Study on hydrogen bonds of carboxymethyl cellulose sodium film with two-dimensional correlation infrared spectroscopy

Carboxymethyl cellulose is widely used in many industrial aspects and also in laboratory due to its good biocompatibility. However, special researches on infrared especially aiming at the hydrogen bonds structure of carboxymethyl cellulose were relatively poor. We demonstrate here a full view of infrared spectroscopy in the temperature range of 40–220 °C, mainly aiming at the hydrogen bonds in the NaCMC film. The two important transition points was defined with DSC and together with Infrared analysis, that is, 100 °C corresponding to the complete loss of water molecules and 170 °C to the starting temperature point the O6H6 being oxidized. The series of IR spectra during heating from 40 to 220 °C was analyzed by the two-dimensional correlation method. We found that the water molecules bound with CO groups and OH groups. With the evaporating of water molecules, the hydrated CO groups gradually transited into non-hydrated CO groups. As the temperature continued to increase, the intrachain hydrogen bonds were weakened and transited into weak hydrogen bonds. When the temperature was higher than 170 °C, the O6H6 groups were gradually oxidized and thus the interchain hydrogen bonds formed between CH₂COONa groups and O6H6 were weakened. In summary, we defined the main sorts of hydrogen bonds in carboxymethyl cellulose and pictured the changes of the hydrogen bonds structure during heating process, which may provide for the further application in both industry aspects and laboratory use.     

Conformation transition in silk protein films monitored by time-resolved Fourier transform infrared spectroscopy: effect of potassium ions on Nephila spidroin films

We used time-resolved Fourier transform infrared spectroscopy (FTIR) to follow a conformation transition in Nephila spidroin film from random coil and/or helical structures to beta-sheet induced by the addition of KCl from 0.01 to 1.0 mol/L in D(2)O. Time series difference spectra showed parallel increases in absorption at 1620 and 1691 cm(-)(1), indicating formation of beta-sheet, together with a coincident loss of intensity of approximately 1650 cm(-)(1), indicating decrease of random coil and/or helical structures. Increase in KCl concentration produced an increased rate of the conformation transition that may attributable to weakening of hydrogen bonds within spidroin macromolecules. The conformation transition was a biphasic process with [KCl] > or = 0.3 mol/L but monophasic with [KCl] < or = 0.1 mol/L. This suggests that, at high KCl concentrations, segments of the molecular chain are adjusted first and then the whole molecule undergoes rearrangement. We discuss the possible significance of these findings to an understanding of the way that spiders spin silk.     

Structural study of the catalytic domain of PKCzeta using infrared spectroscopy and two-dimensional infrared correlation spectroscopy

The secondary structure of the catalytic domain from protein kinase C zeta was studied using IR spectroscopy. In the presence of the substrate MgATP, there was a significant change in the secondary structure. After heating to 80 degrees C, a 14% decrease in the alpha-helix component was observed, accompanied by a 6% decrease in the beta-pleated sheet; no change was observed in the large loops or in 3(10)-helix plus associated loops. The maximum increase with heating was observed in the aggregated beta-sheet component, with an increase of 14%. In the presence of MgATP, and compared with the sample heated in its absence, there was a substantial decrease in the 3(10)-helix plus associated loops and an increase in alpha-helix. Synchronous 2D-IR correlation showed that the main changes occurred at 1617 cm(-1), which was assigned to changes in the intermolecular aggregated beta-sheet of the denaturated protein. This increase was mainly correlated with the change in alpha-helix. In the presence of MgATP, the main correlation was between aggregated beta-sheet and the large loops component. The asynchronous 2D-correlation spectrum indicated that a number of components are transformed in intermolecularly aggregated beta-sheet, especially the alpha-helix and beta-sheet components. It is interesting that changes in 3(10)-helix plus associated loops and in alpha-helix preceded changes in large loops, which suggests that the open loops structure exists as an intermediate state during denaturation. In summary, IR spectroscopy revealed an important effect of MgATP on the secondary structure and on the thermal unfolding process when this was induced, whereas 2D-IR correlation spectroscopy allowed us to show the establishment of the denaturation pathway of this protein.     

