Terahertz Time-Domain Spectroscopy of Four Hydroxycinnamic Acid Derivatives

The well-resolved absorption spectra of the hydroxycinnamic acid (HCA) derivatives, caffeic acid, ferulic acid, sinapic acid and chlorogenic acid, were measured over the frequency region from 0.3 to 2.0 THz at 294 K with terahertz time-domain spectroscopy (THz-TDS). Theoretical calculation was applied to assist the analysis and assignment of the individual THz absorption spectra of the HCA derivatives with density functional theory (DFT). The distinctive spectral features were originated from the collective motion of molecules held together by hydrogen bonds. The real and imaginary parts of dielectric function of the four HCA derivatives were also obtained.     

Thermal denaturation of CP43 studied by Fourier transform-infrared spectroscopy and terahertz time-domain spectroscopy

Thermal denaturation of CP43 was studied by Fourier transform-infrared (FT-IR) spectroscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and terahertz time-domain spectroscopy (THz-TDS). Under heat treatment, the secondary structure of CP43 changed, and the main thermal transition occurred at 59 degrees C. During the process, CP43 aggregated at first, and then with increasing temperature degraded. The low-frequency collective vibrational modes of CP43 changed with increasing temperature and decreasing mass. THz-TDS is a new technique used to study the conformational state of a molecule, and it is the first use of this technique to study the photosynthesis membrane proteins in this paper. The results presented here demonstrate that THz-TDS has both advantages and disadvantages in monitoring the thermal denaturation of membrane proteins, which is important in applying THz-TDS technique to study of biomolecules.     

Vibrational analysis and spectra of orotic acid

The IR and Raman spectra of polycrystalline anhydrous orotic acid and its N1, N3, and O12 trideuterated isotopomer are recorded in the 4000-40 cm(-1) spectral interval as part of a series of vibrational analyses of nucleosides, nucleotides, and related compounds carried out in our laboratory. The frequencies of the fundamental transitions and the potential energy distributions of the 39 normal modes of orotic acid and its isotopomer are calculated by an ab initio density functional theory Becke3P86/6-311G** treatment. Assignments of the vibrational modes are proposed that consider the results of these calculations and the observed spectra. The results of the ab initio treatment are related to crystallographic and spectral data, and they are compared with previous assignments for similar molecules.     

A study of flavin-protein and flavoprotein-ligand interactions. Binding aspects and spectral properties of D-amino acid oxidase and riboflavin binding protein.

To study flavin-protein and flavoprotein-ligand interaction, the absorption, CD and MCD spectra of riboflavin, FAD, roseoflavin, the complexes of riboflavin and roseoflavin with riboflavin binding protein(RBP),D-amino acid oxidase(D-AO) and its complexes with ligands were observed in the spectral region of 310-600 nm and the binding properties of D-AO with di-substituted benzoate derivatives and of RBP with roseoflavin were also measured. The dimer of D-amino acid oxidase has a higher affinity for di-substituted benzoate derivatives than the monomer. The change in the absorption of FAD in D-AO caused by the binding of the first ligand to the dimer, which can bind two ligands, was similar to that caused by the binding of the second ligand. Roseoflavin could bind to RBP in a 1 : 1 ratio and the dissociation constant was 3.8 x 10(-8)M. The protein fluorescence of RBP was quenched by about 86% due to complex formation with roseoflavin. The MCD spectra showed similar patterns for all molecular complexes of riboflavin and FAD, with two negative extrema of ellipticity which probably correspond to the Faraday B-term, but the Faraday A-term could not be observed, suggesting that there was no degeneracy in the excited state of flavins. It is also suggested, based on a comparison of the absorption, CD and MCD spectra, that the vibronic structure of flavin was modified differently by each flavin-protein or flavoprotein-ligand interaction. Comparison of the absorption, CD and MCD spectra(310-600 nm) for roseoflavin and the roseoflavin-RBP complex revealed that there were five spectral components around 320, 340, 400, 500, and 550 nm in roseoflavin.     

