Chao Chengabc,
Zhongjie Zhuac,
Shaoping Lib,
Guanhua Renacd,
Jianbing Zhangac,
Haixia Congc,
Yan Penge,
Jiaguang Hand,
Chao Chang*f and
Hongwei Zhao*ac
aZhangjiang Laboratory, Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai 201210, China
bSchool of Chemical Engineering, East China University of Science and Technology, Shanghai 200237, China
cShanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China. E-mail: zhaohongwei@sinap.ac.cn
dCenter for Terahertz Waves and College of Precision Instrument and Optoelectronics Engineering, Key Laboratory of Optoelectronics Information and Technology, Ministry of Education, Tianjin University, Tianjin 300072, China
eShanghai Key Lab of Modern Optical System, University of Shanghai for Science and Technology, No. 516, Jungong Road, Shanghai 200093, China
fAdvanced Interdisciplinary Technology Research Center, National Innovation Institute of Defense Technology, Beijing 100071, China. E-mail: changc@xjtu.edu.cn
First published on 28th June 2019
γ-Aminobutyric acid (GABA) is the chief inhibitory neurotransmitter in the central nervous system, its conformational behavior is critical for selective biological functions and the process of signal transmission. Although this neuroactive molecule has been extensively studied, its vibrational properties related to the conformation and intermolecular interactions in the terahertz (THz) band have not been identified experimentally yet. In this study, we applied a broadband THz time-domain spectroscopy (THz-TDS) system from 0.5 to 18 THz to characterize a unique THz fingerprint of GABA. The density functional theory calculation results agree well with the THz experimental spectrum. The study shows that the vibrational modes of GABA at 1.15 and 1.39 THz originate from distinct collective vibrations. The absorptions at the higher THz frequencies also carry part of collective vibrations, but more reflect the specific and local vibrational information, including the skeleton deformation and the rocking of the functional groups, which are closely associated with the conformation and flexibility of GABA. This study may help to understand the conformational transitions of neurotransmitter molecules and the resonant response to THz waves.
GABA has an amino group attached to the gamma carbon and a carboxyl group connected to the alpha carbon at the other end of the carbon chain. GABA molecules are zwitterions (with the amino group protonated and the carboxyl group deprotonated), partially folded, flexible, and held in a cross-linked chain arrangement by hydrogen bonds.7 Two different conformations of GABA, gauche and trans, have been reported in previous work.8,9 Besides, its molecular structure may help resolve its mode of action at some regions of the mammalian brain.1,7 Therefore, the study of the conformation and structure of GABA may throw some light on the conformational preference for binding with the receptor and help understand the ability of GABA to activate receptors.1,10
Vibrational spectroscopies, including Fourier-transform infrared (FTIR) spectroscopy and Raman spectroscopy, can provide fundamental and useful information on molecular structure and conformation. Studies have shown that terahertz (THz) spectroscopy can be used to successfully distinguish polymorph pharmaceuticals, research the interactions of chemical materials, and quantitatively detect impurities in organic crystals.11–13 Moreover, THz spectroscopy is sensitive to hydrogen bonding, lattice vibration, water of crystallization, and conformational change.11,14 Hand et al.15 revealed that THz spectroscopy between 1 and 10 THz can obtain more abundant low-frequency vibrational information of molecules, and THz spectra of many biomolecules between 6–9 THz corresponding to the densest transitions between states. Compare with the THz systems equipped with solid state emitters and sensors, gases have no phonon resonances or echoes due to THz or optical reflections, enabling broadband THz spectroscopy free of instrumental artifacts.16 Bartel et al.17 generated single-cycle THz transients in the frequency range 0.3–7 THz by using a Ti:sapphire amplifier in ionized air. Dai et al.18 reported the first demonstration of broadband THz wave detection through third-order nonlinear optical processes using gases as the sensor, and the wider spectral range of their improved system allows spectroscopic measurement across the full THz range and even enter the middle infrared.16 The broadband THz response does not interfere with THz sensitivity to biomolecular binding and the universal THz binding sensitivity has application potential for biosensing.19 Recently, Dampf et al.20 studied temperature-dependent THz-TDS spectra from 10 to 135 cm−1 (1 THz ∼33.33 cm−1) of GABA, they found an anomalous frequency shift upon cooling and further explained it by the quasiharmonic approximation in solid-state density functional theory (DFT) simulations. da Silva et al.2 investigated GABA in the range of 30 to 1700 cm−1 with Raman spectroscopy between 1 atm and 7.1 GPa, and they interpreted the modifications in the Raman spectra mainly on the basis of the conformational changes of the molecules in the unit cell. Du et al.21 studied GABA, benzoic acid (BA), and the corresponding cocrystal by Raman spectroscopy in the range of 150 to 3250 cm−1 and THz spectroscopy from 0.2 to 1.7 THz. They found that the formation of a GABA-BA cocrystal under slow solvent evaporation is affected by the pH value of the aqueous solution. Peng et al.22 researched two typical metabolites and three typical neurotransmitters, including GABA, from 0.9 to 4.5 THz by FTIR and successfully identified the components and proportions of a mixture consisting of neurotransmitters and neurotrophic substances. These works provide useful characteristics of GABA in the THz region and the tentative assignments of its modes. However, the details of the vibrational modes of GABA are still insufficient and the interpretation of the relationship between the THz signal and the biological function of the molecular vibration has not been clarified.