Structure of beta-purothionin in membranes: a two-dimensional infrared correlation spectroscopy study

Two-dimensional infrared correlation spectroscopy has been used to investigate the structure of beta-purothionin, a small basic protein found in the endosperm of wheat seeds, in the absence and presence of dimyristoylphosphatidylglycerol (DMPG) membranes. To generate the two-dimensional synchronous and asynchronous maps, hydrogen-deuterium exchange of the protein amide protons has been used as an external perturbation. This method has allowed us to separate the different secondary structure elements and side chain contributions in the regions of amide I, II, and II' bands to determine that the relative order of deuteration of the beta-purothionin protons is as follows: turns, asparagines, and lysines > unordered structure and tyrosine > beta-sheet > alpha-helices and arginines. The results also indicate that the protein undergoes significant changes both in secondary structure and in deuteration in the presence of DMPG bilayers. The helical content of beta-purothionin is higher in the presence of the lipid, and the relative order of deuteration is as follows: lysines and arginines > asparagines and beta-sheet > unordered structure and alpha-helices. The inversion in the deuteration order of the arginine residues is assigned to a change of the degree of association of the protein in the membrane. In addition, the results reveal that the part of the protein containing the tyrosine residue interacts with the lipid membrane. Our results combined with those previously published suggest that the toxicity of beta-purothionin is more associated with the formation of functional channels in cell membranes rather than with a lytic phenomenon.     

Study of protein aggregation using two-dimensional correlation infrared spectroscopy and spectral simulations

Two-dimensional (2D) correlation spectroscopy establishes correlations between intensity variations in a series of spectra obtained by the application of an external perturbation. However, spectral effects (wavenumber shift or bandwidth change) are known to generate apparent asynchronisms in 2D maps. Surprisingly, spectral effects are often neglected in the literature when interpreting experimental maps, which can lead to erroneous conclusions. In an attempt to evaluate the contribution of these effects and that of true asynchronisms on 2D maps, the heat-induced aggregation of glutamyl-tRNA synthetase (GluRS) was studied as a typical example of the application of Fourier transform infrared (FTIR) spectroscopy in the amide I region. The data were compared with those obtained from a mutant protein that differs by one amino acid. To determine whether the aggregation mechanisms are identical for both proteins, the experimental 2D maps were compared to simulations based on curve fitting of the initial and final spectra of the series, which allows change in position and bandwidth of the components to be taken into account. Intermediate spectra were generated using a convenient function that mimics the spectral evolution. The speed and the delay of each component were controlled. Apart from the appearance of turns that occur for the mutant and not for GluRS, the aggregation mechanisms of both proteins seems to be essentially identical. In particular, the loss of alpha-helices seems to be concomitant with the formation of intermolecular beta-sheets, whereas the loss of intramolecular beta-sheets is delayed. Since the experimental maps are satisfactorily simulated when almost all the components are in phase, it appears that many of the asynchronous features are mainly due to spectral effects. Thus, one has to be aware that true asynchronisms are not necessarily at the origin of peaks observed in asynchronous maps.     

Study on temperature-dependent changes in hydrogen bonds in cellulose Ibeta by infrared spectroscopy with perturbation-correlation moving-window two-dimensional correlation spectroscopy

Infrared (IR) spectra were measured for cellulose Ibeta prepared from the mantle of Halocynthia roretzi over a temperature range of 30-260 degrees C to explore the temperature-dependent changes in hydrogen bonds (H-bonds) in the crystal. Structural changes at the phase transition temperature of 220 degrees C are elucidated at the functional group level by perturbation-correlation moving-window two-dimensional (PCMW2D) correlation spectroscopy. The PCMW2D correlation spectra show that the intensities of bands arising from O3-H3...O5 and O2-H2...O6 intrachain H-bonds dramatically decrease at 220 degrees C, whereas the intensity changes of bands due to interchain H-bonds are not observed adequately. These results suggest that the phase transition is induced by the dissociation of the O3-H3...O5 and O2-H2...O6 intrachain H-bonds. However, the interchain H-bonds are not so much responsible for the transition directly.     

