One-dimensional 1H NMR Spectra Research Articles

Probing the Conformation of a Prion Protein Fibril with Hydrogen Exchange

A fragment of the prion protein, PrP(89-143, P101L), bearing a mutation implicated in familial prion disease, forms fibrils that have been shown to induce prion disease when injected intracerebrally into transgenic mice expressing full-length PrP containing the P101L mutation. In this study, we utilize amide hydrogen exchange measurements to probe the organization of the peptide in its fibrillar form. We determined the extent of hydrogen exchange first by tandem proteolysis, liquid chromatography, and mass spectrometry (HXMS) and then by exchange-quenched NMR. Although single amide resolution is afforded by NMR measurements, HXMS is well suited to the study of natural prions because it does not require labeling with NMR active isotopes. Thus, natural prions obtained from infected animals can be compared with model systems such as PrP(89-143, P101L) studied here. In our study, we find two segments of sequence that display a high level of protection from exchange, residues 102-109 and 117-136. In addition, there is a region that displays exchange behavior consistent with the presence of a conformationally heterogeneous turn. We discuss our data with respect to several structural models proposed for infectious PrP aggregates and highlight HXMS as one of the few techniques well suited to studying natural prions.

Characterization of Phospholipase Cγ Enzymes with Gain-of-Function Mutations

Phospholipase C gamma isozymes (PLC gamma 1 and PLC gamma 2) have a crucial role in the regulation of a variety of cellular functions. Both enzymes have also been implicated in signaling events underlying aberrant cellular responses. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we have recently identified single point mutations in murine PLC gamma 2 that lead to spontaneous inflammation and autoimmunity. Here we describe further, mechanistic characterization of two gain-of-function mutations, D993G and Y495C, designated as ALI5 and ALI14. The residue Asp-993, mutated in ALI5, is a conserved residue in the catalytic domain of PLC enzymes. Analysis of PLC gamma 1 and PLC gamma 2 with point mutations of this residue showed that removal of the negative charge enhanced PLC activity in response to EGF stimulation or activation by Rac. Measurements of PLC activity in vitro and analysis of membrane binding have suggested that ALI5-type mutations facilitate membrane interactions without compromising substrate binding and hydrolysis. The residue mutated in ALI14 (Tyr-495) is within the spPH domain. Replacement of this residue had no effect on folding of the domain and enhanced Rac activation of PLC gamma 2 without increasing Rac binding. Importantly, the activation of the ALI14-PLC gamma 2 and corresponding PLC gamma 1 variants was enhanced in response to EGF stimulation and bypassed the requirement for phosphorylation of critical tyrosine residues. ALI5- and ALI14-type mutations affected basal activity only slightly; however, their combination resulted in a constitutively active PLC. Based on these data, we suggest that each mutation could compromise auto-inhibition in the inactive PLC, facilitating the activation process; in addition, ALI5-type mutations could enhance membrane interaction in the activated state.

Structure-Function Analysis of Inositol Hexakisphosphate-induced Autoprocessing in Clostridium difficile Toxin A

The action of Clostridium difficile toxins A and B depends on inactivation of host small G-proteins by glucosylation. Cellular inositol hexakisphosphate (InsP6) induces an autocatalytic cleavage of the toxins, releasing an N-terminal glucosyltransferase domain into the host cell cytosol. We have defined the cysteine protease domain (CPD) responsible for autoprocessing within toxin A (TcdA) and report the 1.6 A x-ray crystal structure of the domain bound to InsP6. InsP6 is bound in a highly basic pocket that is separated from an unusual active site by a beta-flap structure. Functional studies confirm an intramolecular mechanism of cleavage and highlight specific residues required for InsP6-induced TcdA processing. Analysis of the structural and functional data in the context of sequences from similar and diverse origins highlights a C-terminal extension and a pi-cation interaction within the beta-flap that appear to be unique among the large clostridial cytotoxins.