In this work, GABA was studied with an air-plasma based broadband THz time-domain spectroscopy (THz-TDS) system from 0.5 to 18 THz and was checked by another THz system (TAS7400TS) between 0.5 and 4.0 THz. To make out the difference of the above two systems at about 1 THz, an additional photoconductive-switch-based THz system, whose effective range is 0.2 to 2.5 THz, was used. The temperature effect on GABA from 83 to 363 K was investigated with the TAS7400TS fast scanning THz system. To better interpret the origin of the absorption peaks and corresponding vibrational modes of the experimental THz spectrum, the solid-state density functional theory calculation based on the crystal structure was performed. Considering GABA has polymorphism phenomenon, powder X-ray diffraction (PXRD) was utilized and the result indicated that the GABA we used belonged to the monoclinic system. A mid-infrared FTIR was also carried out to further check the broadband THz spectrum of GABA. The results demonstrate that the absorption of the broadband THz spectrum from 13 to 16 THz is mainly from the substrate and that the contribution of GABA is relatively small.
Drying air was continuously pumped into the THz system to keep the relative humidity under 1.0% during the measurements. The spectrum of each sample was the average of three measurements with the drying air as reference. The measurements with the THz system based on the air-plasma and the system based on the photoconductive switch included 1024 scan numbers. In order to obtain the real-time dynamic data at variable temperatures, 512 scan numbers were selected on the system based on the LiNbO3 nonlinear optical crystal.
The geometry optimization and energy calculations were carried out based on the solid-state DFT using the CASTEP26 program, a part of the Materials Studio package from Accelrys. Grimme's dispersion-corrected method was chose before we conducted geometry optimization. The result was obtained for the crystalline state with the generalized gradient approximation (GGA)-Perdew–Burke–Ernzerhof (PBE) correlation functional.27 Calculations were performed using a norm-conserving pseudopotential in CASTEP. The quality of the energy calculation was ultra-fine. The plane-wave cut-off energy was 830 eV. For GABA, Brillouin zone sampling of electronic states was performed on a 4 × 2 × 4 Monkhorst–Pack grid; the total energy was converged to 5.0 × 10−6 eV per atom and the maximum forces between atoms were less than 0.01 eV Å−1. The grid for the fast Fourier transform was 72 × 80 × 60.
The calculated THz spectrum of GABA is plotted against the experimental one in Fig. 3. It can be seen that the experimental spectrum is roughly in agreement with the calculated one. The temperature difference is main reason of the spectral deviations between the two. The calculated spectrum was performed at 0 K while the experimental broadband THz spectrum in Fig. 3 was measured at 293 K. Meanwhile, the disparity of the crystals conditions in actual experiments and theoretical simulations also need to be taken into consideration.11 The crystal cell parameters used for the calculations are derived from crystals recrystallized from aqueous solution.8 However, the GABA used in our experiment was a commercially obtained powder crystal, and its crystalline quality was not as good as that of recrystallization from solution. In addition, the humidity and pressure are also the factors influencing the experiments.
In order to explain the deviations between the experimental and the calculated spectra, the variable temperature experiment was carried out. Fig. 4 shows the evolution of the THz spectra of GABA from 83 to 363 K. Thirty-six spectra were measured with the TAS7400TS fast-scanning THz system. As we can see in Fig. 4, the broad peak at 3.50 THz splits into shoulder peaks at 3.36 and 3.74 THz upon cooling. An unusual thermal behaviour that occurs at 1.13 THz at 363 K shifts to 0.79 THz at 83 K was exhibited in Fig. 4. The absorption peaks at 1.52, 2.03, and 2.63 THz, at 293 K, blue-shifted with decreasing temperature. All the absorption peaks become more distinct upon cooling. The results of variable temperature experiment agrees well with that of Dampf et al.20 They attributed the unusual thermal behaviour to the distortion of a specific weak intermolecular hydrogen bond in solid GABA, and they explained the spectral features generally narrow and shift to higher frequencies in response to cooling as the crystallographic unit cell changes with temperature. Besides, the degree of shift in different peaks is not the same, this is probably due to different vibrational modes having different degrees of response to the temperature change. The difference between the experimental and calculated THz spectra of GABA is well explained by the variable temperature experiment.
