Structural analysis of DNA-chlorophyll complexes by Fourier transform infrared difference spectroscopy

Porphyrins and metalloporphyrins are strong DNA binders. Some of these compounds have been used for radiation sensitization therapy of cancer and are targeted to interact with cellular DNA. This study was designed to examine the interaction of calf thymus DNA with chlorophyll a (CHL) in aqueous solution at physiological pH with CHL/DNA(phosphate) ratios (r) of 1/160, 1/80, 1/40, 1/20, 1/10, and 1/5. Fourier transform infrared (FTIR) difference spectroscopy was used to characterize the nature of DNA-pigment interactions and to establish correlations between spectral changes and the CHL binding mode, binding constant, sequence selectivity, DNA secondary structure, and structural variations of DNA-CHL complexes in aqueous solution. Spectroscopic results showed that CHL is an external DNA binder with no affinity for DNA intercalation. At low pigment concentration (r = 1/160, 1/80, and 1/40), there are two major binding sites for CHL on DNA duplex: 1) Mg-PO2 and 2) Mg-N7 (guanine) with an overall binding constant of K = 1.13 x 10(4) M-1. The pigment distributions are 60% with the backbone PO2 group and 20% with the G-C base pairs. The chlorophyll interaction is associated with a major reduction of B-DNA structure in favor of A-DNA. At high chlorophyll content (r = 1/10), helix opening occurs, with major spectral alterations of the G-C and A-T bases. At high chlorophyll concentration (1/5), pigment aggregation is observed, which does not favor CHL-DNA complexation.     

Carrageenophyte identification by second-derivative Fourier transform infrared spectroscopy

The second-derivative mode of the Fourier transform I.R. spectra of dried algal material has been applied to distinguish the carrageenans-producingStenogramme interrupta from the isomorphous speciesRhodymenia howeana. Spectra of the tetrasporophyteS. interrupta showed bands assigned to a -carrageenan type polysaccharide, while the gametophytic and cystocarpic plants showed the characteristic absorptions of -and -carrageenans. Results were confirmed by hot water extraction of samples of the three nuclear phases ofS. interrupta and characterization of the extracts by chemical analysis.Author for correspondence     

Fourier transform infrared (FTIR) spectroscopy

Fourier transform infrared (FTIR) spectroscopy probes the vibrational properties of amino acids and cofactors, which are sensitive to minute structural changes. The lack of specificity of this technique, on the one hand, permits us to probe directly the vibrational properties of almost all the cofactors, amino acid side chains, and of water molecules. On the other hand, we can use reaction-induced FTIR difference spectroscopy to select vibrations corresponding to single chemical groups involved in a specific reaction. Various strategies are used to identify the IR signatures of each residue of interest in the resulting reaction-induced FTIR difference spectra. (Specific) Isotope labeling, site-directed mutagenesis, hydrogen/deuterium exchange are often used to identify the chemical groups. Studies on model compounds and the increasing use of theoretical chemistry for normal modes calculations allow us to interpret the IR frequencies in terms of specific structural characteristics of the chemical group or molecule of interest. This review presents basics of FTIR spectroscopy technique and provides specific important structural and functional information obtained from the analysis of the data from the photosystems, using this method.     

Analysis of human tear fluid by Fourier transform infrared spectroscopy

The purpose of this research is to find some useful spectroscopic factors in human tear fluid contents to monitor diurnal changes of the physicochemical ocular conditions noninvasively. All tear fluid samples were collected with glass microcapillary tubes from both eyes of three donors and analyzed by Fourier transform infrared spectroscopy with attenuated total reflectance (FTIR-ATR). We measured the peak intensities at 2852, 1735, 1546, and 1242 cm(-1), and the peak intensity ratios among those peaks in the second derivative spectra. We found significant diurnal and individual variations in those peak intensities for tear fluid obtained from right and left eyes. Among these variations, we observed significant changes in tear samples between right and left eyes. In this case the peak intensity ratio between 1242 (phosphate ester) and 2852 cm(-1) (fatty acid methylene) of right eye tear fluid was increased in the afternoon (1600 to 1900 h), while that of left eye tear fluid did not change significantly. In the ratio between 1242 (phosphate ester) and 1546 cm(-1) (amide II), the difference was not observed between both eyes. We conclude that the difference in diurnal variations of biochemical constituents between right and left eye tear fluids could be monitored noninvasively and nondestructively by FTIR technique and this method could be useful in the future for tear diagnoses.     

