Orientation of the bacteriorhodopsin chromophore probed by polarized Fourier transform infrared difference spectroscopy

Polarized, low-temperature Fourier transform infrared (FTIR) difference spectroscopy has been used to investigate the structure of bacteriorhodopsin (bR) as it undergoes phototransitions from the light-adapted state, bR570, to the K630 and M412 intermediates. The orientations of specific retinal chromophore and protein groups relative to the membrane plane were calculated from the linear dichroism of the infrared bands, which correspond to the vibrational modes of those groups. The linear dichroism of the chromophore C=C and C-C stretching modes indicates that the long axis of the polyene chain is oriented at 20-25 degrees from the membrane plane at 250 K and that it orients more in-plane when the temperature is reduced to 81 K. The polyene plane is found to be approximately perpendicular to the membrane plane from the linear dichroism calculations of the HOOP (hydrogen out-of-plane) wags. The orientation of the transition dipole moments of chromophore vibrations in the K630 and M412 intermediates has been probed, and the dipole moment direction of the C=O bond of an aspartic acid that is protonated in the bR570----M412 transition has been measured.     

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     

Monitoring of phospholipid monolayer hydrolysis by phospholipase A2 by use of polarization-modulated Fourier transform infrared spectroscopy

Polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) was used to follow the hydrolysis of phospholipid monolayers at the air-water interface by phospholipase A2 (PLA2). The decrease in the intensity of the nuC=O ester band of dipalmitoylphosphatidylcholine at 1733 cm(-1) and the appearance of two new infrared bands in the 1530-1580 cm(-1) region allowed to monitor phospholipid hydrolysis by PLA2. Indeed, the decrease in the intensity of the band at 1733 cm(-1) was attributed to the enzymatic hydrolysis of the acyl ester linkage of the sn-2 fatty acid on the glycerol backbone whereas the doublet appearing at 1537 and 1575 cm(-1) was attributed to the nu(a) COO- vibration of the newly formed calcium-palmitate. The presence of this band as a doublet indicates the formation of a crystalline-like calcium-palmitate monolayer. This observation supports our previously postulated mechanism for the formation of PLA2 domains at the air-water interface. Definitive assignment of the infrared bands has been possible by measuring PM-IRRAS spectra of the individual hydrolysis products (palmitic acid and lysopalmitoylphosphatidylcholine) as well as of 1-caproyl-2-palmitoyl-phosphatidylcholine and 1-palmitoyl-2-caproylphosphatidylcholine monolayers before and after hydrolysis by PLA2.     

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.     

Quantitative analysis of cellulose nitrates by Fourier transform infrared spectroscopy

Quantitative express analysis of nitrogen content in cellulose nitrates by Fourier transform infrared spectroscopy has been developed. The slope of the dependence of the ratio of the band intensity (and area) to sample weight in a tablet, on the nitrogen content in a sample was used to find the reduced extinction coefficients for quantitative analysis of nitrogen content in cellulose nitrate samples by IR spectroscopy. The results were compared with the nitrogen content values in the same samples determined by the ferrosulfate 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.     

Application of Fourier transform infrared spectroscopy for monitoring hydrolysis and synthesis reactions catalyzed by a recombinant amidase

This study demonstrates the use of Fourier transform infrared (FTIR) spectroscopy for monitoring both synthesis and hydrolysis reactions catalyzed by a recombinant amidase (EC 3.5.1.4) from Pseudomonas aeruginosa. The kinetics of hydrolysis of acetamide, propionamide, butyramide, acrylamide, benzamide, phenylalaninamide, alaninamide, glycinamide, and leucinamide were determined. This revealed that very short-chain substrates displayed higher amidase activity than did branched side-chain or aromatic substrates. In addition, on reducing the polarity and increasing the substrates' bulkiness, a reduction of the amidase affinity for the substrates took place. Using FTIR spectroscopy it was possible to monitor and quantify the synthesis of several hydroxamic acid derivatives and ester hydrolysis products. These products may occur simultaneously in a reaction catalyzed by the amidase. The substrates used for the study of such reactions were ethyl acetate and glycine ethyl ester. Hydroxylamine was the nucleophile substrate used for the synthesis of acetohydroxamate compounds. Results presented in this article demonstrate the usefulness of FTIR spectroscopy as an important tool for understanding the enzyme structure-activity relationship because it provides a simple and rapid real-time assay for the detection and quantification of amidase hydrolysis and synthesis reactions in situ.     

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.     

Conformation of magainin-2 and related peptides in aqueous solution and membrane environments probed by Fourier transform infrared spectroscopy.

The conformational properties of the magainin family of antimicrobial peptides in aqueous solution and in model membranes have been probed by Fourier transform infrared spectroscopy. The magainins were found to be structureless in aqueous solution at neutral pD, confirming other studies by Raman and circular dichroism spectroscopy. Increasing the pD to 10 induced the formation of predominantly alpha-helical secondary structures, with some beta-sheet. In the presence of negatively charged liposomes (dimyristoylphosphatidylglycerol), the peptides folded into alpha-helical secondary structures with some beta-sheet structure evident. On the other hand, in the presence of zwitterionic phospholipids (dimyristoylphosphatidylcholine), the spectra were identical to those in aqueous solution. For some magainins, the interaction with charged liposomes was modulated by the presence of cholesterol; cholesterol was found to promote the formation of beta-sheet structures, as evidenced by the appearance of amide I bands at 1614 and 1637 cm-1. Differences in structure were observed between the amidated and nonamidated forms of some peptides. From the data, a mechanism of antimicrobial action of the magainin family of peptides is proposed.     

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.     

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.     

The rapid identification of Acinetobacter species using Fourier transform infrared spectroscopy

AIMS: Fourier transform infrared (FT-IR) was used to analyse a selection of Acinetobacter isolates in order to determine if this approach could discriminate readily between the known genomic species of this genus and environmental isolates from activated sludge. METHODS AND RESULTS: FT-IR spectroscopy is a rapid whole-organism fingerprinting method, typically taking only 10 s per sample, and generates 'holistic' biochemical profiles (or 'fingerprints') from biological materials. The cluster analysis produced by FT-IR was compared with previous polyphasic taxonomic studies on these isolates and with 16S-23S rDNA intergenic spacer region (ISR) fingerprinting presented in this paper. FT-IR and 16S-23S rDNA ISR analyses together indicate that some of the Acinetobacter genomic species are particularly heterogeneous and poorly defined, making characterization of the unknown environmental isolates with the genomic species difficult. CONCLUSIONS: Whilst the characterization of the isolates from activated sludge revealed by FT-IR and 16S-23S rDNA ISR were not directly comparable, the dendrogram produced from FT-IR data did correlate well with the outcomes of the other polyphasic taxonomic work. SIGNIFICANCE AND IMPACT OF THE STUDY: We believe it would be advantageous to pursue this approach further and establish a comprehensive database of taxonomically well-defined Acinetobacter species to aid the identification of unknown strains. In this instance, FT-IR may provide the rapid identification method eagerly sought for the routine identification of Acinetobacter isolates from a wide range of environmental sources.     

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.     

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.     

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.     

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.