Enzymic and structural studies on Drosophila alcohol dehydrogenase and other short-chain dehydrogenases/reductases.

Enzymic and structural studies on Drosophila alcohol dehydrogenases and other short-chain dehydrogenases/reductases (SDRs) are presented. Like alcohol dehydrogenases from other Drosophila species, the enzyme from D. simulans is more active on secondary than on primary alcohols, although ethanol is its only known physiological substrate. Several secondary alcohols were used to determine the kinetic parameters kcat and Km. The results of these experiments indicate that the substrate-binding region of the enzyme allows optimal binding of a short ethyl side-chain in a small binding pocket, and of a propyl or butyl side-chain in large binding pocket, with stereospecificity for R(-) alcohols. At a high concentration of R(-) alcohols substrate activation occurs. The kcat and Km values determined under these conditions are about two-fold, and two orders of magnitude, respectively, higher than those at low substrate concentrations. Sequence alignment of several SDRs of known, and unknown three-dimensional structures, indicate the presence of several conserved residues in addition to those involved in the catalyzed reactions. Structural roles of these conserved residues could be derived from observations made on superpositioned structures of several SDRs with known structures. Several residues are conserved in tetrameric SDRs, but not in dimeric ones. Two halohydrin-halide-lyases show significant homology with SDRs in the catalytic domains of these enzymes, but they do not have the structural features required for binding NAD+. Probably these lyases descend from an SDR, which has lost the capability to bind NAD+, but the enzyme reaction mechanisms may still be similar.

Secretory ribonucleases in the primitive ruminant chevrotain (Tragulus javanicus).

Phylogenetic analyses of secretory ribonucleases or RNases 1 have shown that gene duplication events, giving rise to three paralogous genes (pancreatic, seminal and brain RNase), occurred during the evolution of ancestral ruminants. A higher number of paralogous sequences are present in chevrotain (Tragulus javanicus), the earliest diverged taxon within the ruminants. Two pancreatic RNase sequences were identified, one encoding the pancreatic enzyme, the other encoding a pseudogene. The identity of the pancreatic enzyme was confirmed by isolation of the protein and N-terminal sequence analysis. It is the most acidic pancreatic ribonuclease identified so far. Formation of the mature enzyme requires cleavage by signal peptidase of a peptide bond between two glutamic acid residues. The seminal-type RNase gene shows features of a pseudogene, like orthologous genes in other ruminants investigated with the exception of the bovine species. The brain-type RNase gene of chevrotain is expressed in brain tissue. A hybrid gene with a pancreatic-type N-terminal and a brain-type C-terminal sequence has been identified but nothing is known about its expression. Phylogenetic analysis of RNase 1 sequences of six ruminant, three other artiodactyl and two whale species support previous findings that two gene duplications occurred in a ruminant ancestor. Three distinct groups of pancreatic, seminal-type and brain-type RNases have been identified and within each group the chevrotain sequence it the first to diverge. In taxa with duplications of the RNase gene (ruminants and camels) the gene evolved at twice as fast than in taxa in which only one gene could be demonstrated; in ruminants there was an approximately fourfold increase directly after the duplications and then a slowing in evolutionary rate.

Ruminant brain ribonucleases: expression and evolution.

Molecular evolutionary analyses of mammalian ribonucleases have shown that gene duplication events giving rise to three paralogous genes occurred in ruminant ancestors. One of these genes encodes a ribonuclease identified in bovine brain. A peculiar feature of this enzyme and orthologous sequences in other ruminants are C-terminal extensions consisting of 17-27 amino acid residues. Evidence was obtained by Western blot analysis for the presence of brain-type ribonucleases in brain tissue not only of ox, but also of sheep, roe deer and chevrotain (Tragulus javanicus), a member of the earliest diverged taxon of the ruminants. The C-terminal extension of brain-type ribonuclease from giraffe deviates much in sequence from orthologues in other ruminants, due to a change of reading frame. However, the gene encodes a functional enzyme, which could be expressed in heterologous systems. The messenger RNA of bovine brain ribonuclease is not only expressed at a high level in brain tissue but also in lactating mammary gland. The enzyme was isolated and identified from this latter tissue, but was not present in bovine milk, although pancreatic ribonucleases A and B could be isolated from both sources. This suggests different ways of secretion of the two enzyme types, possibly related to structural differences. The sequence of the brain-type RNase from chevrotain suggests that the C-terminal extensions of ruminant brain-type ribonucleases originate from deletions in the ancestral DNA (including a region with stop codons), followed by insertion of a 5-8-fold repeated hexanucleotide sequence, coding for a proline-rich polypeptide.