Rapid identification of coagulase-negative staphylococci by Fourier transform infrared spectroscopy

Coagulase-negative staphylococci (CNS), frequently associated with both community-acquired and nosocomial bloodstream infections, must be distinguished from Staphylococcus aureus for clinical purposes. Conventional methods are too laborious and time-consuming and often lack sensitivity to CNS. Fourier transform infrared (FTIR) spectroscopy combined with the use of a universal growth medium (Que-Bact Universal Medium No. 2) and chemometrics was evaluated for its potential as a rapid and simple clinical tool for making this distinction. FTIR spectra of 11 methicillin-sensitive and 11 methicillin-resistant CNS isolates as well as 25 methicillin-sensitive, 47 methicillin-resistant, 34 borderline oxacillin-resistant and 35 glycopeptide intermediate S. aureus isolates were obtained from dried films of stationary-phase cells grown on the universal medium. Principal component analysis (PCA), self-organizing maps, and the K-nearest neighbor algorithm were employed to cluster the different phenotypes based on similarity of their FTIR spectra. PCA of the first-derivative normalized spectral data from a single narrow region (2888-2868 cm(-1)) yielded complete differentiation of CNS from both methicillin-sensitive and methicillin-resistant S. aureus. The rate of correct classification was somewhat reduced, from 100% to 90%, after inclusion of borderline oxacillin-resistant and glycopeptide intermediate S. aureus strains in the data set. Differentiation based on the data in broader spectral regions was much less reliable. The results of this study indicate that with proper spectral region selection, FTIR spectroscopy and cluster analysis may provide a simple and accurate means of CNS species identification.     

Taxonomic discrimination of flowering plants by multivariate analysis of Fourier transform infrared spectroscopy data

Fourier transform infrared spectroscopy (FTIR) provides biochemical profiles containing overlapping signals from a majority of the compounds that are present when whole cells are analyzed. Leaf samples of seven higher plant species and varieties were subjected to FTIR to determine whether plants can be discriminated phylogenetically on the basis of biochemical profiles. A hierarchical dendrogram based on principal component analysis (PCA) of FTIR data showed relationships between plants that were in agreement with known plant taxonomy. Genetic programming (GP) analysis determined the top three to five biomarkers from FTIR data that discriminated plants at each hierarchical level of the dendrogram. Most biomarkers determined by GP analysis at each hierarchical level were specific to the carbohydrate fingerprint region (1,200–800 cm^–1) of the FTIR spectrum. Our results indicate that differences in cell-wall composition and structure can provide the basis for chemotaxonomy of flowering plants.Abbreviations FTIR Fourier transform infrared spectroscopy - GP Genetic programming - PCA Principal component analysis - PyMS Pyrolysis mass spectrometry     

Characterization of an RNA bulge structure by Fourier transform infrared spectroscopy

There may be several advantages associated with an antisense oligonucleotide that induces a bulged structure into its RNA target molecule. Many structures of RNA bulges are elucidated from single-stranded RNA models. However, a two-component system is the minimum requirement for a realistic antisense model. We have used Fourier transform infrared spectroscopy to investigate a single-stranded RNA oligonucleotide with known NMR solution structure, constructed to model a five nucleotide bulge, and its two-component oligonucleotide counterpart. The infrared spectra show A-helical base-paired stems and non-base-paired loops in both systems. The nucleosides are mainly in an anti-conformation. Both N-type and S-type of sugar puckers can be inferred from the infrared region sensitive to sugar conformations. The S-type of sugar pucker is likely to be associated with the nucleotides in the bulge. The FTIR results display an overall structural similarity between the two model systems.     

