Nuclear magnetic resonance Fourier transform spectroscopy

The development and current status of Fourier transform spectroscopy is described.Nobel Lecture given on December 9, 1991 by Professor R. Ernst and published in Les Prix Nobel 1991, printed in Sweden by Norstedts Tryckeri, Stockholm, Sweden, 1992, republished here with the permission of the Nobel Foundation, the copyright holder.     

Fourier transform infrared (FTIR) spectroscopy for identifying Lactobacillus species

Abstract Fourier Transform infrared (FTIR) spectroscopy can be used to identify microorganisms. This study describes the influence of culture conditions on FTIR spectra and the discrimination of Lactobacillus species found in breweries. Fifty three Lactobacillus strains were analysed by FTIR spectroscopy and identification at the species level was correct for 94% of the strains, and at the strain level for 91% of the strains.     

Probing protonation/deprotonation of tyrosine residues in cytochrome ba3 oxidase from Thermus thermophilus by time-resolved step-scan Fourier transform infrared spectroscopy

Elucidating the properties of the heme Fe-Cu(B) binuclear center and the dynamics of the protein response in cytochrome c oxidase is crucial to understanding not only the dioxygen activation and bond cleavage by the enzyme but also the events related to the release of the produced water molecules. The time-resolved step-scan FTIR difference spectra show the ν(7a)(CO) of the protonated form of Tyr residues at 1247 cm(-1) and that of the deprotonated form at 1301 cm(-1). By monitoring the intensity changes of the 1247 and 1301 cm(-1) modes as a function of pH, we measured a pK(a) of 7.8 for the observed tyrosine. The FTIR spectral changes associated with the tyrosine do not belong to Tyr-237 but are attributed to the highly conserved in heme-copper oxidases Tyr-136 and/or Tyr-133 residue (Koutsoupakis, K., Stavrakis, S., Pinakoulaki, E., Soulimane, T., and Varotsis, C. (2002) J. Biol. Chem. 277, 32860-32866). The oxygenation of CO by the mixed-valence form of the enzyme revealed the formation of the ～607 nm P (Fe(IV)=O) species in the pH 6-9 range and the return to the oxidized form without the formation of the 580 nm F form. The data indicate that Tyr-237 is not involved in the proton transfer pathway in the oxygenation of CO by the mixed-valence form of the enzyme. The implication of these results with respect to the role of Tyr-136 and Tyr-133 in proton transfer/gating along with heme a(3) ring D propionate-H(2)O-ring A propionate-Asp-372 site to the exit/output proton channel (H(2)O pool) is discussed.     

Fast acquisition of NMR spectra using Fourier transform of non-equispaced data

Rapid acquisition of high-resolution 2D and 3D NMR spectra is essential for studying biological macromolecules. In order to minimize the experimental time, a non-linear sampling scheme is proposed for the indirect dimensions of multidimensional experiments. These data can be processed using the algorithm proposed by Dutt and Rokhlin (Appl. Comp. Harm. Anal. 1995, 2, 85–100) for fast Fourier transforms of non equispaced data. Examples of ¹H−¹⁵N HSQC spectra are shown, where crowded correlation peaks can be resolved using non-linear acquisition. Simulated data have been used to analyze the artefacts produced by the Lagrange interpolation. As compared to non-linear processing methods, this algorithm is simple and highly robust since no parameters need to be adjusted by the user.     

Processing of ND NMR spectra sampled in polar coordinates: a simple Fourier transform instead of a reconstruction

In order to reduce the acquisition time of multidimensional NMR spectra of biological macromolecules, projected spectra (or in other words, spectra sampled in polar coordinates) can be used. Their standard processing involves a regular FFT of the projections followed by a reconstruction, i.e. a non-linear process. In this communication, we show that a 2D discrete Fourier transform can be implemented in polar coordinates to obtain directly a frequency domain spectrum. Aliasing due to local violations of the Nyquist sampling theorem gives rise to base line ridges but the peak line-shapes are not distorted as in most reconstruction methods. The sampling scheme is not linear and the data points in the time domain should thus be weighted accordingly in the polar FT; however, artifacts can be reduced by additional data weighting of the undersampled regions. This processing does not require any parameter tuning and is straightforward to use. The algorithm written for polar sampling can be adapted to any sampling scheme and will permit to investigate better compromises in terms of experimental time and lack of artifacts.     