Localization and analysis of nonpolar regions in onconase.

A detailed analysis of the composition and properties of hydrophobic nuclei and microclusters has been carried out for onconase. Two main hydrophobic nuclei in the onconase structure were detected. Their composition and shape were found to be very similar to those of RNase A, in accordance with the predictions made. The nuclei in onconase are more compact, the side-chain atoms of residues included in the nuclei in onconase form more contacts with the environment than in RNase A. The hydrophobic nuclei should be considered as individual structural units along with elements of the secondary structure. Differences in composition and conformation of exposed loops between onconase and RNase A were found. The additional hydrophobic clusters attached to the nuclei in onconase might be involved in the fixation of an appropriate conformation of site(s) for manifestation of the biological activity of onconase. A comparison of amphibian representatives of the RNase A superfamily was also made. The results obtained suggest that the availability of nonpolar residues in established key positions of amino acid sequences determines the characteristic fold of homologous proteins and the structure of the active site cleft.

Enzyme kinetics of hevamine, a chitinase from the rubber tree Hevea brasiliensis.

The enzyme kinetics of hevamine, a chitinase from the rubber tree Hevea brasiliensis, were studied in detail with a new enzyme assay. In this assay, the enzyme reaction products were derivatized by reductive coupling to a chromophore. Products were separated by HPLC and the amount of product was calculated by peak integration. Penta-N-acetylglucosamine (penta-nag) and hexa-N-acetylglucosamine (hexa-nag) were used as substrates. Hexa-nag was more efficiently converted than penta-nag, which is an indication that hevamine has at least six sugar binding sites in the active site. Tetra-N-acetylglucosamine (tetra-nag) and allosamidin were tested as inhibitors. Allosamidin was found to be a competitive inhibitor with a K(i) of 3.1 microM. Under the conditions tested, tetra-nag did not inhibit hevamine.

NMR investigations of protein-carbohydrate interactions: studies on the relevance of Trp/Tyr variations in lectin binding sites as deduced from titration microcalorimetry and NMR studies on hevein domains. Determination of the NMR structure of the complex between pseudohevein and N,N',N"-triacetylchitotriose.

Model studies on lectins and their interactions with carbohydrate ligands in solution are essential to gain insights into the driving forces for complex formation and to optimize programs for computer simulations. The specific interaction of pseudohevein with N,N', N"-triacetylchitotriose has been analyzed by (1)H-NMR spectroscopy. Because of its small size, with a chain length of 45 amino acids, this lectin is a prime target to solution-structure determination by NOESY NMR experiments in water. The NMR-analysis was extended to assessment of the topology of the complex between pseudohevein and N, N',N"-triacetylchitotriose. NOESY experiments in water solution provided 342 protein proton-proton distance constraints. Binding of the ligand did not affect the pattern of the protein nuclear Overhauser effect signal noticeably, what would otherwise be indicative of a ligand-induced conformational change. The average backbone (residues 3-41) RMSD of the 20 refined structures was 1.14 A, whereas the heavy atom RMSD was 2.18 A. Two different orientations of the trisaccharide within the pseudohevein binding site are suggested, furnishing an explanation in structural terms for the lectin's capacity to target chitin. In both cases, hydrogen bonds and van der Waals contacts confer stability to the complexes. This conclusion is corroborated by the thermodynamic parameters of binding determined by NMR and isothermal titration calorimetry. The association process was enthalpically driven. In relation to hevein, the Trp/Tyr-substitution in the binding pocket has only a small effect on the free energy of binding in contrast to engineered galectin-1 and a mammalian C-type lectin. A comparison of the three-dimensional structure of pseudohevein in solution to those reported for wheat germ agglutinin (WGA) in the solid state and for hevein and WGA-B in solution has been performed, providing a data source about structural variability of the hevein domains. The experimentally derived structures and the values of the solvent accessibilities for several key residues have also been compared with conformations obtained by molecular dynamics simulations, pointing to the necessity to further refine the programs to enhance their predictive reliability and, thus, underscoring the importance of this kind of combined analysis in model systems.

Papaya (Carica papaya) lysozyme is a member of the family 19 (basic, class II) chitinases.