Conformational changes of bacteriorhodopsin detected by Fourier transform infrared difference spectroscopy

We report the first Fourier transform infrared difference spectra of purple membrane. Evidence is presented that alterations in the vibrations of both the retinylidene chromophore and the protein groups of bacteriorhodopsin associated with photocycling can be detected. This method provides a new tool for probing the conformational changes occurring in bacteriorhodopsin during the proton pump cycle.     

Molecular surface characterization of oral streptococci by Fourier transform infrared spectroscopy

In order to characterize the molecular composition of oral streptococci, infrared transmission spectroscopy on freeze-dried cells dissolved in KBr was used. All infrared spectra show similar absorption bands for the strains studied with the most important absorption bands located at 2930 cm-1 (CH), 1653 cm-1 (AmI), 1541 cm-1 (AmII) and two bands at 1236 cm-1 and 1082 cm-1, which were assigned to phosphate and sugar groups. However, calculation of absorption band ratios normalized with respect to the integrated intensity of the CH stretching region around 2930 cm-1, show significant differences between the strains. Both Streptococcus mitis strains possess high AmI/CH and AmII/CH absorption band ratios compared to the other strains. Streptococcus salivarius HBC12, a mutant strain devoid of all proteinaceous surface appendages, shows significantly lower AmI/CH and AmII/CH band ratios with respect to its parent strain S. salivarius HB. Two positive relationships could be established both between the AmII/CH absorption band ratio and the N/C elemental surface concentration ratio of the strains previously, determined from X-ray photoelectron spectroscopy (XPS) and also between AmI/CH and the fraction of carbon atoms at the surface involved in amide bonds, determined by XPS as well. From this comparison, it is concluded that transmission infrared spectroscopy can be employed as a technique to study the molecular surface composition of freeze-dried microorganisms.     

Low-temperature Fourier transform infrared spectroscopy of photoactive yellow protein

The photocycle intermediates of photoactive yellow protein (PYP) were characterized by low-temperature Fourier transform infrared spectroscopy. The difference FTIR spectra of PYP(B), PYP(H), PYP(L), and PYP(M) minus PYP were measured under the irradiation condition determined by UV-visible spectroscopy. Although the chromophore bands of PYP(B) were weak, intense sharp bands complementary to the 1163-cm(-1) band of PYP, which show the chromophore is deprotonated, were observed at 1168-1169 cm(-1) for PYP(H) and PYP(L), indicating that the proton at Glu46 is not transferred before formation of PYP(M). Free trans-p-coumaric acid had a 1294-cm(-1) band, which was shifted to 1288 cm(-1) in the cis form. All the difference FTIR spectra obtained had the pair of bands corresponding to them, indicating that all the intermediates have the chromophore in the cis configuration. The characteristic vibrational modes at 1020-960 cm(-1) distinguished the intermediates. Because these modes were shifted by deuterium-labeling at the ethylene bond of the chromophore while labeling at the phenol part had no effect, they were attributed to the ethylene bond region. Hence, structural differences among the intermediates are present in this region. Bands at about 1730 cm(-1), which show that Glu46 is protonated, were observed for all intermediates except for PYP(M). Because the frequency of this mode was constant in PYP(B), PYP(H), and PYP(L), the environment of Glu46 is conserved in these intermediates. The photocycle of PYP would therefore proceed by changing the structure of the twisted ethylene bond of the chromophore.     

Comparative analysis of the amino- and carboxy-terminal domains of calmodulin by Fourier transform infrared spectroscopy

Fourier transform infrared spectra were obtained for mammalian calmodulin and two of its fragments produced by limited proteolysis with trypsin TR₁C (1–77) and TR₂C (78–148). Experiments were done in H₂O, D₂O and D₂O/trifluoroethanol (TFE) mixtures. Information about secondary structure was obtained from analysis of the amide I and II bands; while characteristic absorbances for tyrosine, phenylalanine and carboxylate groups were analyzed for changes in tertiary structure. Our data indicate that the secondary and tertiary structure is preserved in the two half molecules of CaM, both in the apo- and Ca²⁺-saturated state. Addition of the structure-inducing solvent TFE causes marked changes only in the apo-TR₁C domain. The maximum wavenumber for the amide I band of the two domains of CaM in D20 was markedly different (1642 cm^–1 for TR₁C versus 1646/1648 cm^–1 for Ca ²⁺ and apo-TR₂C). This renders the amide I band for the intact protein very broad in comparison to that in other proteins and is indicative of a distribution of -helices with slightly different hydrogen bonding patterns.     

