experimentally obtained THz absorption spectra. The THz waveguide spectroscopy technique can provide information on the orientation-dependent dipole coupling of the vibrational modes, which can aid in validating computational models. Keywords: Terahertz spectroscopy, Far-infrared spectroscopy, density functional theory, DMOL, CHARMM, periodic boundary conditions, peptide nanotubes, crystalline peptides,

1. INTRODUCTION Recent advances in terahertz technology catalyzed exploration in this region of the spectrum. This recently expanded access to the THz region has been of particular interest from a biologist's standpoint because of the high sensitivity of THz spectra to the molecule's surroundings. This sensitivity, engendered by the non-local nature of THz vibrational modes, makes interpretation of of spectral absorption features in this region difficult. For proteins and smaller peptides, these modes are typified by torsional motions or by bending and wagging motions of skeletal backbones - motions which, for biomolecules in vivo, can represent the initiation of conformational and functional changes, processes of great biological interest. THz spectroscopy is a natural tool for probing such forces, but its potential remains largely unrealized due to the lack of theoretical understanding of the physical mechanisms important in this low frequency region. In much recent work directed at developing theoretical underpinnings of the lowest frequency vibrational modes of molecules, crystalline solids have been investigated [1-4]. The highly degenerate vibrational modes of crystalline systems can give rise to sparse, narrow, well-resolved spectral features, data ideally suited for comparisons with and validation of current theoretical models. This recent work has firmly established the non-local nature of THz vibrational modes, and the accompanying sensitivity to the molecular environment. In biological systems, as in crystal structures, the large-scale motions of THz modes take place in a constraining "box" of surrounding molecules, and these motions can depend on concerted action of many molecules. In such a situation the constraining box is an integral part of the system under study. For the purpose of studying complex biological systems, it is ultimately desirable to develop reliable models at the level of empirical force field calculations. However, in spite of the seeming simplicity of THz spectra of crystalline structures, empirically based models are currently not of sufficient quality for definitive THz mode assignments. Indeed, true quantitative agreement may seem beyond reach, given the level of quantum chemical theory required so far to obtain reasonable agreement with experiment [1-4]. Nonetheless, CHARMM predictions have shown more success than might be expected [4,5] and it is hoped that the basis of this apparent success may be uncovered and exploited in establishing a firm basis for relying on empirical calculations.

Biomedical Optical Spectroscopy, edited by Anita Mahadevan-Jansen, Wolfgang Petrich, Robert R. Alfano, Alvin Katz, Proc. of SPIE Vol. 6853, 685302, (2008) · 1605-7422/08/$18 · doi: 10.1117/12.780580 Proc. of SPIE Vol. 6853 685302-1 2008 SPIE Digital Library -- Subscriber Archive Copy

At present, the most successful models of solid state systems couple a high degree of verisimilitude in the theoretical framework with high numerical precision of the density functional theory (DFT) calculations. Hence computational explorations aimed at discovering the requirements of a good model in the THz regime are necessarily limited in system size by the need to first develop accurate quantum chemical models. Significant progress has been demonstrated recently in reproducing the experimentally observed vibrational spectra for biomolecular crystalline systems using ab initio level theory. Recent contributions from several groups, as well as our own prior work [1-4], have shown reasonable agreement between computational and experimental results, and have made it clear that the full periodic structure is unequivocally necessary (if not sufficient) condition, for accurate models of strongly hydrogenbonded crystalline systems. While these recent results have defined some of the necessary attributes of good theoretical models for this very low energy region, many questions remain. The present work extends previous investigations [4] directed at the characterization of THz spectra of small crystalline peptides using experimentally measured spectra as a test of computational methodology. This work has involved two approaches to the problem of developing the theoretical underpinning necessary to interpret spectral information in the terahertz range. First, we have aimed to develop reliable computational methods for the calculation of biomolecular terahertz spectra. In addition, however, we have been implementing recently developed experimental techniques [6-8] to increase sensitivity and resolution of the THz spectrometer. In this work, we demonstrate improved sensitivity of the spectrometer and more clearly resolved THz spectral features by using a planar support for growing thin films of crystalline dipeptides. The planar support then constitutes one half of a plane parallel THz waveguide into which terahertz radiation is coupled. We present calculations at DFT and empirical force field levels to model the spectra of four isostructural dipeptides.

