Nuclear Overhauser Effect Experiments Research Articles

Properties and one-step synthesis of (2-acetylpyridine)tetraammineruthenium(II), [Ru II(2-acpy)(NH 3) 4] 2+ and tetraammine(2-benzoylpyridine)ruthenium(II), [Ru II(NH 3) 4(2-bzpy)] 2+: Redox potentials, UV–Vis and NMR spectra

A more direct and efficient route to the syntheses of [Ru(NH 3) 4(X–Y)](BF 4) 2, where X–Y can be 2-acetylpyridine (2-acpy) or 2-benzoylpyridine (2-bzpy), based on the reactions of [RuCl(NH 3) 5]Cl 2 with these ortho-substituted azines is described. The [Ru(2-acpy)(NH 3) 4](BF 4) 2 and [Ru(NH 3) 5(2-bzpy)](BF 4) 2 complexes have a molar conductance of 328 and 292 Ω −1 cm 2 mol −1, respectively, corresponding to a 1:2 species in solution. These complexes showed two intense absorption bands around 620–650 and 380 nm, the energies of which are solvent dependent, decreasing with the increase of the Gutman’s donor number of the solvent, and were assigned as metal-to-ligand charge transfer (MLCT). The complexes have oxidation potentials (Ru II/III) of +0.380 V vs. Ag/AgCl (2-acpy) and +0.400 V vs. Ag/AgCl (2-bzpy), and reduction potentials (X–Y 0/−) of −1.10 V vs. Ag/AgCl (2-acpy) and −0.950 V vs. Ag/AgCl (2-bzpy) on CF 3COOH/NaCF 3COO at pH=3.0, scan rate 100 mV s −1, [Ru]=1.0×10 −3 mol l −1. Both processes show a coupled chemical reaction. Upon oxidation of the metal center, the MLCT absorption bands are bleached and restored upon subsequent reduction. In order to confirm the structure of the complexes a detailed 1H NMR investigation was performed in d 6-acetone. Further confirmation of the structure was obtained by recording the 15N NMR spectrum of [Ru(NH 3) 4(2-bzpy)] 2+ in d 6-DMSO using the INEPT pulse sequence improving the sensitivity of 15N by polarization transfer from the protons to the 15N. The Nuclear Overhauser Effect (NOE) experiments were made qualitatively for [Ru(NH 3) 4(2-acpy)] 2+, and showed that H 6 of the pyridine is close to a NH 3 proton, which should then be in a cis position, and, hence, confirming that acpy is acting as a bidentate ligand.

Solution Structure of Substrate-based Ligands When Bound to Hepatitis C Virus NS3 Protease Domain

The interactions of the NS3 protease domain with inhibitors that are based on N-terminal cleavage products of peptide substrates were studied by NMR methods. Transferred nuclear Overhauser effect experiments showed that these inhibitors bind the protease in a well defined, extended conformation. Protease-induced line-broadening studies helped identify the segments of inhibitors which come into contact with the protease. A comparison of the NMR data of the free and protease-bound states suggests that these ligands undergo rigidification upon complexation. This work provides the first structure of an inhibitor when bound to NS3 protease and should be valuable for designing more potent inhibitors.

Analysis of the (Trimethylsilyl)propionic Acid−β(12−28) Peptide Binding Equilibrium with NMR Spectroscopy

The binding of a small molecule, (trimethylsilyl)propionic acid (TSP), to a 17-residue peptide, β(12-28), is examined using (1)H NMR spectroscopy. β(12-28) (VHHQKLVFFAEDVGSNK) is a central fragment of the 40-42-residue Alzheimer's-associated Aβ peptide. This peptide has been previously shown to form soluble aggregates in low-pH aqueous solution. The TSP resonance is broadened appreciably in solutions containing relatively high concentrations (∼2 mM) of the peptide. The changes in TSP line width measured by titration of a peptide solution with TSP indicate a 1:1 binding stoichiometry. If the concentrations of both the peptide and TSP are reduced by 1 order of magnitude, the resonances of both species are sharp, suggesting that TSP binds predominately to the aggregated peptide. Nuclear Overhauser effect experiments indicate that the TSP interacts predominately with the side chains of the aliphatic peptide residues Leu(17) and Val(18). Pulsed-field gradient NMR measurements of TSP and peptide diffusion coefficients provide a more quantitative picture of the TSP-peptide binding equilibrium. The measured diffusion coefficients were used to calculate the fractions of the free and bound TSP. These results substantiate the conclusion that the stoichiometry of the TSP-peptide binding equilibrium is essentially 1:1 and further indicate anticooperative behavior in solutions containing an excess of TSP resulting in a dissociation of the peptide aggregates.