S-state dependent Fourier transform infrared difference spectra for the photosystem II oxygen evolving complex

Vibrational spectroscopy provides a means to investigate molecular interactions within the active site of an enzyme. We have applied difference FTIR spectroscopy coupled with a flash turnover protocol of photosystem II (PSII) to study the oxygen evolving complex (OEC). Our data show two overlapping oscillatory patterns as the sample is flashed through the four-step S-state cycle that produces O(2) from two H(2)O molecules. The first oscillation pattern of the spectra shows a four-flash period four oscillation and reveals a number of new vibrational modes for each S-state transition, indicative of unique structural changes involved in the formation of each S-state. Importantly, the first and second flash difference spectra are reproduced in the 1800-1200 cm(-)(1) spectral region by the fifth and sixth flash difference spectra, respectively. The second oscillation pattern observed is a four-flash, period-two oscillation associated with changes primarily to the amide I and II modes and reports on changes in sign of these modes that alternate 0:0:1:1 during S-state advance. This four-flash, period-two oscillation undergoes sign inversion that alternates during the S(1)-to-S(2) and S(3)-to-S(0) transitions. Underlying this four-flash period two is a small-scale change in protein secondary structure in the PSII complex that is directly related to S-state advance. These oscillation patterns and their relationships with other PSII phenomena are discussed, and future work can initiate more detailed vibrational FTIR studies for the S-state transitions providing spectral assignments and further structural and mechanistic insight into the photosynthetic water oxidation reaction.     

Chlorophyll a and carotenoid triplet states in light-harvesting complex II of higher plants.

Laser-flash-induced transient absorption measurements were performed on trimeric light-harvesting complex II to study carotenoid (Car) and chlorophyll (Chl) triplet states as a function of temperature. In these complexes efficient transfer of triplets from Chl to Car occurs as a protection mechanism against singlet oxygen formation. It appears that at room temperature all triplets are being transferred from Chl to Car; at lower temperatures (77 K and below) the transfer is less efficient and chlorophyll triplets can be observed. In the presence of oxygen at room temperature the Car triplets are partly quenched by oxygen and two different Car triplet spectral species can be distinguished because of a difference in quenching rate. One of these spectral species is replaced by another one upon cooling to 4 Ki demonstrating that at least three carotenoids are in close contact with chlorophylls. The triplet minus singlet absorption (T-S) spectra show maxima at 504-506 nm and 517-523 nm, respectively. In the Chl Qy region absorption changes can be observed that are caused by Car triplets. The T-S spectra in the Chl region show an interesting temperature dependence which indicates that various Car's are in contact with different Chl a molecules. The results are discussed in terms of the crystal structure of light-harvesting complex II.     

New estimation method for fatty acid composition in oil using near infrared spectroscopy

The absorption bands of cis-unsaturation and the carbon chain length of the fatty acid moieties in oil appear in the near infrared (NIR) wavelength region, especially around 1600-1800 nm. Using this region, a new estimation method for fatty acid composition analysis is proposed. Because the differences of the original NIR spectra are miniscule even in this region, the second derivative NIR spectra were examined in order to estimate the fatty acid composition in oil exclusively from the spectral patterns obtained. The parameters for calculating the second derivative NIR spectra were examined to make the spectral difference clearer. In any parameter, the absorption band was shifted to the shorter wavelength region when the unsaturation in fatty acid moieties increased, and it was shifted to the longer wavelength region when the carbon chain length increased. When the parameters were correct, this NIR method can estimate the fatty acid composition roughly, but simply, easily, and sometimes nondestructively.     

Vibrational circular dichroism of A-, B-, and Z-form nucleic acids in the PO2-stretching region.