The most comprehensive studies on a plant lysozyme (EC 3.2.1.17) are those on the enzyme from papaya (Carica papaya) latex, published in 1967 and 1969. However, the N-terminal amino acid sequence of five amino acid sequence of this enzyme, determined by manual Edman degradation, did not allow assignment to any of the much later-classified families of glycosyl hydrolases. N-Terminal sequence analysis of 22 residues of papaya lysozyme now shows unambiguously that the enzyme belongs to the family 19 chitinases. It has properties similar to those of basic class I chitinases with lysozyme activity, such as cleavage specificity at the C-1 of N-acetylmuramic acid with inversion of configuration, but as it lacks an N-terminal hevein domain, it should be classified as a class II chitinase.

The phylogenetic position of "Acomyinae" (Rodentia, Mammalia) as sister group of a Murinae + Gerbillinae clade: evidence from the nuclear ribonuclease gene.

The phylogenetic relationships of Acomys and Uranomys within Muridae were investigated using nuclear pancreatic ribonuclease A gene sequences. The various kinds of substitutions in the data matrix (15 taxa x 375 nucleotides) were examined for saturation, in order to apply a weighted parsimony approach. Phylogenies were derived by maximum parsimony (weighted and unweighted) and maximum likelihood procedures, using a dormouse (Gliridae) as outgroup. Maximum likelihood gave the most robust results. All analyses cluster some traditional taxa with a strong robustness, such as three species of the genus Mus, two South-East Asian rats, and two genera in each of the gerbil and vole families. When analyzed with those of other murid rodents representing Murinae, Gerbillinae, Arvicolinae, Cricetinae, and Sigmodontinae, sequences of the ribonuclease gene suggest that Acomys and Uranomys constitute a monophyletic clade at the subfamily level, denoted "Acomyinae." The relationships between the six subfamilies of Muridae appear poorly resolved, except for a clade uniting Murinae, Acomyinae, and Gerbillinae. Within this clade, the sister group of Acomyinae could not be identified, as the branch length defining a Gerbillinae + Murinae cluster is extremely short. The poor resolution of our phylogenetic inferences is probably the result of two confounding factors, namely the limited size of the pancreatic ribonuclease sequence and the probable short time intervals during the radiation of the six murid subfamilies involved in this study.

Seminal-type ribonuclease genes in ruminants, sequence conservation without protein expression?

Bovine seminal ribonuclease (BS-RNase) is an interesting enzyme both for functional and structural reasons. The enzyme is the product of a gene duplication that occurred in an ancestral ruminant. It is possible to demonstrate the presence of seminal-type genes in all other investigated ruminant species, but they are not expressed and show features of pseudogenes. In this paper we report the determination of two pancreatic and one seminal-type ribonuclease gene sequences of swamp-type water buffalo (Bubalus bubalis). The two pancreatic sequences encode proteins with identical amino acid sequences as previously determined for the enzymes isolated from swamp-type and river-type water buffalo, respectively. The seminal-type sequence has no pseudogene features and codes for an enzyme with no unusual features compared with the active bovine enzyme, except for the replacement of one of the cysteines which takes part in the two intersubunit disulfide bridges. However, Western blotting demonstrates the presence of only small amounts of the pancreatic enzymes in water buffalo semen, suggesting that also in this species the seminal-type sequence is not expressed. But it is still possible that the gene is expressed somewhere else in the body or during development. Reconstruction of seminal-type ribonuclease sequences in ancestors of Bovinae and Bovidae indicates no serious abnormalities in the encoded proteins and leads us to the hypothesis that the ruminant seminal-type ribonuclease gene has not come to expression during most of its evolutionary history, but did not exhibit a high evolutionary rate that is generally observed in pseudogenes.

Eosinophil derived neurotoxin (EDN) levels in commercial human urinary preparations of glycoprotein hormones.

Eosinophil derived neurotoxin (EDN) is a ubiquitous human ribonuclease, occurring not only in eosinophils, but also in many tissues and body fluids. It may be a contaminant of commercial human urinary preparations of chorionic gonadotropin (hCG) and other glycoprotein hormones. Here we describe the use of a fast commercial assay to quantify this contaminant and demonstrate that the content varies much between different commercial glycoprotein hormone preparations. As this ribonuclease may have a cytotoxic activity on certain cells, it is useful to be able to determine its quantity in a fast and reliable way in these preparations.