Synthesis, Conformational Analysis, and Phase Characterization of a Versatile Self-Assembling Monoglucosyl Diacylglycerol Analog

Glycosyl diacylglycerols are excellent lipids for the formation of both bi- and monolayer lamellar systems but they are generally not commercially available, and the synthesis of optically pure glycosyl diacylglycerols inevitably involves protection and deprotection of glycerol linkers. A novel glycolipid designed to be an easily accessible structural surrogate of monoglucosyl diacylglycerol (MGDG) has been synthesized. In this molecule, glycerol is replaced with (S)-1,2,4-trihydroxybutane. Instead of the fatty acyl moieties found in MGDG, a 2,2-dialkyl-1,3-dioxolane function provides the hydrophobic moiety of the molecule. This different functionality affords chemically and physically tunable new properties in a glycolipid. These include base stability, increased mobility of the headgroup, possibilities of new packing arrangements, and the potential for use in encapsulation strategies using liposomes where a decrease in pH is used as the environmental cue for release. The acetal linkage also makes the molecule unsusceptible to degradation by phospholipase A and other esterase activities found in biological systems thus further extending their utility. The choice of the butane triol linker removes the common problem of racemization of protected glycerol by acetal and ester migration. It also affords synthetic simplicity since only one dioxolane acetal is formed on reaction of butane-1,2,4-triol with ketones, whereas in the case of glycerol, one hydroxyl group has to be selectively protected to avoid the formation of both enantiomers. 2-D NMR homonuclear and heteronuclear correlation spectroscopy together with nuclear Overhauser effect experiments and molecular mechanics calculation were used to obtain information on the headgroup orientation and on the configuration of the trialkoxybutane backbone. These supported a structure in which the alkyl chains were extended in a parallel fashion and the headgroup, although free to rotate along C2−C3 and C3−C4 of the trialkoxybutane substructure, extended away in the other direction in a manner similar to that observed in the case of MGDG. X-ray powder diffraction and optical microscopy data both supported a lamellar phase behavior of this amphiphilic molecule in water. 1H NMR experiments monitoring the rate of acetal cleavage of this amphiphile revealed its tunable acid susceptibility. The unique structural feature, phase behavior, and controllable acid susceptibility of this glycolipid is potentially useful in many applications.

NMR spectroscopy in structure-based drug design

NMR methods for the study of motion in proteins continue to improve, and a number of studies of protein–ligand complexes relevant to drug design have been reported over the past year, for example, studies of fatty-acid-binding protein and SH2 and SH3 domains. These studies have begun to give a picture of the structural dynamics of protein–ligand complexes and to relate the changes in dynamics on ligand binding to the origins of specificity. NMR is also valuable in locating binding sites, both qualitatively from changes in chemical shift and more precisely from distances measured from relaxation effects. The conformation of the bound ligand can provide useful information for drug design, and over the past year improvements in methods have made it easier to obtain quantitative information from transferred nuclear Overhauser effect experiments.

A molecular dynamics study of the 41-56 beta-hairpin from B1 domain of protein G.

The structural and dynamical behavior of the 41-56 beta-hairpin from the protein G B1 domain (GB1) has been studied at different temperatures using molecular dynamics (MD) simulations in an aqueous environment. The purpose of these simulations is to establish the stability of this hairpin in view of its possible role as a nucleation site for protein folding. The conformation of the peptide in the crystallographic structure of the protein GB1 (native conformation) was lost in all simulations. The new equilibrium conformations are stable for several nanoseconds at 300K (>10 ns), 350 K (>6.5 ns), and even at 450 K (up to 2.5 ns). The new structures have very similar hairpin-like conformations with properties in agreement with available experimental nuclear Overhauser effect (NOE) data. The stability of the structure in the hydrophobic core region during the simulations is consistent with the experimental data and provides further evidence for the role played by hydrophobic interactions in hairpin structures. Essential dynamics analysis shows that the dynamics of the peptide at different temperatures spans basically the same essential subspace. The main equilibrium motions in this subspace involve large fluctuations of the residues in the turn and ends regions. Of the six interchain hydrogen bonds, the inner four remain stable during the simulations. The space spanned by the first two eigenvectors, as sampled at 450 K, includes almost all of the 47 different hairpin structures found in the database. Finally, analysis of the hydration of the 300 K average conformations shows that the hydration sites observed in the native conformation are still well hydrated in the equilibrium MD ensemble.