Vibrational circular dichroism (VCD) spectra were measured for H2O solutions of several natural and model DNAs (single and double strands, oligomers and polymers) in the B-form, poly(dG-dC)-poly(dG-dC) in the Z-form, and various duplex RNAs in an A-form over the PO2-stretching region. Only the symmetric PO2 stretch at approximately 1075 cm-1 yields a significant intensity VCD signal. Differences of the PO2-stretching VCD spectra found for these conformational types are consistent with the spectral changes seen in the base region, but no sequence dependence was seen in contrast to VCD for base modes. The B to Z transition is accompanied by an inversion of the PO2- VCD spectra, which is characteristic of the change in the helical sense of the nucleic acid backbone. A-RNAs give rise to the same sense of couplet VCD as do B-DNAs but have a somewhat different shape because of overlapping ribose modes. These PO2- VCD spectral characteristics have been successfully modeled using simple dipole coupling calculations. The invariability of the symmetric PO2- stretching mode VCD spectra to the base sequence as opposed to that found for the C = O stretching and base deformation modes is evidence that this mode will provide a stable indication of the DNA helical sense.     

Circular dichroism of helical polynucleotides calculated by the linear response method

Linear response theory in the decorrelation or random-phase approximation is used to calculate the absorption and CD spectra of model helical polymers, including single-stranded polyadenylic acid. The method, which makes use of infinite polymer selection rules for the linear response tensor, has the advantages that (1) only a few three-dimensional matrices need be inverted; (2) spectral band shapes of the polymer arise naturally from those of the monomer, as well as from the geometry-dependent interactions in the helix; and (3) the spectral dependence on geometrical factors of the helix is made transparent. It is found that the structure of the polymer CD spectrum depends critically on monomer bandshape. An asymmetric CD spectrum, similar to some experimental spectra, arises from either a Gaussian or a composite monomer band. Single-stranded polyadenylic acid spectra are sensitive to helix geometry in the region 200–240 nm, in reasonable agreement with experimental spectra. This sensitivity arises from the 207-nm monomer transition, and the results suggest that this region of the spectrum should be more fully exploited as a tool for helix geometry studies.     

Probing the collective vibrational dynamics of a protein in liquid water by terahertz absorption spectroscopy

Biological polymers are expected to exhibit functionally relevant, global, and subglobal collective modes in the terahertz (THz) frequency range (i.e., picosecond timescale). In an effort to monitor these collective motions, we have experimentally determined the absorption spectrum of solvated bovine serum albumin (BSA) from 0.3 to 3.72 THz (10-124 cm(-1)). We successfully extract the terahertz molar absorption of the solvated BSA from the much stronger attenuation of water and observe in the solvated protein a dense, overlapping spectrum of vibrational modes that increases monotonically with increasing frequency. We see no evidence of distinct, strong, spectral features, suggesting that no specific collective vibrations dominate the protein's spectrum of motions, consistent with the predictions of molecular dynamics simulations and normal mode analyses of a range of small proteins. The shape of the observed spectrum resembles the ideal quadratic spectral density expected for a disordered ionic solid, indicating that the terahertz normal mode density of the solvated BSA may be modeled, to first order, as that of a three-dimensional elastic nanoparticle with an aperiodic charge distribution. Nevertheless, there are important detailed departures from that of a disordered inorganic solid or the normal mode densities predicted for several smaller proteins. These departures are presumably the spectral features arising from the unique molecular details of the solvated BSA. The techniques used here and measurements have the potential to experimentally confront theoretical calculations on a frequency scale that is important for macromolecular motions in a biologically relevant water environment.     

Circular dichroism and absorbance properties of nitrotyrosyl chromophores in staphylococcal nuclease and in a model diketopiperazine.