Use of prior information in simple random sampling

Is is assumed that the population mean, to be estimated, is less than or equal to a known constant. An estimator is suggested which takes into account this information to advantage. It is also suggested that stratification coupled with the modified estimator can be best suited in such situations.     

Second-derivative Fourier transform infrared spectra of seaweed galactans

The Fourier transform infrared spectra of agar, agarose, -, -, and -carrageenan, and ofChondrus canaliculatus, Iridaea ciliata, I. membranacea, I. laminarioides andGracilaria chilensis polysaccharides were recorded in the 4000–400 cm^-1 region. The bands in the second derivative mode are sharper and more bands are resolved than in the normal spectra.Agar, agarose andG. chilensis phycocolloids exhibit diagnostic bands at 790 and 713 cm^-1. -, - and -carrageenans, and native carrageenan-type polysaccharides fromC. canaliculatus andIridaea species exhibit bands at around 1160, 1140, 1100, 1070, 1040, 1008, 610, and 580 cm^-1. Therefore, FT-IR spectroscopy in the second-derivative mode may be applied to differentiate between agar- and carrageenan-types seaweed galactans.     

Application of Fast Fourier Transform in Electroencephalography

Today the Fourier Transform is also used in the analysis of biological time series with periodical parts. For N = 2^M EEG sampling values the Fast Fourier Transform algorithm of COOLEY and TUKEY is applicable. For this a FORTRAN IV program EEG-FFT has been developed for computation of power spectrum. At the data processing equipment EC 1040 the CPU-time for 2¹⁰ = 1024 EEG sampling values is smaller than 1.5 s. The FORTRAN IV program can be applied both in offline and online data processing.     

Fourier transform infrared spectroscopy as a tool to characterize molecular composition and stress response in foodborne pathogenic bacteria

Vibrational spectroscopy techniques have shown capacity to provide non-destructive, rapid, relevant information on microbial systematics, useful for classification and identification. Infrared spectroscopy enables the biochemical signatures from microbiological structures to be extracted and analyzed, in conjunction with advanced chemometrics. In addition, a number of recent studies have shown that Fourier Transform Infrared (FT-IR) spectroscopy can help understand the molecular basis of events such as the adaptive tolerance responses expressed by bacteria when exposed to stress conditions in the environment (e.g. those that cells confront in food and during food processing). The current review gives an overview of the published experimental techniques, data-processing algorithms and approaches used in FT-IR spectroscopy to assess the mechanisms of bacterial inactivation by food processing technologies and antimicrobial compounds, to monitor the spore and membrane properties of foodborne pathogens in changing environments, to detect stress-injured microorganisms in food-related environments, to assess dynamic changes in bacterial populations, and to study bacterial tolerance responses.     

PR-CALC: A Program for the Reconstruction of NMR Spectra from Projections

Projection-reconstruction NMR (PR-NMR) has attracted growing attention as a method for collecting multidimensional NMR data rapidly. The PR-NMR procedure involves measuring lower-dimensional projections of a higher-dimensional spectrum, which are then used for the mathematical reconstruction of the full spectrum. We describe here the program PR-CALC, for the reconstruction of NMR spectra from projection data. This program implements a number of reconstruction algorithms, highly optimized to achieve maximal performance, and manages the reconstruction process automatically, producing either full spectra or subsets, such as regions or slices, as requested. The ability to obtain subsets allows large spectra to be analyzed by reconstructing and examining only those subsets containing peaks, offering considerable savings in processing time and storage space. PR-CALC is straightforward to use, and integrates directly into the conventional pipeline for data processing and analysis. It was written in standard C+ + and should run on any platform. The organization is flexible, and permits easy extension of capabilities, as well as reuse in new software. PR-CALC should facilitate the widespread utilization of PR-NMR in biomedical research. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Liposome-based formulations for the antibiotic nonapeptide Leucinostatin A: Fourier transform infrared spectroscopy characterization and in vivo toxicologic study