Conformational analysis of josamycin, a 16-membered macrolide free in solution and bound to bacterial ribosomes

The potent 14-membered macrolide antibiotics displayed a strong NMR response in the TRNOESY experiments whereas their metabolites which do not retain antimicrobial activity gave essentially blank TRNOESY spectra. These TRNOESY experiments are here extended to a macrolide (josamycin) belonging to the 16-atom macrolide class in order to compare responses according to the macrocycle size. Analysis of transferred nuclear Overhauser effect (TRNOE) experiments resulted in a set of constraints for all proton pairs. These constraints were used in structure determination procedures based on molecular modelling to obtain a bound structure compatible with the experimental NMR data. This study allowed us to identify the binding structure at the ribosome for the active 16-membered macrolide. The different conformations existing in solution for this antibiotic in the free state will be compared and correlated with the binding conformation at the ribosome obtained with TRNOE experiments so as to establish a structure–activity relationship. The comparative results indicate that one conformation (called S3) pre-existing in the conformational equilibrium of macrocycle in solution and close to the major one S5, is preferred in the bound state.

Preferred Conformation of C-Lactose at the Free and Peanut Lectin Bound States

A conformational analysis of methyl -C-lactoside (5) is carried out (Table 1), which suggests the preferred solution conformation of 5 to be described as a mixture of conformers A ( = 300; = 60) and B ( = 300; = 300) to a first approximation. Applying a modified Karplus equation for the vicinal coupling constants observed for the H.a and H.1' protons of 5, the dihedral angle ( = O.5'-C.1'-C.a-C.4) was deduced to be approximately +280. Nuclear Overhauser effect (NOE) experiments were then conducted on 5-D2 and its parent methyl -O-lactoside in a 7:3 mixture of pyridine-d5 and methanol-d4, qualitatively demonstrating that the conformational characteristics of both methyl -C- and -O-lactosides at the free states are represented as a mixture of two conformers A and B, but their relative population may be different. Through X-ray analysis, it has been definitively established that the conformation ( = 297(7) and = 120(2)) of C-lactose bound to peanut lectin is practically identical to the conformation ( = 291(6) and = 118(9)) of its parent O-lactose bound to the same protein.

Stereospecific 1H and 13C NMR Assignments of Crotonyl CoA and Hexadienoyl CoA: Conformational Analysis and Comparison with Protein−CoA Complexes

Stereospecific 1H and 13C resonance assignments of hexadienoyl-CoA and crotonyl-CoA have been determined. The two diastereotopic methyl groups at the C2‘‘ carbon were assigned via transferred nuclear Overhauser effect experiments on the complex of hexadienoyl-CoA with the enzyme enoyl-CoA hydratase. The two diastereotopic 1‘‘ protons were assigned using heteronuclear multiple-bond correlation experiments in conjunction with rotating frame nuclear Overhauser spectroscopy. This represents the first set of complete stereospecific assignments for any CoA derivative. The assignments allow a detailed quantitative conformational analysis of the uncomplexed form of the molecule. In particular, the relative population of the various rotamers about the C1‘‘−C2‘‘ and the C2‘‘−C3‘‘ bonds have been determined. The database of protein-bound CoA structures has been surveyed and used to compare the structure(s) of CoA in protein−CoA complexes with the conformational preferences of free CoA.

Molecular dynamic simulation and docking energy to forecast the stability of βCyD-complexes in water solution

Establishment of molecular modelling able to simulate the behaviour of the βCyD-complexes in aqueous solution was studied by applying the fundamentals of classic mechanics. The existence of stable complexes was experimentally determined by the findings of 1H NMR NOE (Nuclear Overhauser Effect) experiments in aqueous solution. A `training set' of molecules was chosen to test the ability of the mathematic models to describe the behaviour of βCyD-complexes. A `test set' group of molecules was defined to better confirm the validity of the proposed models. Based on the results, molecular modelling seems to be a useful tool for forecasting the behaviour of βCyD-complexes in water solution since the results of the mathematic models are close to the experimental findings obtained from NOE experiments.

Solution structures of the fibronectin-like Leishmania gp63 SRYD-containing sequence in the free and antibody-bound states--transferred NOE and molecular dynamics studies.