An active derivative of staphylococcal nuclease, in which only tyrosine residue 115 has been nitrated with use of tetranitromethane, has been characterized using absorbance, circular dichroism, and fluorescence spectroscopy. The results show that nitrotyrosine-115 nuclease is indistinguishable from native nuclease with regard to the average secondary structure of the folded polypeptide chain, the susceptibility of the enzyme to heat denaturation, and the local tertiary structure around tryptophan residue 140. Inasmuch as optical properties of nitrotyrosine-115 nuclease from 300 to 500 nm can be unambiguously assigned to nitrotyrosine residue 115 in the active site region, this modified enzyme presents a good model system for studying the circular dichroism properties of this aromatic amino acid in a protein. The spectral properties of nitrotyrosine-115 nuclease have been compared to those of the model compounds, cyclo-(-Gly-Tyr(3NO2)-) and Tyr(3NO2). Circular dichroism spectral changes in nitrotyrosine-115 nuclease due to the binding of deoxythymidine 3',5'-diphosphate and Ca-2+ have been compared to the corresponding nitrotyrosyl-115 absorption spectral changes. This comparison shows that the circular dichroism difference spectrum exhibits an over-all change in the intensity of the observed Cotton effects, whereas the absorption difference spectrum exhibits a blue shift. This finding supports the suggestion that perturbations of aromatic amino acid chromophores in proteins due to ligand binding result in red or blue shifts in absorption difference spectra, but in over-all changes of intensity in circular dichroism difference spectra.     

Mid- to low-frequency Fourier transform infrared spectra of S-state cycle for photosynthetic water oxidation in Synechocystis sp. PCC 6803

Flash-induced Fourier transform infrared (FTIR) difference spectra for the four-step S-state cycle and the effects of global (15)N- and (13)C-isotope labeling on the difference spectra were examined for the first time in the mid- to low-frequency (1200-800 cm(-1)) as well as the mid-frequency (1700-1200 cm(-1)) regions using photosystem (PS) II core particles from cyanobacterium Synechocystis sp. PCC 6803. The difference spectra clearly exhibited the characteristic vibrational features for each transition during the S-state cycling. It is likely that the bands that change their sign and intensity with the S-state advances reflect the changes of the amino acid residues and protein matrices that have functional and/or structural roles within the oxygen-evolving complex (OEC). Except for some minor differences, the trends of S-state dependence in the 1700-1200 cm(-1) frequency spectra of the PS II cores from Synechocystis were comparable to that of spinach, indicating that the structural changes of the polypeptide backbones and amino acid side chains that occur during the oxygen evolution are inherently identical between cyanobacteria and higher plants. Upon (13)C-labeling, most of the bands, including amide I and II modes and carboxylate stretching modes, showed downward shifts; in contrast, (15)N-labeling induced isotopic shifts that were predominantly observed in the amide II region. In the mid- to low-frequency region, several bands in the 1200-1140 cm(-1) region were attributable to the nitrogen- and/or carbon-containing group(s) that are closely related to the oxygen evolution process. Specifically, the putative histidine ligand exhibited a band at 1113 cm(-1) which was affected by both (15)N- and (13)C-labeling and showed distinct S-state dependency. The light-induced bands in the 900-800 cm(-1) region were downshifted only by (13)C-labeling, whereas the bands in the 1000-900 cm(-1) region were affected by both (15)N- and (13)C-labeling. Several modes in the mid- to low-frequency spectra were induced by the change in protonation state of the buffer molecules accompanied by S-state transitions. Our studies on the light-induced spectrum showed that contributions from the redox changes of Q(A) and the non-heme iron at the acceptor side and Y(D) were minimal. It was, therefore, suggested that the observed bands in the 1000-800 cm(-1) region include the modes of the amino acid side chains that are coupled to the oxidation of the Mn cluster. S-state-dependent changes were observed in some of the bands.     

High-resolution waveguide THz spectroscopy of biological molecules

Low-frequency vibrational modes of biological molecules consist of intramolecular modes, which are dependent on the molecule as a whole, as well as intermolecular modes, which arise from hydrogen-bonding interactions and van der Waals forces. Vibrational modes thus contain important information about conformation dynamics of biological molecules, and can also be used for identification purposes. However, conventional Fourier transform infrared spectroscopy and terahertz time-domain spectroscopy (THz-TDS) often result in broad, overlapping features that are difficult to distinguish. The technique of waveguide THz-TDS has been recently developed, resulting in sharper features. For this technique, an ordered polycrystalline film of the molecule is formed on a metal sample plate. This plate is incorporated into a metal parallel-plate waveguide and probed via waveguide THz-TDS. The planar order of the film reduces the inhomogeneous broadening, and cooling of the samples to 77K reduces the homogenous broadening. This combination results in the line-narrowing of THz vibrational modes, in some cases to an unprecedented degree. Here, this technique has been demonstrated with seven small biological molecules, thymine, deoxycytidine, adenosine, D-glucose, tryptophan, glycine, and L-alanine. The successful demonstration of this technique shows the possibilities and promise for future studies of internal vibrational modes of large biological molecules.     