Structural study of the C2 domains of the classical PKC isoenzymes using infrared spectroscopy and two-dimensional infrared correlation spectroscopy

The secondary structure of the C2 domains of the classical PKC isoenzymes, alpha, betaII, and gamma, has been studied using infrared spectroscopy. Ca(2+) and phospholipids were used as protein ligands to study their differential effects on the isoenzymes and their influence on thermal protein denaturation. Whereas the structures of the three isoenzymes were similar in the absence of Ca(2+) and phospholipids at 25 degrees C, some differences were found upon heating in their presence, the C2 domain of the gamma-isoenzyme being better preserved from thermal denaturation than the domain from the alpha-isoenzyme and this, in turn, being better than that from the beta-isoenzyme. A two-dimensional correlation study of the denaturation of the three domains also showed differences between them. Synchronous 2D-IR correlation showed changes (increased aggregation of denaturated protein) occurring at 1616-19 cm(-1), and this was found in the three isoenzymes. On the other hand, the asynchronous 2D-IR correlation study of the domains in the absence of Ca(2+) showed that, in all cases, the aggregation of denaturated protein increased after changes in other structural components, an increase perhaps related with the hard-core role of the beta-sandwich in these proteins. The differences observed between the three C2 domains may be related with their physiological specialization and occurrence in different cell compartments and in different cells.     

Sequential events in ribonuclease A thermal unfolding characterized by two-dimensional infrared correlation spectroscopy

The conformational changes in the thermal denaturation of bovine pancreatic ribonuclease A was followed with infrared spectra and analyzed by second derivative and two-dimensional correlation techniques. By analyzing the sequential events in each transition stage, the results were consistent with a step-wise thermal denaturation mechanism in which the structural adjustment of the N-terminal and the opening of the central structure of the protein come before the main unfolding process. Non-native turns were found to form along with the unfolding of the native structures. The central region that is composed of some beta-sheet and alpha-helical structures was found to be the most stable part that might form the residual structure at high temperatures.     

Molecular conformation of gonadoliberin using two-dimensional NMR spectroscopy

Complete resonance assignments of the proton NMR spectrum of gonadoliberin (in its native amide and free acid forms) have been obtained using two-dimensional nuclear magnetic resonance spectroscopy under three different environmental conditions, namely, dimethyl sulphoxide solution, aqueous solution and lipid-bound form in model membranes. The proton chemical shifts in the three cases have been compared to derive information about inherent conformational characteristics of the molecule. It has been inferred that the molecule possesses no short-range or long-range order under any of the three solvent conditions. However, there is a nonspecific increase in the linewidths when gonadoliberin is bound to model membranes, indicating a reduced internal motion in the molecule due to lipid-peptide interactions.     

The conformation of porcine-brain natriuretic peptide by two-dimensional NMR spectroscopy

Two-dimensional (2D) 1H-NMR spectra of porcine-brain natriuretic peptide (pBNP) have been recorded at 300 MHz and 400 MHz. Peak assignments have been made and the combined information from chemical shifts, coupling constants, temperature coefficients and NOEs have been used to determine the conformational properties of pBNP in (C2H3)2SO. Overall the peptide appears to be flexible, with the possibility of some beta-type structure near the C terminus. Some of the assignments and deduced structural features in the current study differ from those in a recent report by Inooka et al. [Inooka, H., Kikuchi, T., Endo, S., Ishibashi, Y., Wakimasu, M. and Mizuta, E. (1990) Eur. J. Biochem. 193, 127-134] which may indicate the sensitivity of the structure of this peptide to differences in solution conditions.     

Temperature-dependent changes in hydrogen bonds in cellulose Ialpha studied by infrared spectroscopy in combination with perturbation-correlation moving-window two-dimensional correlation spectroscopy: comparison with cellulose Ibeta

Our recent IR study demonstrated that hydrogen-bond structure in cellulose Ibeta drastically changes around 220 degrees C (Watanabe et al. Biomacromolecules 2006, 7, 3164). In the present study, temperature-dependent IR spectra of cellulose Ialpha from 30 to 260 degrees C were analyzed by use of perturbation-correlation moving-window two-dimensional correlation spectroscopy. It was observed that as in the case of cellulose Ibeta abrupt changes in the hydrogen-bond structure occur around 220 degrees C in cellulose Ialpha. It was also revealed that although weakly hydrogen-bonded OH groups in Ibeta are stable below 230 degrees C thermal oxidation of those in Ialpha is accelerated around 220 degrees C. In this way, the present study has clarified a difference between the thermal behavior of Ialpha and that of Ibeta at the functional group level. Our result suggests that the drastic change in the hydrogen-bond structure around 220 degrees C makes cellulose Ialpha much more unstable than Ibeta.     