2. INSTRUMENTATION A schematic of the continuous wave spectrometer, which has been described in detail elsewhere [9,10], is given in Figure 1. Briefly, the system is built around an InAs photomixer driven at the difference frequency of two infrared lasers. Output from a fixed frequency laser at 840 nm is combined with the output from a grating-tuned standing wave cavity Ti:sapphire laser having a resolution of 0.04 cm-1 (1.2 GHz) and the two collinear beams are focused onto the photomixer to produce the THz difference frequency, which sweeps through 3 THz (100 cm-1) as the grating tunes the Ti:sapphire laser . A maximum power estimated at 5 µW is obtained near 0.6 Thz (20 cm-1) and THz output falls off with frequency as ω-4 beyond this peak. The THz radiation, confined within a vacuum chamber to avoid water-vapor absorption, is collimated by a silicon lens and then focused by off-axis parabolic mirrors. A sample, fixed to the cold finger of a cryostat in the vacuum chamber, is placed at the THz focus, and transmitted power is detected by a heliumcooled silicon composite bolometer having a power detection sensitivity of < 1 nW up to 3 THz in a 400 Hz bandpass (NEP of the bolometer is 1 pW/Hz1/2). For normalization purposes, voltage proportional to the photomixer photocurrent is recorded simultaneously with the detected THz power. For the commonly used method of preparation, dipeptides obtained from Bachem at >99% purity were diluted with fine grain polyethylene (PE) powder (average particle diameter 4 µm), and 100 mg of a 10% to 15% concentration mixture were pressed at 2 × 107 Pa (3000 psi) to form a disk ≈750 µm thick and 13 mm in diameter. This disk was placed in a brass holder attached to the cryostat coldfinger. A second pure polyethylene sample was used to obtain background power transmission. Waveguide spectroscopy experiments using a plane-parallel waveguide [6] require only minor modification to this system. The inset of Figure 1 shows the plane-parallel waveguide assembly, fabricated of copper and coated with gold to minimize surface reactivity. Hyperhemispherical silicon lenses couple the THz radiation into and out of the waveguide. The waveguide path length is 2.5 cm, and the hyperhemispherical lenses have a diameter of 12 mm with the shoulder offset placing the focal point at the silicon surface nearly abutting the waveguide; a small (50 µm) gap exists between the lens and the guide. A thin film of crystalline dipeptide was prepared on the surface of one thoroughly cleaned plate of the waveguide. Acetone was added to a nearly saturated aqueous solution of dipeptide, to aid the wetting of the gold surface (which is prone to induce hydrophobic beading of the solution), and a few hundred microliters of solution were spread on the gold surface and evaporated at 75 to 80 °C. A visually uniform crystalline

Proc. of SPIE Vol. 6853 685302-2

film, composed of a patchwork of oriented crystallites, was formed in this manner. At the optimum THz absorption strength, dipeptide films are estimated to be 1 µm to 2 µm thick.