Inclusion of cetaceans within the order Artiodactyla based on phylogenetic analysis of pancreatic ribonuclease genes.

Mammalian secretory ribonucleases (RNases 1) form a family of extensively studied homologous proteins that were already used for phylogenetic analyses at the protein sequence level previously. In this paper we report the determination of six ribonuclease gene sequences of Artiodactyla and two of Cetacea. These sequences have been used with ruminant homologues in phylogenetic analyses that supported a group including hippopotamus and toothed whales, a group of ruminant pancreatic and brain-type ribonucleases, and a group of tylopod sequences containing the Arabian camel pancreatic ribonuclease gene and Arabian and Bactrian camel and alpaca RNase 1 genes of unknown function. In all analyses the pig was the first diverging artiodactyl. This DNA-based tree is compatible to published trees derived from a number of other genes. The differences to those trees obtained with ribonuclease protein sequences can be explained by the influence of convergence of pancreatic RNases from hippopotamus, camel, and ruminants and by taking into account the information from third codon positions in the DNA-based analyses. The evolution of sequence features of ribonucleases such as the distribution of positively charged amino acids and of potential glycosylation sites is described with regard to increased double-stranded RNA cleavage that is observed in several cetacean and artiodactyl RNases which may have no role in ruminant or ruminant-like digestion.

Introduction: the ribonuclease A superfamily.

In this multi-author issue several aspects of the ribonuclease A superfamily are reviewed. This superfamily can be subdivided in a number of mammalian and other vertebrate ribonuclease families. In the introduction chapter the titles of the other contributions are presented. There is little uniformity in the nomenclature of ribonucleases, caused in part by gene duplications, which have occurred independently in several mammalian lineages, and which are nice examples for explaining orthology and paralogy in molecular evolution.

The ribonuclease A superfamily: general discussion.

Enzymic properties of members of the ribonuclease A superfamily, like the activity on RNA, the preference for either cytosine or uracil in the primary binding site B1, the preference for the other side of the cleaved phosphodiester bond, the B2 site, and features of the two noncatalytic phosphate-binding sites P0 and P2 are discussed in several articles in this multi-author review, and are summarized in this closing article. A special feature of members of the ribonucleases 1 family is their destabilizing action on double-stranded nucleic acid structures. A feature of the ribonuclease A superfamily is the frequent occurrence of gene duplications, both in ancestral vertebrate lineages and in recently evolved taxa. Three different bovine ribonucleases 1 have been identified in pancreas, semen and brain, respectively, which are the result of two gene duplications in an ancestral ruminant. Similar gene duplications have been identified in other ribonuclease families in several mammalian and other vertebrate taxa. The ribonuclease family, of which the human members have been assigned numbers 2, 3 and 6, underwent a still mysterious pattern of gene duplications and functional expression as proteins with ribonuclease activity and other physiological properties.

Secretory ribonuclease genes and pseudogenes in true ruminants.

Mammalian pancreatic ribonucleases (RNase) form a family of extensively studied homologous proteins. Phylogenetic analyses, based on the primary structures of these enzymes, indicated that the presence of three homologous enzymes (pancreatic, seminal and brain ribonucleases) in the bovine species is due to gene duplication events, which occurred during the evolution of ancestral ruminants. In this paper the sequences are reported of the coding regions of the orthologues of the three bovine secretory ribonucleases in hog deer and roe deer, two deer species belonging to two different subfamilies of the family Cervidae. The sequences of the 3' untranslated regions of the three different secretory RNase genes of these two deer species and giraffe are also presented. Comparison of these and previously determined sequences of ruminant ribonucleases showed that the brain-type enzymes of giraffe and these deer species exhibit variations in their C-terminal extensions. The seminal-type genes of giraffe, hog deer and roe deer show all the features of pseudogenes. Phylogenetic analyses, based on the complete coding regions and parts of the 3' untranslated regions of the three different secretory ribonuclease genes of ox, sheep, giraffe and the two deer species, show that pancreatic, seminal- and brain-type RNases form three separate groups.

Ribonucleases from rat and bovine liver: purification, specificity and structural characterization.