TβR-II Discriminates the High- and Low-Affinity TGF-β Isoforms via Two Hydrogen-Bonded Ion Pairs

The TGF-beta isoforms, TGF-beta1, -beta2, and -beta3, share greater than 70% sequence identity and are almost structurally identical. TGF-beta2 differs from the others, however, in that it binds the TGF-beta type II receptor (TbetaR-II) with much lower affinity than either TGF-beta1 or -beta3. It has been previously shown that three conserved interfacial residues, Arg25, Val92, Arg94, in TGF-beta1 and -beta3 are responsible for their high-affinity interaction with TbetaR-II. In this study, the role of each of these residues was examined by creating single, double, and triple substitutions resulting in both TGF-beta3 loss-of-function and TGF-beta2 gain-of-function variants. One-dimensional 1H NMR spectra of the variants confirmed a lack of large structural perturbations. Affinities, kinetics, and thermodynamics for TbetaR-II binding were determined by surface plasmon resonance biosensor analysis. Double substitutions revealed that nearly all of the high-affinity binding is contributed by Arg25 and Arg94. Single site substitutions showed that Arg94 makes the greatest contribution. Substitution of Arg25 and Arg94 with alanine verified the requirement of the arginine guanidinium functional groups for the highly specific hydrogen-bonded ion pairs formed between Arg25 and Arg94 of TGF-beta1 and -beta3, and Glu119 and Asp32 of TbetaR-II. Further kinetic and thermodynamic analyses confirmed that Arg25 and Arg94 are primarily responsible for high-affinity binding and also revealed that noninterfacial longer range effects emanating from the TGF-beta structural framework contribute slightly to TbetaR-II binding. Growth inhibition assays showed that binding changes generally correlate directly with changes in function; however, a role Val92 in this cellular context was uncovered.

Identification of botanical biomarkers found in Corsican honey

Honeys from specified botanical sources often command a premium price due to their organoleptic or pharmacoactive properties. To prevent the fraudulent marketing of honey, analytical techniques are required to confirm its origin. NMR spectroscopy has been used to identify biomarkers of botanical and geographical origin for European honey. One-dimensional 1H NMR spectra were acquired from 374 authentic European honeys collected during 2 years, with the majority of these (220) taken from the island of Corsica. Biomarkers of sweet chestnut, Corsican spring Maquis and Arbousier (strawberry-tree) honeys were identified. Kynurenic acid was found to be a biomarker of sweet chestnut honey. α-Isophorone and 2,5-dihydroxyphenylacetic acid were confirmed as markers of strawberry-tree honey. Additional compounds specific to strawberry-tree and Corsican spring Maquis honey were partially characterised.

1H NMR determination of hypericin and pseudohypericin in complex natural mixtures by the use of strongly deshielded OH groups

The 1H NMR spectra of the commercially available compounds hypericin and its derivative pseudohypericin in CD 3OH solutions indicate significantly deshielded signals in the region of 14–15 ppm. These resonances are attributed to the peri hydroxyl protons OH(6), OH(8) and OH(1), OH(13) of hypericins which participate in a strong six-membered ring intramolecular hydrogen bond with CO(7) and CO(14), respectively, and therefore, they are strongly deshielded. In the present work, we demonstrate that one-dimensional 1H NMR spectra of hypericin and pseudohypericin, in Hypericum perforatum extracts show important differences in the chemical shifts of the hydroxyl groups with excellent resolution in the region of 14–15 ppm. The facile identification and quantification of hypericin and its derivative compound pseudohypericin was achieved, without prior HPLC separation, for two H. perforatum extracts from Greek cultivars and two commercial extracts: a dietary supplement, and an antidepressant medicine. The results were compared with those obtained from UV–vis and LC/MS measurements.

On the structure and synthesis of neuroprotectin D1, a novel anti-inflammatory compound of the docosahexaenoic acid family

Potato tuber lipoxygenase was shown to convert 17(S)-hydro(pero)xydocasahexaenoic acid in 10,17(S)-dihydro(pero)xydocosahexa-4Z,7Z,11E,13Z,15E,19Z-enoic acid [10,17(S)-diHDHA] which was formed apparently through a double lipoxygenation mechanism. No traces of 10,17(S)-dihydro(pero)xydocosahexa-4Z,7Z,11E,13E,15Z,19Z-enoic acid were found among the reaction products. It is very likely that a described earlier "neuroprotectin D1" [or "10,17(S)docosatriene"], a novel and potent anti-inflammatory compound derived from docosahexaenoic acid, was, in fact, 10,17(S)-dihydroxydocosahexa-4Z,7Z,11E,13Z,15E,19Z-enoic acid formed through a double lipoxygenation mechanism instead of a previously thought epoxidation/isomerization mechanism.