Grating-tuned Ti:Sap Laser

Waveguide

Pump Laser

Laser Calibration & Stabilization 60 mW total

Diode Amplifier

Chopper Amplitude Modulation ~ 400Hz

Photomixer and Si lens

Cryostat

Diode Laser

Evacuated Sample Chamber

Bolometer

Lock-in PhotoCurrent

Computer Lock-in

Fig. 1. Schematic of the continuous wave THz spectrometer. The sample is placed in a holder on the cold finger of the cryostat. For thin film samples, the parallel plate waveguide and coupling lens assembly replaces the standard sample holder, as diagrammed in the inset. The waveguide assembly was fixed to the cold finger of the cryostat and inserted into the beam path at nearly the same position occupied by the sample holder in prior experiments. For our incompletely optimized system, only minor adjustments were made to the THz beam collimation to improve coupling into the waveguide. A waveguide plate separation of 250 µm was maintained using Teflon spacers at the edges of the 3.75 cm (1.5 inch) wide assembly and this rather large gap gave sufficient sensitivity for the approximately 1 mg of dipeptide film. Fine adjustment of these short focal length lenses is necessary to achieve good parallel alignment with the gap at the correct vertical position. The assembly allows adjustment via set screws. A place-holding bracket was then locked in place to fix the correct lens position, which allowed disassembly and reassembly of the waveguide without losing lens alignment. An additional positioning system was used to improve the stability and repeatability of the cryostat position in the beam path. During acquisition of spectra, the helium-cooled sample assembly was held at 4.2 K in vacuum for the polyethylene-diluted disk-shaped samples in the smaller brass assembly. The larger gold-coated copper waveguide assembly was held at 4.6 K, the minimum temperature which could be achieved due to the necessary removal of one radiation shield. It is possible that the temperature of the sample is higher than the readout indicates, since the thermistor is placed at the base of the cold finger, and not at the sample location. However, since the temperature is not observed to rise immediately after cessation of helium flow, it is assumed to be within 1K of the nominal 4.6 K indicated on the control box.

3. THEORETICAL MODELING DFT calculations performed with periodic boundary conditions in DMOL3 [11, 12] were done using the same parameters previously found to be successful in reproducing the THz spectra of three different crystal structures of


trialanine [4]. The calculations use the PW91 exchange correlation functional based on the generalized gradient approximation (GGA) in the parameterization of Perdew and Wang. A double numerical plus polarization atomic orbital basis set, equivalent to double-ζ, was used, and all electrons were included in the calculations. The minimum-energy geometry was optimized with a convergence criteria of 10-6 hartrees (Ha) for energy, 2 × 10-4 Ha/Å for the maximum gradient, and 5 × 10-4 Å for maximum displacement, and with the atomic orbital cutoff for integration set at the default value, Rc = 3.7 Å. All the DMOL3 calculations were performed with the lengths of the unit cell axes held fixed at the values taken from x-ray crystallographic data. Harmonic frequencies and normal-mode displacements were obtained at the Γ-point by a two-point numerical differentiation of the forces, following diagonalization of the mass-weighted Hessian matrix [13] of the unit cell. The intensity for each normal mode, Qi, is proportional to the square of the transition dipole moment derivative, δµi/δQi as calculated using the approximation

∂µ i Ii ∝ ∂Qi

2

≅

3N

∑ j

e j Qi , j − e oj Qio, j ∆Qi

2

3N

≅∑

e oj (Qi , j − Qio, j )

j

∆Qi

2

(1)

where the sum runs over the 3N Cartesian coordinate displacements. The static charges were set to the Mulliken atomic charges of the optimized structure. An additional correction was made for electrical anharmonicity by recalculating the equilibrium charges after displacement by the normal modes. Eqn. 1 gives integrated intensity, therefore, for comparison with experiment, the vibrational lines were all convolved with a Gaussian line shape function of fixed width of 1.5 cm-1 (45 GHz). In addition to the DFT calculations, empirical force-field calculations were done using periodic boundary conditions in CHARMM22 [14, 15]. Periodic structures are created using a set of image cells surrounding a core block of one unit cell. The core atom cutoff radius for including pairwise non-bonded interactions, which determines the number of image cells generated, was set at 15 Å since changes in energy were negligible for larger cutoff radii. Threeatom terms to describe hydrogen bonds between core and image cells, which can incorporate angular dependence, could not be included, hence all intermolecular hydrogen bonds are treated with two-atom terms. This is a severe limitation to the present calculations, but one which is challenging to address. The Ewald approximation [16-18] was used to ensure convergence in the electrostatic energy sums, and a Gaussian width of 0.34 cm-1 was used in transitioning from real to kspace sums. The number of grid points in k-space was set to twice the unit-cell dimensions. Normal mode frequencies were found from the eigenvalues of the mass-weighted Hessian matrix using a two-point finite difference method, and normal-mode displacements of 0.01 Å were used in the final expression of Eqn 1. For all the CHARMM calculations, unlike the DMOL3 calculations, both the unit cell and individual molecular geometries were optimized. Gaussian lines of width 1.5 cm-1 were used for comparison of experimental and computational spectra, as was done for the DMOL3 results.