Gamma irradiation and cellular damage in Kocuria rosea: investigation by one- and two-dimensional infrared spectroscopy

Fourier transform infrared (FT-IR) spectroscopy was used to investigate the radiation-induced effects on Kocuria rosea. Bacterial suspensions at the stationary phase were exposed to increasing doses of gamma radiation. In the region 1350-840cm(-1), assigned to phosphodiester backbone, nucleic acids, and sugar rings, the radical damaging effects were dose-dependent, with the first threshold at 2.75kGy and the second at 13.75kGy inducing more striking spectral variations. Postirradiation reincubation did not significantly affect the biomolecular response, except in the spectral range 1100-1000cm(-1). These observations suggest the occurrence of new phylogenetic characteristics for K. rosea following irradiation. Moreover, two-dimensional analysis was used to highlight correlated evolutions of molecular species as radical aggression increased. The results point to an evolutionary scheme during the time course of irradiation. Thus, one- and two-dimensional IR analyses are convenient means of investigating the metabolic events following oxidative stress generated by either chemical or physical agents.     

Monitoring a complex medium fermentation with sample-sample two-dimensional FT-NIR correlation spectroscopy

The possibility of monitoring state transitions during the time course of a fermentation through the analysis of the on-line collected near-infrared (NIR) spectra with sample-sample two-dimensional correlation spectroscopy (SS-2DCoS) was investigated. SS-2DCoS has proved to be useful for extracting process information directly from the spectra collected on-line. The complexity of the system studied prevented the extraction of concentration profiles, but nevertheless, the application of SS-2DCoS enables the identification of fermentation state transitions due to metabolic and morphological changes of the microorganism.     

Multidimensional spectroscopic data correlation in the conformation transition of biological macromolecules

A computerized spectrophotometer system which is capable of simultaneously obtaining three spectral dimensions, that is, the absorbance, the circular dichroism and the fluorescence intensity, from one sample solution has been developed for the purpose of attaining a higher resolving power in the study of conformational transitions of biological macromolecules. Measurement conditions, such as the wavelength, the temperature, pH or the concentration of reagents in the sample solution, can be scanned according to a sequence that is set just prior to the measurement. A computer-driven micro-injector and a pH electrode directly immersed in the sample solution make it possible to obtain a titration curve in parallel to the optical measurement. All the data taken are stored on a magnetic disk for later retrieval. They can be processed and displayed in any required form. The helix-coil transitions of a polynucleotide caused by temperature and those of a polypeptide caused by pH, and the denaturation of proteins caused by guanidine hydrochloride, were studied by this measuring system. The continuous plotting of transition profiles and the correlation diagrams among different spectral dimensions has proved to be a good way of demonstrating the existence of different modes of transition.     

Study of bradykinin conformation in a dimethylsulfoxide solution by two-dimensional 1H-NMR spectroscopy

The role of charged groups of the nonapeptide bradykinin in stabilization of its spatial structure in dimethyl sulfoxide solution was investigated. The signal assignment in the 1H-NMR spectra was achieved by means of two dimensional correlated spectroscopy (COSY) and nuclear Overhauser enhancement spectroscopy (NOESY). The changes in the NH and C alpha H proton chemical shifts of the Arg1 and Arg9 residues, variations both in temperature coefficients of chemical shifts of NH-resonances and coupling constants, as well as the appearance of additional NOE cross-peaks in NOESY spectra for d alpha N and d beta N 1H-1H distances were revealed by comparing the NMR spectra for two states--with the protonated C-terminal carboxyl group and deprotonated one. The experimental results are in agreement with the assumption that the conformation of the peptide in (CD3)2SO is stabilized by electrostatic interaction between the oppositely charged N- and C-terminal groups. The conformation with deprotonated alpha-carboxyl group is characterized by two beta-turns in the sequences Pro2-Pro-Gly-Phe5 and Ser6-Pro-Phe-Arg9.     

Optical fingerprinting of peptides using two-dimensional infrared spectroscopy: proof of principle

We employ a particular form of two-dimensional infrared four-wave mixing (2DIR FWM) as a vibrational spectroscopic tool to quantify the amino acid content of a number of peptides. Vibrational features corresponding to ring modes of the aromatic groups of phenylalanine (Phe) and tyrosine (Tyr), as well as a methylene mode that is used as an internal reference, are identified. We show that the ratios of the integrated intensities, and the amplitudes, of the aromatic peaks of Phe and Tyr relative to the methylene integrated intensity, and amplitude, are proportional to the actual ratio of Phe and Tyr to CH₂ in the samples within a precision of ±12.5%. This precision is shown to be sufficient to use this form of 2DIR spectroscopy as a possible proteins fingerprinting tool.     