Neutral glycolipids of the filamentous fungus Neurospora crassa:altered expression in plant defensin-resistant mutants

To defend themselves against fungal pathogens, plants produce numerous antifungal proteins and peptides, including defensins, some of which have been proposed to interact with fungal cell surface glycosphingolipid components. Although not known as a phytopathogen, the filamentous fungus Neurospora crassa possesses numerous genes similar to those required for plant pathogenesis identified in fungal pathogens (Galagan, J. E., et al. 2003. Nature 422: 859-868), and it has been used as a model for studying plant-phytopathogen interactions targeting fungal membrane components (Thevissen, K., et al. 2003. Peptides. 24: 1705-1712). For this study, neutral glycolipid components were extracted from wild-type and plant defensin-resistant mutant strains of N. crassa. The structures of purified components were elucidated by NMR spectroscopy and mass spectrometry. Neutral glycosphingolipids of both wild-type and mutant strains were characterized as beta-glucopyranosylceramides, but those of the mutants were found with structurally altered ceramides. Although the wild type expressed a preponderance of N-2'-hydroxy-(E)-Delta3-octadecenoate as the fatty-N-acyl component attached to the long-chain base (4E,8E)-9-methyl-4,8-sphingadienine, the mutant ceramides were found with mainly N-2'-hydroxyhexadecanoate instead. In addition, the mutant strains expressed highly increased levels of a sterol glucoside identified as ergosterol-beta-glucoside. The potential implications of these findings with respect to defensin resistance in the N. crassa mutants are discussed.

Metabonomic Deconvolution Of Embedded Toxicity: Application To Thioacetamide Hepato- and Nephrotoxicity

We present here the potential of an integrated metabonomic strategy to deconvolute the biofluid metabolic signatures in experimental animals following multiple organ toxicities, using the well-known hepato- and nephrotoxin, thioacetamide. Male Han-Wistar rats were dosed with thioacetamide (150 mg/kg, n = 25), and urine, plasma, liver, and kidney samples were collected postdose for conventional NMR and magic angle spinning (MAS) NMR spectroscopy. These data were correlated with histopathology and plasma clinical chemistry collected at all time points. 1H MAS NMR data from liver and kidney were related to sequential 1H NMR measurements in urine and plasma using pattern recognition methods. One-dimensional 1H NMR spectra were data-reduced and analyzed using principal components analysis (PCA) to show the time-dependent biochemical variations induced by thioacetamide toxicity. From the eigenvector loadings of the PCA, those regions of the 1H NMR spectra, and hence the combinations of endogenous metabolites marking the main phase of the toxic episode, were identified. The thioacetamide-induced biochemical manifestations included a renal and hepatic lipidosis accompanied by hypolipidaemia; increased urinary excretion of taurine and creatine concomitant with elevated creatine in liver, kidney, and plasma; a shift in energy metabolism characterized by depleted liver glucose and glycogen; reduced urinary excretion of tricarboxylic acid cycle intermediates and raised plasma ketone bodies; increased levels of tissue and plasma amino acids leading to amino aciduria verifying necrosis-enhanced protein degradation and renal dysfunction; and elevated hepatic and urinary bile acids indicating secondary damage to the biliary system. This integrated metabonomic approach has been able to identify the tissue of origin for biomarkers present in the metabolic profiles of biofluids, following the onset and progression of a multiorgan pathology, and as such highlights its potential in the evaluation of embedded toxicity in novel drug candidates.

NMR structure of the conserved hypothetical protein TM0979 from Thermotoga maritima

NMR structure of the conserved hypothetical protein TM0979 from <i>Thermotoga maritima</i>

A 700 MHz 1H-NMR study reveals apoptosis-like behavior in human K562 erythroleukemic cells exposed to a 50 Hz sinusoidal magnetic field

Purpose: To study cell damage and possible apoptosis in K562 human erythroleukemic cells exposed for 2 h to an extremely low frequency (ELF) 50 Hz sinusoidal magnetic field with a magnetic induction of either 1 or 5 mT using high resolution proton nuclear magnetic resonance (1H-NMR) spectroscopy.Materials and methods: One-dimensional 1H-NMR spectra were obtained on whole K562 cells and perchloric acid extracts of these cells. In addition, two-dimensional 1H-NMR spectra were also acquired. Cell damage was examined by lactate dehydrogenase release and changes in cell growth were monitored by growth curve analyses, bromodeoxyuridine incorporation and Ki67 antigen localization. Cell death (necrosis and apoptosis) were also studied by using the chromatin dye Hoechst 33258.Results: The variations in numerous metabolites observed with 1H-NMR reveal apoptosis-like behavior in response of K562 cells to ELF fields.Conclusion: 1H-NMR can be extremely useful in studying the effects of ELF fields on cells. In particular, the variations in metabolites which suggest apoptosis-like behavior occur when the cells are not identifiable as apoptotic by more traditional techniques.