Fig. 4 Evolution of the THz spectra of GABA from 83 to 363 K (The color bar indicates the magnitude of the absorption coefficient). |
Fig. 5 (a) Carbon atomic nomenclature of the GABA molecule in the lattice, C(1) = Cγ, C(2) = Cβ, C(3) = Cα. (b) The hydrogen bonds network (black dashed lines) of GABA lattices. |
Four typical vibrational modes of GABA (1.15, 1.39, 8.53, and 17.05 THz) are presented in Fig. 6. The experimental peaks at 1.09 and 1.52 THz correspond to the calculated modes at 1.15 and 1.39 THz, arising from rotation and translation of GABA, respectively, with all atoms involved. They indicate that at 1.09 and 1.52 THz, the characteristic features of GABA come from distinct collective vibrations of the molecule or lattice mode. The broad 3.48 THz peak at 293 K, split into shoulder peaks at 83 K, which can be attributed to the calculated modes at 3.73 and 4.06 THz, comes from the CαCβCγN skeleton deformation, translation of the COO− group at 3.73 THz and rocking of the COO− group at 4.06 THz. In addition, the measured feature at 8.26 THz corresponds to the vibrational mode calculated at 8.53 THz, mainly from the wagging of the CαCβCγ skeleton and rocking of the NH3+ group. These results suggest that at higher THz frequencies, the absorptions also take along part of collective vibrations but mainly from the specific and local movements of the molecule. The descriptions of the calculated vibrational modes of GABA are shown in Table 1. By analyzing the vibrational modes of GABA between 0.5 and 18 THz, it was found that most of the vibrations are collective, but there were differences in the contribution of the skeleton and functional groups. Hand et al.15 revealed that THz spectra of many biomolecules above 6 THz corresponding to available conformational states and dense transitions between phonon modes. As a functional group of GABA, amino is closely related to the biological effects of the molecule. Taking the amino group as an example, its motions after 5.80 THz make a more prominent contribution to the vibration of GABA. This may indicate the structural or even conformational transformation of GABA molecule after 6 THz. Besides, although the frequencies of the absorption peaks at 8.17 and 8.53 THz are similar, their vibrational modes are quite different, which further indicates the complexity of the vibration of GABA in the THz band.
Fig. 6 Calculated vibrational modes of GABA at 1.15, 1.39, 8.53, and 17.05 THz (The blue arrows represent the direction of the molecular vibration). |
Exp./THz | Cal./THz | Vibrational mode assignment | |
---|---|---|---|
293 K | 83 K | 0 K | |
a ν, rotation; t, translation; ω, wagging; r, rocking; δtw, twisting; δ, bending. | |||
1.09 | 0.79 | 1.15 | Collective |
1.52 | 1.57 | 1.39 | Collective |
2.03 | 2.17 | 2.68 | t(COO−) + ν(CαCβCγ) + t(NH3+) |
2.58 | 2.75 | 3.16 | Mainly from r(CαCβCγ) + t(NH3+) |
3.37 | Mainly from t(CαCβCγ) | ||
3.48 | 3.36 | 3.73 | t(COO−) + δtw(CαCβCγN) |
3.74 | 4.06 | r(COO−) + ω(CαCβCγN) | |
4.34 | 4.72 | r(COO−) + δtw(CαCβCγN) | |
5.08 | Mainly from ν(CβCγ) | ||
5.53 | 5.59 | Mainly from ν(CβCγ) | |
5.80 | Mainly from r(CαCβCγ) + t(NH3+) | ||
5.92 | r(COO−) + r(CαCβCγN) | ||
7.80 | 8.17 | Mainly from δ(CαCβCγ) + r(NH3+) | |
8.26 | 8.53 | Mainly from ω(CαCβCγ) + r(NH3+) | |
9.63 | 9.85 | r(COO−) + δ(CαCβCγN) | |
10.09 | r(COO−) + δ(CαCβCγN) | ||
12.0 | 11.89 | r(COO−) + δ(CαCβCγ) + r(NH3+) | |
12.16 | r(COO−) + δ(CαCβCγ) + r(NH3+) | ||
16.7 | 17.05 | t(COO−) + δ(CαCβCγN) |
Chou31 used a basic natural law to elucidate some important biological functions of an IgG antibody molecule. He suggested that when the dominant low-frequency amplitude of a domain is larger, the energy barrier which must be surmounted for a successful biochemical reaction is more easily overcome.32 Consequently, the variation of the dominant low-frequency amplitudes of various domains in an antibody molecule will reflect not only the effects of physical coupling but, more importantly, their mutual influence in the biological function.30 Recently, Woods et al.33 found that during the photo-isomerization process, the retinal low-frequency motion is directly tied with the overall protein configuration. Balu et al.34 demonstrated that low-frequency vibrations are related to the conformational changes in bacteriorhodopsin and rhodopsin. GABA ligand can interact with diverse receptors. The resonant coupling at different frequencies corresponds to the specific molecular structures and interactions. The structural fluctuations of the flexible ligand play a critical role in the conformational selection in proteins. GABA functions appear to be triggered by binding of GABA to its ionotropic and metabotropic receptors, and the diversity in the pharmacologic properties of the receptor subtypes is clinically important.35 Structural or conformational fluctuations and microenvironment changes of the neurotransmitter may affect the mutual recognition and interactions between the ligand and the receptors, which have potential impact on biological events such as specific selection, signal transmission, and activation of biomolecules. Therefore, the properties of the low-frequency vibrational modes in a wide THz region provide useful information for understanding the neurotransmitter molecule and may also throw some light on the biological function of GABA in the synapse.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c9ra02971k |
This journal is © The Royal Society of Chemistry 2019 |