4. RESULTS AND DISCUSSION A series of spectra from valyl alanine (VA) appear in Figure 2, from 1 to 2.5 THz, obtained from a thin crystalline film of VA using the parallel plate waveguide. In Figure 1a on the left, the power transmission spectra are shown. A background transmission spectrum taken using the empty cold waveguide appears at top and the remaining four spectra were taken, in descending order, as temperature dropped from 30 K at the start of the first sweep, to 7 K at the beginning of the third sweep, and to 4.6 K by the start of the fourth. A full spectrum from 0 to 3 THz requires about 8 minutes to complete, so sample temperature is not constant over the spectra shown, except for the final, sharpest spectrum which was taken entirely at 4.6 K. As noted previously, this was the minimum nominal temperature that could be reached in this configuration. Figure 1b shows the corresponding absorbance spectra of the four single-sweep spectra, ratioed to the background spectrum of Fig. 1a. Temperature dependent broadening is strikingly evident in this


series. In particular, the line narrowing evident in the fourth spectrum at 4.6 K compared to the third spectrum just above it is due to a nominal temperature difference of less than 3 K. All of the thin dipeptide layers investigated showed similar strong temperature-dependent line-narrowing with the onset of significant narrowing at nominal temperatures below 30 K. In contrast, for the dipeptide-PE samples studied at 4.2 K, significant differences in spectra were observed only for much higher temperatures of about 100 K.

1.5

THz

2

2.5

transmission (arbitrary units)

a)

empty waveguide 30 K at start of sweep

7 K at start of sweep 4.6 K throughout sweep

1 absorbance (arbitrary units)

1

1.5

THz

2

30 K at start of sweep

2.5

b)

7K at start of sweep

4.6 K throughout sweep

0 40

60 cm-1

80

40

60 cm-1

80

Fig. 2. Spectra of VA taken as the crystalline film cools from 30K to 4.6K. Spectra are offset for clarity, with descending order corresponding to descending temperature. a) power transmission spectra, with a background spectrum of the empty waveguide shown at top, followed by a series of VA spectra. b) The corresponding absorbance spectra for the VA transmission curves. Temperature is not constant through acquisition, but sensitivity to temperature is nonetheless extremely clear. In particular, the third VA spectrum was taken with a temperature reading dropping from 7K to 4.7K throughout acquisition, but is markedly broader than the final spectrum acquired at a constant temperature reading of 4.6 K.

Figures 3 through 6 show spectra of alanyl isoleucine (AI), isoleucyl alanine (IA), alanyl valine (AV), and valyl alanine (VA) respectively. Figures 3a through 6a show the spectra of the four isostructural dipeptides obtained from pressed polyethylene-dipeptide disk samples, at 4.2 K. Figures 3b through 6b show spectra of the same dipeptides acquired by waveguide spectroscopy of thin crystalline films of 1 to 2 µm thickness, at 4.6 K. The latter spectra suffer from significantly higher noise levels, first due to the reduced power transmitted by the waveguide (about 30% of incident, at 1 THz) and second, due to the difficulty of obtaining background spectra with good repeatability. That is to say, the waveguide must be removed, disassembled, cleaned of dipeptide, reassembled and replaced in the chamber to obtain a background spectrum. However, the system is quite sensitive to small changes in position, especially of the tightly focused silicon coupling lenses. It should be noted that spectra obtained from thin films in the waveguide required about 1 mg of dipeptide sample, compared to 10 to15 mg for the method of diluting the dipeptide with polyethylene to obtain a pressed disk sample thick enough to be durable. The waveguide technique therefore demonstrates a factor of ten increase in sensitivity.


THz

1

0

3

2

absorbance (arbitrary units)

d)

c)

b)

a)

0

20

40

cm-1

60

80

100

Fig 3. THz spectra of alanyl isoleucine. a) crystalline AI pressed with polyethylene. b) thin crystalline film, 1 µm to 2 µm thick. c) calculated using periodic boundary conditions in DMOL3 with the PW91 functional. d) calculated using periodic boundary conditions in CHARMM22.

THz

1

0

3

2


d)

c)

b)

a)

0

20

40

cm-1

60

80

100

Fig 4. THz spectra of isoleucyl alanine. a) crystalline IA pressed with polyethylene. b) thin crystalline film, 1µm to 2 µm thick. c) calculated using periodic boundary conditions in DMOL3 with the PW91 functional. d) calculated using periodic boundary conditions in CHARMM22.