Trypanosoma brucei Glycoproteins Contain Novel Giant Poly-N-acetyllactosamine Carbohydrate Chains

The flagellar pocket of the bloodstream form of the African sleeping sickness parasite Trypanosoma brucei contains material that binds the beta-d-galactose-specific lectin ricin (Brickman, M. J., and Balber, A. E. (1990) J. Protozool. 37, 219-224). Glycoproteins were solubilized from bloodstream form T. brucei cells in 8 M urea and 3% SDS and purified by ricin affinity chromatography. Essentially all binding of ricin to these glycoproteins was abrogated by treatment with peptide N-glycosidase, showing that the ricin ligands are attached to glycoproteins via N-glycosidic linkages to asparagine residues. Glycans released by peptide N-glycosidase were resolved by Bio-Gel P-4 gel filtration into two fractions: a low molecular mass mannose-rich fraction and a high molecular mass galactose and N-acetylglucosamine-rich fraction. The latter fraction was further separated by high pH anion exchange chromatography and analyzed by gas chromatography mass spectrometry, one- and two-dimensional NMR, electrospray mass spectrometry, and methylation linkage analysis. The high molecular mass ricin-binding N-glycans are based on a conventional Manalpha1-3(Manalpha1-6)Manbeta1-4-GlcNAcbeta1-4GlcNAc core structure and contain poly-N-acetyllactosamine chains. A significant proportion of these structures are extremely large and of unusual structure. They contain an average of 54 N-acetyllactosamine (Galbeta1-4GlcNAc) repeats per glycan, linked mostly by -4GlcNAcbeta1-6Galbeta1-interrepeat linkages, with an average of one -4GlcNAcbeta1-3(-4GlcNAcbeta1-6)Galbeta1- branch point in every six repeats. These structures, which also bind tomato lectin, are twice the size reported for the largest mammalian poly-N-acetyllactosamine N-linked glycans and also differ in their preponderance of -4GlcNAcbeta1-6Galbeta1- over -4GlcNacbeta1-3Galbeta1- interrepeat linkages. Molecular modeling suggests that -4GlcNAcbeta1-6Galbeta1- interrepeat linkages produce relatively compact structures that may give these giant N-linked glycans unique physicochemical properties. Fluorescence microscopy using fluorescein isothiocyanatericin indicates that ricin ligands are located mainly in the flagellar pocket and in the endosomal/lysosomal system of the trypanosome.

Quantification of organic and amino acids in beer by 1H NMR spectroscopy.

The quantification of organic and amino acids in beer using 1H NMR spectroscopy is demonstrated. Quantification was made both by integration of signals in the spectra together with use of calibration references and by use of partial least-squares (PLS) regression. Results from the NMR quantifications were compared with those obtained from determinations by amino acid analysis on HPLC and organic acid analysis by capillary electrophoresis. The described NMR-based methods could satisfactorily be used for quantification of several of the investigated metabolites in beer down to approximately 10 mg/L and for most with a good to high accuracy compared to results obtained by HPLC and capillary electrophoresis (R2 0.90-0.99). This was achieved with a simple sample preparation and one-dimensional 1H NMR spectra obtained in a few minutes. The use of PLS clearly improves the accuracy of the quantifications, based on comparison to results obtained by HPLC and capillary electrophoresis, and furthermore permits the determination of components with partially overlapped signals in the spectrum. NMR spectroscopy in combination with PLS will be a useful tool for the quantification of metabolites, not only in beer but also in other beverages and biofluids.