Protein structure by Fourier transform infrared spectroscopy: second derivative spectra

Second derivative Fourier transform infrared spectra of the proteins ribonuclease A, hemoglobin, and beta-lactoglobulin A (native and denatured) have been obtained in deuterium oxide solution from 1350 to 1800 cm-1. The relationship of the original spectra to their second derivatives is briefly discussed. In the second derivative spectra, clearly resolved peaks are observed which can be associated with the alpha-helix, beta-strands, and turns. No protein spectra with such resolution have heretofore been reported. Tentative assignments are proposed, and the observed peaks are related to the secondary structure of the proteins studied. The data appear to present the first direct spectroscopic evidence of turns in a native protein.     

Enzymatic transesterification monitored by an easy-to-use Fourier transform infrared spectroscopy method

Transesterification of triglycerides with short chain alcohols is the key reaction in biodiesel production, in addition to other applications in chemical synthesis. However, it is crucial to optimize reaction conditions to make enzymatic transesterification a cost-effective and competitive process. In this work, a new, easy Fourier transform infrared (FTIR) spectroscopic approach for monitoring the transesterification reaction is reported and compared with a gas-chromatographic method. The concentration of the total methyl esters in the reaction mixture is determined from the peak intensity at ∼1435 cm^–1 in the second derivatives of the FTIR absorption spectra using a linear regression calibration. Interestingly, we found that the use of second derivatives allows an accurate determination of the methyl esters without the interference of free fatty acids. Moreover, information on substrate hydrolysis can be obtained within the same measurement by the infrared absorption at ∼1709 cm^–1. We applied this approach to monitor methanolysis and hydrolysis reactions catalyzed by different commercial lipases, which displayed different sensitivities to methanol inhibition. Therefore, the FTIR approach reported in this work represents a rapid, inexpensive, and accurate method to monitor enzymatic transesterification, requiring very limited sample preparation and a simple statistical analysis of the spectroscopic data.     

Fibrinogen-catecholamine interaction as observed by NMR and Fourier transform infrared spectroscopy

In this work, the interactions between the main catecholamines-epinephrine and norepinephrine-and fibrinogen were investigated by NMR and Fourier transform infrared spectroscopies. The two hormones were found to interact with fibrinogen and to affect the protein secondary structure to a different extent. In particular, the protein selectively binds epinephrine at both the basal and stress concentrations, while it shows a weak nonspecific interaction with norepinephrine. The interaction with the stress level of epinephrine leads to drastic protein conformational changes, whereas norepinephrine does not affect fibrinogen secondary structure, even at stress concentration.     

Water structural changes in the bacteriorhodopsin photocycle: analysis by Fourier transform infrared spectroscopy.

The Fourier transform infrared difference spectra between light-adapted bacteriorhodopsin (BR) and its photointermediates, L and M, were analyzed for the 3750-3450-cm-1 region. The O-H stretching vibrational bands were identified from spectra upon substitution with 2H2O. Among them, the 3642-cm-1 band of BR was assigned to water by substitution with H2(18)O. By a comparison with the published infrared spectra of the water in model systems [Mohr, S.C., Wilk, W.D., & Barrow, G.M. (1965) J. Am. Chem. Soc. 87, 3048-3052], it is shown that the O-H bonds of the water in BR interact very weakly. Upon formation of L, the interaction becomes stronger. The O-H bonds of the protein side chain undergo similar changes. On the other hand, M formation further weakens the interaction of the same water molecules in BR. The appearance of a sharp band at 3486 cm-1, which was assigned tentatively to the N-H stretching vibration of the peptide bond, is unique to L. The results suggest that the water molecules are involved in the perturbation of Asp-96 in the L intermediate and that they are exerted from the protonated Schiff base which changes position upon the light-induced reaction.     