THz

1

0

3

2


d)

c)

b)

a)

0

20

40

cm-1

60

80

100

Fig 5. THz spectra of alanyl valine. a) crystalline AV pressed with polyethylene. b) thin crystalline film, 1 µm to 2 µm thick. c) calculated using periodic boundary conditions in DMOL3 with the PW91 functional. d) calculated using periodic boundary conditions in CHARMM22.

THz

1

0

3

2


d)

c)

b)

a)

0

20

40

cm-1

60

80

100

Fig 6. THz spectra of valyl alanine. a) crystalline VA pressed with polyethylene. b) thin crystalline film, 1 to 2 µm thick. c) calculated using periodic boundary conditions in DMOL3 with the PW91 functional. d) calculated using periodic boundary conditions in CHARMm22.


All the spectra have been fit using Gaussian lineshapes, allowing line frequency, width, and intensity to vary in an iterative least-squares fit, as an aid in determining the minimum number of features that can account for the observed intensity. Certain of the less resolved shoulders and low intensity peaks have had widths fixed at values commensurate with the widths of nearby well-resolved features. Two features of the spectra obtained from thin crystalline films contrast strongly with the results obtained from disks made of mixed dipeptide and polyethylene. Most obviously, the spectral features from the thin film data are much narrower, by a factor of 2 to 4. Similar line narrowing in the spectra of thin films of tetracyanoquinodimethane and 1,3dicyanobenzene has previously been observed by Melinger et al. [7] using waveguide spectroscopy. Table 1 summarizes a comparison of widths obtained for thin film samples and for pressed PE-dipeptide disk samples, for some of the strong absorption lines in each of the four dipeptides. Second, in addition to the line-narrowing effects, differences in the relative line intensities are significant for some dipeptides. Relative intensity differences are especially evident in the spectra of IA, and also in the extremely strong, narrow lowest frequency feature seen in the VA thin film spectrum. This 0.40 cm-1 wide line is one of the strongest in the thin film spectrum, but is barely apparent as a broad low-intensity bump in the spectrum from a PE-dipeptide mixture.

Table 1. Gaussian widths of certain lines from fits to spectra of the four dipeptides, comparing results for the thin film samples versus pressed PE-dipeptide disk samples. All units are cm-1. Linewidths marked by “*” were not well-resolved, so were held fixed in fitting the spectra.