Adsorption of Bovine α-Lactalbumin on Suspended Solid Nanospheres and Its Subsequent Displacement Studied by NMR Spectroscopy

Detailed knowledge of the adsorption-induced conformational changes of proteins is essential to understand the process of protein adsorption. However, not much information about these conformational changes is available. Here, the adsorption of calcium-depleted (APO)- and calcium-containing (HOLO)-bovine alpha-lactalbumin (BLA) on suspended solid polystyrene nanospheres and their subsequent displacement by a surfactant are studied by NMR spectroscopy. To our knowledge, this is the first time that adsorption of proteins on solid nanospheres, with both components present in the NMR sample, is studied by this method. High-quality one-dimensional and two-dimensional 1H NMR spectra of nonadsorbed APO- and HOLO-BLA in the presence of BLA- and/or surfactant-covered solid polystyrene nanospheres in suspension are obtained using standard NMR procedures. BLA and surfactant molecules that are adsorbed on the polystyrene nanospheres give rise to extremely broadened proton resonances. This can be exploited to determine the amount of adsorbed protein and of adsorbed surfactant in a system containing protein, nanospheres, and surfactant, without disturbing the equilibrium of the system. Two-dimensional 1H NMR spectroscopy shows that the chemical shifts of the backbone amide protons of HOLO-BLA after its adsorption and subsequent displacement from polystyrene nanospheres by the surfactant 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) are identical to those of native HOLO-BLA. The adsorption-induced unfolding of BLA to a molten globule state on polystyrene nanospheres is thus fully reversible at the residue level upon CHAPS-induced displacement of BLA. The latter is the now fulfilled essential requirement that enables the future indirect study, at the residue level, of the conformational characteristics of BLA adsorbed on polystyrene nanospheres by hydrogen/deuterium exchange and NMR spectroscopy. The results presented show that NMR spectroscopy is clearly feasible to study the adsorption of BLA on suspended polystyrene nanospheres. This technique should be applicable to the study of the adsorption of other proteins on other surfaces as well.

The 2D-J-DOSY Experiment: Resolving Diffusion Coefficients in Mixtures

Many diffusion-ordered spectroscopy (DOSY) NMR techniques have recently been developed to aid in the deconvolution of complex mixtures. Spectroscopic separation based on chemical and physical properties facilitates the identification of mixture components while eliminating time-consuming separation steps and preserving the chemical environment. One way to improve resolution in such experiments is to spread the spectroscopic information into two dimensions. The 2D-J-DOSY experiment has been designed to resolve mixture components in terms of a chemical shift and proton coupling constant as well as distinguishing them on the basis of translational diffusion. Acquiring a series of spectra as a function of gradient amplitude permits the determination of diffusion coefficients for components that cannot be resolved in the one-dimensional (1D) 1H NMR spectrum. Comparison of the resulting values with those obtained through the traditional 1D diffusion experiment for a mixture of sugars validates The 2D-J-DOSY technique.

Determination of the absolute configuration of 2-hydroxyglutaric acid and 5-oxoproline in urine samples by high-resolution NMR spectroscopy in the presence of chiral lanthanide complexes

Determination of the absolute configuration of some metabolites in body fluids is important for the diagnosis of some inborn errors of metabolism. Presently available methods of such determinations are tedious and usually require highly specialized instrumentation. In this work, an alternative method, based on high-resolution nuclear magnetic resonance spectroscopy in the presence of the chiral lanthanide shift reagent as an auxiliary additive, has been proposed (NMR/LSR). The method involves the lineshape analysis of a chosen multiplet of the one-dimensional 1H NMR spectrum or application of the two-dimensional 1H- 13C correlation spectroscopy (HSQC). In order to confirm the resonance assignments and to boost the signal–noise ratio, the addition of an amount of racemic analyte to the urine sample is recommended. The entire procedure is simple in application and demands minimal or no preprocessing of urine samples. The effectiveness of the method has been confirmed by finding the expected forms of 2-hydroxyglutaric acid and 5-oxoproline in the urine samples of an independently diagnosed patient with 2- d-hydroxyglutaric aciduria and 5- l-oxoprolinuria, respectively.

Data mining and knowledge discovery in proton nuclear magnetic resonance (1H-NMR) spectra using frequency to information transformation (FIT)

Recent rapid development of research in the fields of structural genomics and bioinformatics has stressed the need for the development of effective methods of data mining and knowledge extraction from complex and convoluted signals. In this paper we introduce frequency to information transformation (FIT) as a novel method of extracting information content of complex signals. Because FIT uses a priori knowledge and is a comparative technique, it is well suited for data mining and knowledge discovery from complex data. In this paper, we introduce FIT and compare it to established methods used in automated conditioning and knowledge discovery in proton-nuclear magnetic resonance (1H-NMR) spectra. FIT transformation was applied to a collection of 80 one-dimensional (1D) 1H-NMR spectra of 23 N-linked oligosaccharides. Three classification methods, namely, cluster analysis, Bayesian analysis and artificial neural networks (ANN) were used to demonstrate the advantages of FIT in information and knowledge extraction in comparison with classical methods such as frequency-based filtering, nonlinear and piecewise linear curve fitting, and correlation coefficient analysis.