The anti-SRYD monoclonal antibody (mAbSRYD) raised against the IASRYDQL synthetic octapeptide, the 250-257 sequence of the Leishmania major surface glycoprotein gp63 recognizes both SRYD-containing peptides and the whole cognate major surface protein on intact parasites. Two SRYD-containing peptides, which antigenically and functionally mimic the RGDS sequence of fibronectin and efficiently inhibit parasite attachment to the macrophage receptors, were studied by two-dimensional transferred nuclear Overhauser effect experiments in the presence of mAbSRYD. The antibody-bound IASRYDQL octapeptide solution conformation was determined on the basis of 55 interproton-distance restraints, derived from NMR measurements. Eighteen structures which were first generated using an approach combining distance geometry and molecular dynamics, converge by energy minimization toward a folded structure with an average rmsd from the experimental data of less than 0.05 nm for the overall backbone and 0.025 nm for the SRYD motif. A distorted gamma-turn was found, stabilized by the backbone-backbone D255-NH to R253-CO hydrogen bond, while the R253 and D255 side chains are pointing in opposite directions. This latter antibody-bound structure is compared with that of the free octapeptide in dimethylsulfoxide solution, and with the crystal structure of the RYD fragment in OPG2 Fab, an antireceptor antibody that mimics the RGD cell adhesion site. On this basis, a mechanism for IASRYDQL-receptor interaction is discussed.

Transferred nuclear Overhauser effect study of macrolide–ribosome interactions: correlation between antibiotic activities and bound conformations

The study of macrolide–ribosome interactions has been investigated using two-dimensional transferred nuclear Overhauser effect spectroscopy (TRNOESY). A new medically important macrolide antibiotic, roxithromycin, with the replacement of the 9-keto group in erythromycin by a 9-oxime chain, was studied in the complex state with the bacterial ribosome. Analysis of transferred nuclear Overhauser effect (TRNOE) experiment resulted in a set of constraints for all protons pairs. These constraints were used in structure determination procedures based on molecular modelling to obtain a bound structure compatible with the experimental NMR data. The results compared with the conformational analysis of the substrate in solution indicate that only one specific conformation is preferred in the bound state while in the free state the sugar ring moities were relatively disordered. The bioactive macrolide antibiotics studied roxithromycin and erythromycin which displayed a strong NMR response, are metabolized in RU39001 and erythralosamine respectively which do not retain antimicrobial activity. The inactive major metabolites were used to define if TRNOEs observation may be characteristic of a biological activity. These control experiments gave essentially blank TRNOESY spectra. This study shows that Mg 2+ does not play a direct role for the low affinity binding site studied by TRNOE what is in agreement with an hypothesis of two distinct binding levels, with a low affinity binding level necessary for the tight binding one.

Constituents of Domestic Citrus Plants. PartXXXII. Four New Acridone-Coumarin Dimers from a Citrus Plant.

Four new acridone-coumarin dimers, neoacrimarines-H (1), -I (2), -J (3) and -K (4), were isolated from the roots of Marsh grapefruit (Citrus paradisi MACF.). The structures of these new compounds were elucidated by spectral analysis especially using heteronuclear multiple-bond correlation (HMBC) and nuclear Overhauser effect experiments.

NMR Verification of Helical Conformations of Phycocyanobilin in Organic Solvents

AbstractSelective NMR decoupling and nuclear Overhauser effect (NOE) experiments with phycocyanobilin (PCB) show proton‐proton interactions between the terminal rings A and D, viz. the chiral C(2) methine center and the ethyl substituent at C(18), as a result of the helical conformation of this open‐chain tetrapyrrole in solution. Quantitative NOE measurements and a combination of force‐field and semiempirical calculations (FSC) afford inter‐proton distances across the helical gap of 4.2–4.6 (NOE) and 3.2–4.2 A° (FSC). The NOE and FSC, in conjuction with a qualitative evaluation of the steric interactions in the two optimized helices, suggest furthermore that, in solution, the helix M is somewhat more stable than P. The coexistence of at least two diastereoisomers is corroborated also by the circular dichroism (CD) spectra of PCB in MeOH/EtOH which point to a temperature‐dependent equilibrium in solution, and by a considerable increase of this CD upon changing the solvent from the achiral alcohols to ethyl (−)‐(S)‐lactate which reflects a selective solvent‐induced CD differentiating between diastereoisomers.