thin film frequency

PE-dipeptide mix

linewidth

frequency

linewidth

44.04 61.91 80.32 93.27

3.29 3.07 3.89 3.99

35.69 49.09 69.28 77.85 85.72

3.50* 4.10 4.15 3.04 4.57

52.23 59.93 64.18 83.02 89.41

2.21 2.80 2.43 2.89 4.00*

38.77 46.91 57.40 63.27

4.00* 3.45 3.77 4.20

AI 44.12 62.09 81.19 92.56

0.81 1.18 1.49 1.66

36.73 48.59 70.58 78.48 86.43

0.69 0.86 1.58 2.09 1.06

IA

AV 52.38 60.35 64.91 85.50 90.96

1.05 1.12 1.16 1.43 0.88

38.84 47.66 57.12 64.15

0.40 0.89 0.92 2.01

VA


We attribute the line narrowing to the improved crystal quality achievable by growing thin dipeptide layers. We have found that thicker films give broader spectral features. For one thin film sample which was estimated at 5-8 µm thick, spectral lines were overall as broad as those in the spectrum from the PE-dipeptide mixture. Also, the dipeptides which form good crystals most easily are VA and AV, in that order. In looking at Figs 3a,b through 6a,b, it is clear that these are also the dipeptides which show the most extreme line-narrowing, again, in that order. While not conclusive, these observations suggest that higher quality crystals with fewer lattice defects are the cause of the narrower spectral features. Differences in relative line intensity are due to the difference in orientational information contained in the spectra acquired by the two different methods. Where the photomixer is known to produce circularly polarized radiation varying with frequency [20], the electric field within the waveguide is linearly polarized. In addition, x-ray crystallographic interrogation of a VA film grown on a gold-coated silicon wafer showed that the nanotube-forming dipeptides grow with the c-axis normal to the surface; ie, with the nanotubes standing on end. Hence, spectra from the pressed PE-dipeptide disks were obtained with a varyingly circularly polarized electric field incident on randomly oriented crystallites in the sample disk. Conversely, spectra from the thin film samples are the result of a well-defined linearly polarized THz field, perpendicular to the waveguide plates, incident on crystallites which are also oriented with nanotube axis normal to the waveguide plate. Figures 3c through 6c show spectra calculated using the quantum chemical program DMOL3 with periodic boundary conditions, while Fig 3d through 6d show spectra calculated using periodic boundary conditions in CHARMM. The calculated spectra have not been separated into the different contributions to absorption from coupling to different polarizations, and should therefore most reasonably be compared to the data acquired from PE-dipeptide disk samples shown in Fig. 3a-6a. The computed spectra agree moderately well with the experimental spectra shown for the PE-dipeptide disk samples, for both DMOL3 or CHARMM calculations. The calculations are most successful for the two dipeptides IA and VA which have a single strong absorption peak at the lowest frequency, near 50 cm-1 and 47 cm-1 respectively, which is accompanied by a series of lower-intensity features at higher frequencies. The DMOL3 and CHARMM spectra both reproduce this pattern, although there is a very large red-shift of the IA peak to 20 cm-1 in CHARMM. Less dramatic red shifts of ~5 cm-1 for the main peak appear in the DMOL3 spectra of both IA and VA. The experimental spectra for the remaining two dipeptides, AI and AV, are reproduced less well, by both computational methods. The computed DMOL3 spectra for AI and AV, in Figures 3c and 5c, show significant redshifting, with the lowest frequency mode appearing 5 to 10 cm-1 lower than in the experimental data. For CHARMM, the red-shift is again very large, with the lowest modes appearing 15 to 20 cm-1 too low, compared to the experimental spectra. The poorer quality of agreement for these two dipeptides may be related to structural differences between the dipeptides. IA and VA are known from thorough crystallographic investigation by Gorbitz [20-22] to have qualitative structural differences compared to the retroanalogue peptides, AI and AV. First, IA has a very narrow pore, which can not accommodate any guest molecules whereas AI is known to accommodate guest water molecules in its roomier nanotube. Second, the structure of VA is more rigid than that of AV. AV is known to change pore size to accommodate several different guest alcohol molecules, whereas VA, having nearly the same pore size, was found not to accommodate such alcohol guests in the pores. The more constarined IA and VA structures might be expected to be less computationally problematic than the comparatively flexible hydrogen-bonding networks of AV and AI which may require greater computational accuracy. A number of factors can contribute to the disparity between experiment and calculations. Anharmonic effects have been calculated to cause deviations of up to 11% in the skeletal vibrations of gas-phase peptide molecules in this frequency region [23], and these are not considered in the harmonic frequency calculations performed in both DMOL3 and CHARMM here. Polarization effects of long-wavelength lattice modes have also been noted by other investigators as a critical correction in crystalline systems at THz frequencies [1]. Furthermore, as noted earlier, DMOL3 calculations have been done without optimizing the full unit cell. Further calculations implementing this step are under way. Finally, the limitations in the CHARMM description of hydrogen bonding which were mentioned earlier also contribute to error in the CHARMM calculations.


5. CONCLUSION We have demonstrated a factor of 10 increase in sensitivity using a plane parallel waveguide to perform waveguide absorption spectroscopy on four isostructural nanotube-forming dipeptides. Where PE-dipeptide disks required 10 to15 mg of sample for absorption spectroscopy, 1 mg of a thin dipeptide film is sufficient using the waveguide spectrometer. In addition, thin films of dipeptides produced well-resolved spectral features with a factor of 2 to 4 line-narrowing compared to spectra obtained from the PE-dipeptide disks. This experimental technique can be applied to obtain orientational information on the coupling of THz radiation to the molecular dipoles since the polarization in the waveguide is well-defined, and the sample orientation can also be well-defined. Such information can be usefully compared with the results of calculations as a further means of investigating possible improvements to current computational methods and validating theoretical models.

ACKNOWLEDGEMENTS The authors gratefully acknowledge the help of M. D. Vaudin at NIST in determining dipeptide orientation on gold surfaces.

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