Solution conformation of the antitumor antibiotic chromomycin A3 determined by two-dimensional NMR spectroscopy

A conformational analysis and a complete assignment of the nonexchangeable proton resonances of chromomycin A3, dechromose-A chromomycin A3, and deacetylchromose-B chromomycin A3 were carried out in organic solvents. The resulting conformation in methanol has the three side chains of chromomycin A3 fully extended, away from one another and from the aglycon. In dichloromethane on the other hand, the drug was shown to adopt a highly compact conformation in which most of the 26 oxygen atoms in the molecule point out toward the solvent. The two carbohydrate side chains extend parallel to each other on the same side of the aglycon. Two intramolecular nuclear Overhauser enhancement contacts have been observed between different sugar units on these side chains, indicating close proximity for these moieties. In addition, the aliphatic side chain is folded toward the aglycon, parallel to the two oligosaccharide side chains. The overall conformation has a wedge-like shape with the two phenoxy groups exposed at the pointed edge. The presence of some exchange cross-peaks in the NOESY spectra suggests the presence of intramolecular hydrogen bonds that probably help to maintain the compact conformation. The derivatives of chromomycin A3 have qualitatively similar conformations, though their respective conformations are not as compact as the parent drug. The significance of these results is discussed in terms of a model of chromomycin A3 binding to DNA in the major groove.     

An investigation into the effect of potassium ions on the folding of silk fibroin studied by generalized two-dimensional NMR-NMR correlation and Raman spectroscopy

We used generalized two-dimensional NMR-NMR correlation to examine the effect of potassium ions on the conformation transition in silk fibroin to investigate the possibility that the fairly high K+ ion content found in the distal end of silk-secreting ducts in the silkworms could have a bearing on natural formation of the silk fiber. This has enabled us to propose a detailed mechanism for the transition process. Our evidence indicates that increasing the [K+] from 0 to 3.7 mg.g(-1) in the silk fibroin, as is thought to occur as the silk fibroin moves through the secretory pathway to the spigot, produces a sequence of secondary structural changes: helix and/or random coil-->helix-like-->beta-sheet-like-->beta-sheet. The sequence is the same as that produced in silk fibroin films by decreasing the pH of fibroin from 6.8 to 4.8. In addition, we used Raman spectroscopy to study the effect of K+ ions on the Fermi doublet resonance of the tyrosyl phenolic ring at 850 and 830 cm(-1). The intensity ratio I(850)/I(830) at these wave numbers indicated that the hydrogen bonding formed by the tyrosyl phenolic-OH becomes more stable with an increase in the K+ ion concentration as above. Our investigation on the effect of K+ ions on fibroin may help provide a theoretical basis for understanding the natural silk-spinning process and the conditions required for biomimetic spinning. It may also have relevance to the aggregation of other beta-sheet proteins, including prion proteins, neurofibrillary proteins and amyloid plaques.     

Novel solution conformation of DNA observed in d(GAATTCGAATTC) by two-dimensional NMR spectroscopy

Resonance assignments of nonexchangeable base and sugar protons of the self-complementary dodecanucleotide d(GAATTCGAATTC) have been obtained by using the two-dimensional Fourier transform NMR methods correlated spectroscopy and nuclear Overhauser effect spectroscopy. Conformational details about the sugar pucker, the glycosidic dihedral angle, and the overall secondary structure of the molecule have been derived from the relative intensities of cross peaks in the two-dimensional NMR spectra in aqueous solution. It is observed that d(GAATTCGAATTC) assumes a novel double-helical structure. The solution conformations of the two complementary strands are identical, unlike those observed in a related sequence in the solid state. Most of the five-membered sugar rings adopt an unusual O1'-endo geometry. All the glycosidic dihedral angles are in the anti domain. The AATT segments A2-T5 and A8-T11 show better stacking compared to the rest of the molecule. These features fit into a right-handed DNA model for the above two segments, with the sugar geometries different from the conventional ones. There are important structural variations in the central TCG portion, which is known to show preferences for DNase I activity, and between G1-A2 and G7-A8, which are cleavage points in the EcoRI recognition sequence. The sugar puckers for G1 and G7 are significantly different from the rest of the molecule. Further, in the three segments mentioned above, the sugar phosphate geometry is such that the distances between protons on adjacent nucleotides are much larger than those expected for a right-handed DNA. We suggest that such crevices in the DNA structure may act as "hot points" in initiation of protein recognition.