Spectral studies of human erythrocyte catalase.

The optical absorption and circular dichroic spectra of human erythrocyte catalase (EC 1.11.1.6) and its cyanide, azide, and fluoride derivatives over the wavelength range of 210 to 700 nm are reported. Treatment with acid or alkaline solutions causes spectral changes which may be due to dissociation of the enzyme into subunits and removal of the heme group from the protein. The fractions of the protein structure present as alpha helix, beta pleated sheet, and unordered structure have been estimated from the CD spectrum in the far-ultraviolet region. The CD spectra also indicate that the protein conformation does not change appreciably after cyanide binding. The epr spectroscopy of the native enzyme and its cyanide complex are reported. The spectral results are compared with catalase obtained from other mammalian and bacterial sources.     

Optical effects of sodium dodecyl sulfate treatment of the isolated light harvesting complex of higher plants

The light-harvesting complex (LHC) of higher plants isolated using Triton X-100 has been studied during its transformation into a monomeric form known as CPII. The change was accomplished by gradually increasing the concentration of the detergent, sodium dodecyl sulfate (SDS). Changes in the red spectral region of the absorption, circular dichroism (CD), and linear dichroism spectra occurring during this treatment have been observed at room temperature. According to a current hypothesis the main features of the visible region absorption and CD spectra of CPII can be explained reasonably successfully in terms of an exciton coupling among its chlorophyll (Chl) b molecules. We suggest that the spectral differences between the isolated LHC and the CPII may be understood basically in terms of an exciton coupling between the Chl b core of a given CPII unit and at least one of the Chla's of either the same or the adjacent CPII. We propose that this Chl a-Chl b coupling existing in LHC disappears upon segregation into CPII, probably as a result of a detergent-related overall rotation of the strongly coupled Chl b core which changes the relative orientations of the two types of pigments and thus the nature of their coupling.Abbreviations Chl Chlorophyll - CD Circular dichroism - LD Linear dichroism - LHC Light-harvesting complex - SDS Sodium dodecyl sulfate - CPII A solubilized form of LHC obtained with SDS polyacrylamide gel electrophoresis Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement     

Redox-induced conformational changes in myoglobin and hemoglobin: electrochemistry and ultraviolet-visible and Fourier transform infrared difference spectroscopy at surface-modified gold electrodes in an ultra-thin-layer spectroelectrochemical cell.

Using suitable surface-modified electrodes, we have developed an electrochemical system which allows a reversible heterogeneous electron transfer at high (approximately 5 mM) protein concentrations between the electrode and myoglobin or hemoglobin in an optically transparent thin-layer electrochemical (OTTLE) cell. With this cell, which is transparent from 190 to 10,000 nm, we have been able to obtain electrochemically-induced Fourier-transform infrared (FTIR) difference spectra of both proteins. Clean protein difference spectra between the redox states were obtained because of the absence of redox mediators in the protein solution. The reduced-minus-oxidized difference spectra are characteristic for each protein and arise from redox-sensitive heme modes as well as from polypeptide backbone and amino acid side chain conformational changes concomitant with the redox transition. The amplitudes of the difference bands, however, are small as compared to the total amide I absorbance, and correspond to approximately 1% (4%) of the reduced-minus-oxidized difference absorbance in the Soret region of myoglobin (hemoglobin) and to less than 0.1% of the total amide I absorbance. Some of the bands in the 1560-1490-cm-1 spectral regions could be assigned to side-chain vibrational modes of aromatic amino acids. In the conformationally sensitive spectral region between 1680 and 1630 cm-1, bands could be attributed to peptide C = O modes because of their small (2-5 cm-1) shift in 2H2O. A similar assignment could be achieved for amide II modes because of their strong shift in 2H2O.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes of absorption spectra during heat-induced denaturation of Photosystem II core antenna complexes CP43 and CP47: revealing the binding states of chlorophyll molecules in these two complexes