Leucinostatin-A is a nonapeptide isolated fromPaecilomyces marquandii Paecilomyces lilacinus A257, andAcremonium sp., exerting remarkable phytotoxic, antibacterial (especially against Gram-positive) and antimycotic activities. With the aim to find alternative formulation for in vivo administration, a number of Leucinostatin-A—loaded liposomal formulations have been prepared and characterized. Both large unilamellar vesicles and multilamellar vesicles consisting of synthetic and natural lipids were evaluated. In addition, to determine the nature of peptide-membrane interactions and the stability of liposomes loaded with Leucinostatin-A, a Fourier Transform Infrared Spectroscopy study was performed. The results suggest that the mode of interaction of the peptide is dependent on its concentration, on bilayer fluidity, and on liposome type. Finally, the LD₅₀ of both free and liposome-delivered Leucinostatin-A was determined in mice. These results suggest that the incorporation of Leucinostatin-A into liposomes may result in decreased Leucinostatin-A toxicity, as the intraperitoneal administration of Leucinostatin-A—loaded liposomes reduced the LD₅₀ of Leucinostatin-A 15-fold.     

High resolution 4-D spectroscopy with sparse concentric shell sampling and FFT-CLEAN

Recent efforts to reduce the measurement time for multidimensional NMR experiments have fostered the development of a variety of new procedures for sampling and data processing. We recently described concentric ring sampling for 3-D NMR experiments, which is superior to radial sampling as input for processing by a multidimensional discrete Fourier transform. Here, we report the extension of this approach to 4-D spectroscopy as Randomized Concentric Shell Sampling (RCSS), where sampling points for the indirect dimensions are positioned on concentric shells, and where random rotations in the angular space are used to avoid coherent artifacts. With simulations, we show that RCSS produces a very low level of artifacts, even with a very limited number of sampling points. The RCSS sampling patterns can be adapted to fine rectangular grids to permit use of the Fast Fourier Transform in data processing, without an apparent increase in the artifact level. These artifacts can be further reduced to the noise level using the iterative CLEAN algorithm developed in radioastronomy. We demonstrate these methods on the high resolution 4-D HCCH-TOCSY spectrum of protein G's B1 domain, using only 1.2% of the sampling that would be needed conventionally for this resolution. The use of a multidimensional FFT instead of the slow DFT for initial data processing and for subsequent CLEAN significantly reduces the calculation time, yielding an artifact level that is on par with the level of the true spectral noise.     

Determination of solid-state fungal growth by Fourier transform infrared-photoacoustic spectroscopy

Abstract Photoacoustic spectroscopy (PAS) does not require optically transparent samples and is, therefore, well suited for analysis of solid-state samples. Fourier transform infrared (FTIR)-PAS of solid materials containing protein exhibited strong absorption in the amide I and amide II regions of the IR spectrum. Growth of a filamentous fungus, Phanerochaete chrysosporium , on cellulose discs was quantitatively determined by monitoring amide I absorption with FTIR-PAS.     

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.     

Time-resolved Fourier transform infrared spectroscopy of the nucleotide-binding domain from the ATP-binding Cassette transporter MsbA: ATP hydrolysis is the rate-limiting step in the catalytic cycle

MsbA is an essential Escherichia coli ATP-binding cassette (ABC) transporter involved in the flipping of lipid A across the cytoplasmic membrane. It is a close homologue of human P-glycoprotein involved in multidrug resistance, and it similarly accepts a variety of small hydrophobic xenobiotics as transport substrates. X-ray structures of three full-length ABC multidrug exporters (including MsbA) have been published recently and reveal large conformational changes during the transport cycle. However, how ATP hydrolysis couples to these conformational changes and finally the transport is still an open question. We employed time-resolved FTIR spectroscopy, a powerful method to elucidate molecular reaction mechanisms of soluble and membrane proteins, to address this question with high spatiotemporal resolution. Here, we monitored the hydrolysis reaction in the nucleotide-binding domain of MsbA at the atomic level. The isolated MsbA nucleotide-binding domain hydrolyzed ATP with V(max) = 45 nmol mg(-1) min(-1), similar to the full-length transporter. A Hill coefficient of 1.49 demonstrates positive cooperativity between the two catalytic sites formed upon dimerization. Global fit analysis of time-resolved FTIR data revealed two apparent rate constants of ~1 and 0.01 s(-1), which were assigned to formation of the catalytic site and hydrolysis, respectively. Using isotopically labeled ATP, we identified specific marker bands for protein-bound ATP (1245 cm(-1)), ADP (1101 and 1205 cm(-1)), and free phosphate (1078 cm(-1)). Cleavage of the β-phosphate-γ-phosphate bond was found to be the rate-limiting step; no protein-bound phosphate intermediate was resolved.     