Prolidase deficiency diagnosed by 1H NMR spectroscopy of urine.

Three urine samples from two prolidase-deficient patients were analysed using 1H NMR spectroscopy. One-dimensional 1H NMR spectra showed a characteristic pattern of overlapping resonances of the proline and hydroxyproline protons of the imidodipeptides. The model compounds Ala-Pro, Gly-Pro, Phe-Pro, Leu-Pro, Val-Pro, Gly-Hyp and Pro-Hyp were measured as well. The non-proline resonances of Val-Pro, Ala-Pro and Gly-Pro could be assigned in the urine spectra. These resonances could then be used for quantification of the corresponding imidodipeptids. The presence of Leu-Pro in the patients' urine was demonstrated by the results of COSY experiments. However, this imidodipeptide could not be quantified owing to overlap of the resonaces in the one-dimensional 1H NMR spectrum of the patients' urine. Phe-Pro, Pro-Hyp and Gly-Hyp could not be assigned in the spectrum of the patient's urine. The characteristic resonances in the urine from a prolidase-deficient patient, i.e. Ala-Pro, Val-Pro, Gly-Pro, and resonances of the (hydroxy)proline part of the imidodipeptides can be used to diagnose this disease.

Epitope Diversity of N-Glycans from Bovine Peripheral Myelin Glycoprotein P0 Revealed by Mass Spectrometry and Nano Probe Magic Angle Spinning 1H NMR Spectroscopy

The carbohydrate structures present on the glycoproteins in the central and peripheral nerve systems are essential in many cell adhesion processes. The P0 glycoprotein, expressed by myelinating Schwann cells, plays an important role during the formation and maintenance of myelin, and it is the most abundant constituent of myelin. Using monoclonal antibodies, the homophilic binding of the P0 glycoprotein was shown to be mediated via the human natural keller cell (HNK)-1 epitope (3-O-SO(3)H-GlcUA(beta1-3)Gal(beta1-4)GlcNAc) present on the N-glycans. We recently described the structure of the N-glycan carrying the HNK-1 epitope, present on bovine peripheral myelin P0 (Voshol, H., van Zuylen, C. W. E. M., Orberger, G., Vliegenthart, J. F. G., and Schachner, M. (1996) J. Biol. Chem. 271, 22957-22960). In this study, we report on the structural characterization of the detectable glycoforms, present on the single N-glycosylation site, using state-of-the-art NMR and mass spectrometry techniques. Even though all structures belong to the hybrid- or biantennary complex-type structures, the variety of epitopes is remarkable. In addition to the 3-O-sulfate present on the HNK-1-carrying structures, most of the glycans contain a 6-O-sulfated N-acetylglucosamine residue. This indicates the activity of a 6-O-sulfo-GlcNAc-transferase, which has not been described before in peripheral nervous tissue. The presence of the disialo-, galactosyl-, and 6-O-sulfosialyl-Lewis X epitopes provides evidence for glycosyltransferase activities not detected until now. The finding of such an epitope diversity triggers questions related to their function and whether events, previously attributed merely to the HNK-1 epitope, could be mediated by the structures described here.

WebSpectra: Online NMR and IR Spectra for Students

WebSpectra is a World Wide Web site at UCLA through which organic chemistry students have convenient access to a library of problems in NMR and IR spectroscopy, ranging in difficulty from introductory to advanced. Students are presented with high-resolution spectra of unknown compounds in addition to the molecular formula. Expandable one-dimensional 1H and 13C NMR spectra are available for all problems and many also include IR, DEPT NMR, and/or COSY NMR spectra. In addition to the spectral problems, WebSpectra provides instructional documents on basic NMR and IR spectroscopy concepts. There is also an IR comparison tool that allows overlays of spectra and a search engine that allows students to locate specific types of compounds based upon name, formula, or functional group. The design and capabilities of this Web-based educational tool are described in this paper.