The Photosystem II (PSII) core antenna complexes, CP43 and CP47, were prepared from spinach (Spinacia oleracea L.). The absorption spectra in the red region at room temperature were recorded for the PSII core antenna samples after increased temperature treatment (up to 80 degrees C). Derivative and difference spectra revealed the existence of two groups of chlorophyll a (Chl a) molecules in both CP43 and CP47. The one with the absorption peak in the shorter wavelength region was designated as CP43-669 and CP47-669, while the other with the absorption peak in the longer wavelength region was designated as CP43-682 and CP47-680. The results of the thermal treatment experiment demonstrated that CP43-669 and CP47-669 may exist as monomers of Chl a and that their binding sites on the polypeptides are insensitive to thermal treatment, whereas CP43-682 and CP47-680 may exist as dimers or multimers of Chl a and their binding regions in the polypeptide chains are more sensitive to heat treatment. The excitation energy transfer mechanism between these two different groups of Chl a molecules is also analyzed.     

Spectroscopic properties of the main-form and high-salt peridinin-chlorophyll a proteins from Amphidinium carterae

The main-form (MFPCP) and high-salt (HSPCP) peridinin-chlorophyll a proteins from the dinoflagellate Amphidinium carterae were investigated using absorption, fluorescence, fluorescence excitation, two-photon, and fast-transient optical spectroscopy. Pigment analysis has demonstrated previously that MFPCP contains eight peridinins and two chlorophyll (Chl) a molecules, whereas HSPCP has six peridinins and two Chl a molecules [Sharples, F. P., et al. (1996) Biochim. Biophys. Acta 1276, 117-123]. Absorption spectra of the complexes were recorded at 10 K and analyzed in the 400-600 nm region by summing the individual 10 K spectra of Chl a and peridinin recorded in 2-MTHF. The absorption spectral profiles of the complexes in the Q(y) region between 650 and 700 nm were fit using Gaussian functions. The absorption and fluorescence spectra from both complexes exhibit several distinguishing features that become evident only at cryogenic temperatures. In particular, at low temperatures the Q(y) transitions of the Chls bound in the HSPCP complex are split into two well-resolved bands. Fluorescence excitation spectroscopy has revealed that the peridinin-to-Chl a energy transfer efficiency is high (>95%). Transient absorption spectroscopy has been used to measure the rate of energy transfer between the two bound Chls which is a factor of 2.9 slower in HSPCP than in MFPCP. The kinetic data are interpreted in terms of the F?rster mechanism describing energy transfer between weakly coupled, spatially fixed, donor-acceptor Chl a molecules. The study provides insight into the molecular factors that control energy transfer in this class of light-harvesting pigment-protein complexes.     

龙须菜及其色素突变体藻胆蛋白的光谱及分子特征的比较(英)

对龙须菜 (GracilarialemaneiformisGreville)及其色素突变体藻胆蛋白吸收光谱进行了比较研究 ,结果显示不同藻株藻红蛋白的吸收光谱有显著的变化 ,而藻蓝蛋白和别藻蓝蛋白的基本相同。我们克隆了龙须菜及其色素突变体的藻红蛋白亚基的部分基因序列 ,用该基因序列推导出的氨基酸序列进行分析以揭示这一变化的分子机理 ,结果显示除几个氨基酸残基的替换外 ,几株藻间的藻红蛋白的氨基酸序列十分相似 ,一些氨基酸的替换发生在决定藻红蛋白二级结构及亚基间相互作用的区域 ,可能会影响藻胆蛋白的构型及相互作用 ,导致光谱性质的变化。