The use of Fourier transform infrared spectroscopy to assay for urease from Pseudomonas aeruginosa and Canavalia ensiformis

A novel assay method was investigated for urease (EC 3.5.1.5) from Pseudomonas aeruginosa and Canavalia ensiformis by Fourier transform infrared spectroscopy. This enzyme catalyzed the hydrolysis of urea in phosphate buffer in deuterium oxide (²H₂O). The intensities of the bicarbonate bands maxima at 1625 and 1365 cm⁻¹ and of the amide I band at 1605 cm⁻¹ were measured as a function of time to study the kinetics of urea hydrolysis. The extinction coefficients ε of urea and bicarbonate were determined to be 0.72, 0.48, and 0.56 mM⁻¹ cm⁻¹ at 1625, 1605, and 1365 cm⁻¹, respectively. The initial velocity is proportional to the enzyme concentration by using the ureases from both C.ensiformis and P. aeruginosa. The kinetic constants (V_max, K_m, and K_cat) determined by Lineweaver-Burk plot were 532.2  U mg⁻¹ protein, 6.4 mM, and 806.36 s⁻¹, respectively. These data are in agreement with the results obtained by a spectrophotometric method using a linked assay based on glutamate dehydrogenase in aqueous media. Therefore, this spectroscopic method is highly suited to assay for urease activity and its kinetic parameters by using either cell-free extracts or purified enzyme preparations with an additional advantage of performing a real-time measurement of urease activity.     

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.     

Multidimensional vector space representation for convergent evolution and molecular phylogeny

With growing amounts of genome data and constant improvement of models of molecular evolution, phylogenetic reconstruction became more reliable. However, our knowledge of the real process of molecular evolution is still limited. When enough large-sized data sets are analyzed, any subtle biases in statistical models can support incorrect topologies significantly because of the high signal-to-noise ratio. We propose a procedure to locate sequences in a multidimensional vector space (MVS), in which the geometry of the space is uniquely determined in such a way that the vectors of sequence evolution are orthogonal among different branches. In this paper, the MVS approach is developed to detect and remove biases in models of molecular evolution caused by unrecognized convergent evolution among lineages or unexpected patterns of substitutions. Biases in the estimated pairwise distances are identified as deviations (outliers) of sequence spatial vectors from the expected orthogonality. Modifications to the estimated distances are made by minimizing an index to quantify the deviations. In this way, it becomes possible to reconstruct the phylogenetic tree, taking account of possible biases in the model of molecular evolution. The efficacy of the modification procedure was verified by simulating evolution on various topologies with rate heterogeneity and convergent change. The phylogeny of placental mammals in previous analyses of large data sets has varied according to the genes being analyzed. Systematic deviations caused by convergent evolution were detected by our procedure in all representative data sets and were found to strongly affect the tree structure. However, the bias correction yielded a consistent topology among data sets. The existence of strong biases was validated by examining the sites of convergent evolution between the hedgehog and other species in mitochondrial data set. This convergent evolution explains why it has been difficult to determine the phylogenetic placement of the hedgehog in previous studies.     

Metabolomic profiling of Drosophila using liquid chromatography Fourier transform mass spectrometry

Hydrophilic interaction chromatography (HILIC) interfaced with an Orbitrap Fourier transform mass spectrometer (FT-MS) was used to carry out metabolomic profiling of the classical Drosophila mutation, rosy (ry). This gene encodes a xanthine oxidase/dehydrogenase. In addition to validating the technology by detecting the same changes in xanthine, hypoxanthine, urate and allantoin that have been reported classically, completely unsuspected changes were detected in each of the tryptophan, arginine, pyrimidine and glycerophospholipid metabolism pathways. The rosy mutation thus ramifies far more widely than previously detected.