Fourier transform infrared spectroscopy in high-pressure studies on proteins

Several aspects of the application of Fourier transform infrared spectroscopy (FTIR) in high-pressure studies on proteins are reviewed. Basic methodological considerations regarding spectral band assignments, quantitative analysis, and choice of pressure calibrants are also placed within the scope of this paper. This work attempts to evaluate recent developments in the field of high-pressure FTIR of proteins and its prospects for future. Particular attention is paid to the phenomenon of protein aggregation.     

Rhodopsin-lumirhodopsin phototransition of bovine rhodopsin investigated by Fourier transform infrared difference spectroscopy

The rhodopsin-lumirhodopsin transition has been investigated by Fourier transform infrared difference spectroscopy using isotope-labeled retinals. In the transition, two protonated carboxyl groups are involved. Another carbonyl band, located at 1725 cm-1 in rhodopsin, is shifted to 1731.5 cm-1 in lumirhodopsin. This line is tentatively assigned to a carbonyl stretching vibration of a peptide bond adjacent to the nitrogen of a proline residue. The C=N stretching vibration of rhodopsin could unequivocally be assigned to a band at 1659 cm-1. In contrast to rhodopsin and bathorhodopsin, the C=N stretching vibration of lumirhodopsin is at a low position, i.e., at 1635 cm-1, and exhibits only a downshift of 4 cm-1 upon deuteriation of the nitrogen. The C15-H rocking vibration of rhodopsin is assigned to the unusual high position of 1456 cm-1 and shifts into the normal region upon formation of lumirhodopsin. From these results, it is concluded that, whereas the environment of the Schiff base in rhodopsin, bathorhodopsin, and isorhodopsin is approximately the same, large changes occur with the formation of lumirhodopsin. From the assignment of the C10-C11 stretching vibration in bathorhodopsin and lumirhodopsin, a 10-s-cis geometry of lumirhodopsin can be excluded.     

Solvent denaturation of proteins as observed by resolution-enhanced Fourier transform infrared spectroscopy

Fourier self-deconvolution of Fourier transform infrared (FTIR) spectra and second derivative FTIR spectroscopy were applied to study solvent-induced conformational changes in globular proteins. For beta-lactoglobulin a total of three different denatured forms were identified in alkaline solution and in aqueous methanol-d1 and isopropanol-d1. In isopropanol-d1 solution a new conformation was identified which appears to resemble, but is not identical with, the beta-structure of native proteins. This conformation is characterized by absorption bands around 1615-1618 and 1684-1688 cm-1, and is also observed for concanavalin A and chymotrypsinogen A in aqueous isopropanol-d1 solution.     

Epidemiological typing of methicillin-resistant Staphylococcus aureus strains by Fourier transform infrared spectroscopy

A rapid and simple typing system is needed for controlling the spread of epidemic methicillin-resistant Staphylococcus aureus (MRSA), currently one of the most widespread multi-resistant nosocomial pathogens in Canadian hospitals. Fourier transform infrared (FTIR) spectroscopy was used to subtype 85 isolates representing five strains of epidemic Canadian MRSA (CMRSA). Spectral fingerprints of whole cells grown on Que-Bact(R) Universal Medium No. 2 were transformed to first derivative peak-height normalized files and examined visually and by singular-value decomposition (SVD). Distinguishing spectral regions were processed by principal component analysis (PCA), self-organizing map and K-nearest neighbor supervised cluster analysis. Among the visually identified regions, 1070-1050 and 1155-1137 cm(-1) were found suitable for discrimination of CMRSA-4 and CMRSA-2 respectively, while CMRSA-1, CMRSA-3, and CMRSA-5 each exhibited distinctive spectral profiles in the 1123-1094 cm(-1) region. The combination, 1123-1094, 1174-1154 and 2904-2864 cm(-1) separated the five CMRSA with 84.6% correct classification by PCA. Five clusters were also obtained using the SVD-selected regions 1096-1066, 1118-1090 and 2914-2880 cm(-1), with 87.8% correct classification based on visual examination of the PCA scores plot and 97% based on supervised cluster analysis. These results demonstrate that FTIR spectroscopy has considerable potential as a rapid (1-hour) and simple method for MRSA strain typing and monitoring in clinical settings.