The presence of four members of the pyrimidine-specific ribonuclease superfamily was demonstrated in rat liver. Three of them (RL1, RL2 and RL3) were purified and showed ribonuclease activity at pH 7.5 with yeast RNA as substrate. RL1 is identical to rat pancreatic ribonuclease (ribonuclease 1). N-terminal sequence analysis showed the presence of the native protein and several N-terminally degraded components. RL2 and RL3 were N-terminally blocked proteins. After acidic cleavage or CNBr digestion, several parts of their sequences were determined. RL2 has high sequence similarity with neurotoxin-type ribonucleases (ribonucleases 2, 3 and 6). The amino acid sequence of rat liver-type ribonuclease (ribonuclease 4) was determined from a liver cDNA library. It differs at about 20% of the amino acid positions from other mammalian liver-type ribonucleases. The sequence of a peptide of RL3 was identical to that derived from the cDNA sequence of the liver-type ribonuclease. A contaminant of the RL3 fraction had a high sequence similarity with mouse and other mammalian angiogenins. Bovine, porcine and rat liver-type ribonucleases showed a strong preference for poly(U) over poly(C). This preference is a unique property of the liver-type enzymes of the ribonuclease superfamily.

Isolation of protease-free alcohol dehydrogenase (ADH) from Drosophila simulans and several homozygous and heterozygous Drosophila melanogaster variants.

The enzyme alcohol dehydrogenase (ADH) from several naturally occurring ADH variants of Drosophila melanogaster and Drosophila simulans was isolated. Affinity chromatography with the ligand Cibacron Blue and elution with NAD+ showed similar behavior for D. melanogaster ADH-FF, ADH-71k, and D. simulans ADH. Introduction of a second Cibacron Blue affinity chromatography step, with gradient elution with NAD+, resulted in pure and stable enzymes. D. melanogaster ADH-SS cannot be eluted from the affinity chromatography column at a high concentration of NAD+ and required a pH gradient for its purification, preceded by a wash step with a high concentration of NAD+. Hybrid Drosophila melanogaster alcohol dehydrogenase FS has been isolated from heterozygous flies, using affinity chromatography with first elution at a high concentration NAD+, directly followed by affinity chromatography elution with a pH gradient. Incubation of equal amounts of pure homodimers of Drosophila melanogaster ADH-FF and ADH-SS, in the presence of 3 M urea at pH 8.6, for 30 min at room temperature, followed by reassociation yielded active Drosophila melanogaster ADH-FS heterodimers. No proteolytic degradation was found after incubation of purified enzyme preparations in the absence or presence of SDS, except for some degradation of ADH-SS after very long incubation times. The thermostabilities of D. melanogaster ADH-71k and ADH-SS were almost identical and were higher than those of D. melanogaster ADH-FF and D. simulans ADH. The thermostability of D. melanogaster ADH-FS was lower than those of D. melanogaster ADH-FF and ADH-SS. D. melanogaster ADH-FF and ADH-71k have identical inhibition constants with the ligand Cibacron Blue at pH 8.6, which are two times higher at pH 9.5. The Ki values for D. simulans ADH are three times lower at both pH values. D. melanogaster ADH-SS and ADH-FS have similar Ki values, which are lower than those for D. melanogaster ADH-FF at pH 8.6. But at pH 9.5 the Ki value for ADH-FS is the same as at pH 8.6, while that of ADH-SS is seven times higher. Kinetic parameters of Drosophila melanogaster ADH-FF, ADH-SS, and ADH-71k and Drosophila simulans ADH, at pH 8.6 and 9.5, showed little or no variation in K(m)eth values. The K(m)NAD values measured at pH 9.5 for Drosophila alcohol dehydrogenases are all lower than those measured at pH 8.6. The rate constants (kcat) determined for all four Drosophila alcohol dehydrogenases are higher at pH 9.5 than at pH 8.6. D. melanogaster ADH-FS showed nonlinear kinetics.

Modeling studies of conformational changes in the substrate-binding loop in Drosophila alcohol dehydrogenase.

Three-dimensional structures of seven short-chain dehydrogenases/reductases show that these enzymes share common structural features. Sequence alignment studies of Drosophila alcohol dehydrogenase (DADH), with an unknown 3D-structure, and four short-chain dehydrogenases/reductases with known X-ray structures suggest that DADH shares the same structural features. However, the substrate binding regions, which are located in the C-terminal region of these enzymes, share little sequence homology, because of the wide variety of substrates used. X-ray structures of short-chain dehydrogenases/reductases indicate that conformational changes occur in a loop, in the C-terminal region, upon substrate binding. This substrate-binding loop is located between a strand and a helix and may contain one or two small helices. Secondary structure predictions and modeling studies of this substrate-binding loop in DADH predict that the two helices may also be present in this enzyme. The naturally occurring variants of Drosophila melanogaster alleloenzymes ADH-S and ADH-F differ in a replacement of threonine by lysine at position 192, which is located at a central position in the substrate-binding loop. The positive charge of lysine may move significantly on substrate binding, resulting in a direct charge interaction with NAD+ in the enzyme-substrate complex, explaining a very strong influence of pH on the binding of ADH-S for the NAD+ analogue Cibacron Blue. This indicates that the ADH S/F polymorphism has a direct influence on the catalytic properties of the enzyme.

Molecular evolution of hemoglobins of Antarctic fishes (Notothenioidei).

Amino acid sequences of alpha- and beta-chains of human hemoglobin and of hemoglobins of coelacanth and 24 teleost fish species, including 11 antarctic and two temperate Notothenioidei, were analyzed using maximum parsimony. Trees were derived for the alpha- and beta-chains separately and for tandemly arranged sequences, using the human and coelacanth sequences as outgroups in all analyses. The topologies of the trees of the alpha- and beta-chains are highly congruent and indicate a specific pattern of gene duplications and gene expression of teleost hemoglobins which has not yet been investigated into more detail. The Notothenioid fish generally contain a single major hemoglobin and often a second minor component. The alpha- and beta-chains of the major components form a monophyletic group in all investigated trees, with the nonantarctic Pseudaphritis as their sister taxon. The minor chains also are a monophyletic group and form an unresolved cluster with the major chains and the hemoglobins of tuna and red gurnard. The Notothenioid families Nototheniidae and Bathydraconidae appear to be paraphyletic.

Hevamine, a chitinase from the rubber tree Hevea brasiliensis, cleaves peptidoglycan between the C-1 of N-acetylglucosamine and C-4 of N-acetylmuramic acid and therefore is not a lysozyme.

Hevamine is a chitinase from the rubber tree Hevea brasiliensis and belongs to the family 18 glycosyl hydrolases. In this paper the cleavage specificity of hevamine for peptidoglycan was studied by HPLC and mass-spectrometry analysis of enzymatic digests. The results clearly showed that the enzyme cleaves between the C-1 of a N-acetylglucosamine and the C-4 of a N-acetylmuramate residue. This means that hevamine, and very likely also other family 18 glycosyl hydrolases which cleave peptidoglycan, cannot be classified as lysozymes.

Role of aromatic amino acids in carbohydrate binding of plant lectins: laser photo chemically induced dynamic nuclear polarization study of hevein domain-containing lectins.

Carbohydrate recognition by lectins often involves the side chains of tyrosine, tryptophan, and histidine residues. These moieties are able to produce chemically induced dynamic nuclear polarization (CIDNP) signals after laser irradiation in the presence of a suitable radical pair-generating dye. Elicitation of such a response in proteins implies accessibility of the respective groups to the light-absorbing dye. In principle, this technique is suitable to monitor surface properties of a receptor and the effect of ligand binding if CIDNP-reactive amino acids are affected. The application of this method in glycosciences can provide insights into the protein-carbohydrate interaction process, as illustrated in this initial study. It focuses on a series of N-acetylglucosamine-binding plant lectins of increasing structural complexity (hevein, pseudohevein, Urtica dioica agglutinin and wheat germ agglutinin and its domain B), for which structural NMR- or X-ray crystallographic data permit a decision of the validity of the CIDNP method-derived conclusions. On the other hand, the CIDNP data presented in this study can be used for a rating of our molecular models of hevein, pseudohevein, and domain B obtained by various modeling techniques. Experimentally, the shape and intensity of CIDNP signals are determined in the absence and in the presence of specific glycoligands. When the carbohydrate ligand is bound, CIDNP signals of side chain protons of tyrosine, tryptophan, or histidine residues are altered, for example, they are broadened and of reduced intensity or disappear completely. In the case of UDA, the appearance of a new tryptophan signal upon ligand binding was interpreted as an indication for a conformational change of the corresponding indole ring. Therefore, CIDNP represents a suitable tool to study protein-carbohydrate interactions in solution, complementing methods such as X-ray crystallography, high-resolution multidimensional nuclear magnetic resonance, transferred nuclear Overhauser effect experiments, and molecular modeling.