A simple method for the assignment of the relative stereochemistry of 2-substituted clavams

Bentley and Hunt’s empirical rule for the determination of the anti/syn relationship of protons attached to C-2 and C-5 atoms in 2-substituted clavams does not predict the correct stereochemistry in a series of 2-substituted clavams synthesised by us. In this paper we propose a simple method for determination of the relative stereochemistry of 2-substituted clavams based on nuclear Overhauser effect (NOE) experiments and assignment of the diastereotopicity of protons attached to the C-3 atom. Circular dichroism (CD) measurements and X-ray crystallography provide stereochemical and spectral assignments.

Structural, Spectroscopic, and Theoretical Study of 1-Acetylvinyl p-Nitrobenzoate, a Highly Reactive and Selective Captodative Olefin in Cycloaddition Reactions

X-ray crystallography, variable temperature 1H NMR and nuclear Overhauser effect experiments, and ab initio calculations provided evidence of the s-trans preferential conformation of captodative olefin 1-acetylvinyl p-nitrobenzoate (1a) in solid, solution, and gas phases. The reactivity of 1a in cycloaddition reactions was rationalized under the basis of experimental ionization energy and electron affinity parameters. Correlation of the latter with the frontier molecular orbitals, calculated at the ab initio 3-21G and 6-31G* levels, suggests a nonsignificant effect of the electron-donor group upon control of the reactivity and regioselectivity of these olefins in Diels−Alder reactions.

Ligand preferences of kringle 2 and homologous domains of human plasminogen: canvassing weak, intermediate, and high-affinity binding sites by 1H-NMR.

The interaction of various small aliphatic and aromatic ionic ligands with the human plasminogen (HPg) recombinant kringle 2 (r-K2) domain has been investigated by 1H-NMR spectroscopy at 500 MHz. The results are compared against ligand-binding properties of the homologous, lysine-binding HPg kringle 1 (K1), kringle 4 (K4), and kringle 5 (K5). The investigated ligands include the omega-aminocarboxylic acids 4-aminobutyric acid (4-ABA), 5-aminopentanoic acid (5-APA), 6-aminohexanoic acid (6-AHA), 7-aminoheptanoic acid (7-AHA), lysine and arginine derivatives with free and blocked alpha-amino and/or carboxylate groups, and a number of cyclic analogs, zwitterions of similar size such as trans-(aminomethyl)cyclohexanecarboxylic acid (AMCHA) and p-benzylaminesulfonic acid (BASA), and the nonzwitterions benzylamine and benzamidine. Equilibrium association constant (Ka) values were determined from 1H-NMR ligand titration profiles. Among the aliphatic linear ligands, 5-APA (Ka approximately 3.4 mM-1) shows the strongest interaction with r-K2 followed by 6-AHA (Ka approximately 2.3 mM-1), 7-AHA (Ka approximately 0.45 mM-1), and 4-ABA (Ka approximately 0.22 mM-1). In contrast, r-K1, K4, and K5 exhibit a preference for 6-AHA (Ka approximately 74.2, 21.0, and 10.6 mM-1, respectively), a ligand approximately 1.14 A longer than 5-APA. Mutations R220G and E221D increase the affinity of r-K2 for these ligands but leave the selectivity profile essentially unaffected: 5-APA > 6-AHA > 7-AHA > 4-ABA (Ka approximately 6.5, 3.9, 1.8, and 0.74 mM-1, respectively). We find that, while r-K2 definitely interacts with Nalpha-acetyl-L-lysine and L-lysine (Ka approximately 0.96 and 0.68 mM-1, respectively), the affinity for analogs carrying a blocked carboxylate group is relatively weak (Ka approximately 0.1 mM-1). We also investigated the interaction of r-K2 with L-arginine (Ka approximately 0.31 mM-1) and its derivatives Nalpha-acetyl-L-arginine (Ka approximately 0.55 mM-1), Nalpha-acetyl-L-arginine methyl ester (Ka approximately 0.07 mM-1), and L-arginine methyl ester (Ka approximately 0.03 mM-1). Zwitterionic gamma-guanidinobutyric acid, containing one less methylene group than arginine, exhibits a Ka of approximately 0.28 mM-1. The affinity of r-K2 for lysine and arginine derivatives suggests that K2 could play a role in intermolecular as well as intramolecular interactions of HPg. As is the case for the HPg K1, K4, and K5, among the tested ligands, AMCHA is the one which interacts most firmly with r-K2 (Ka approximately 7.3 mM-1) while the aromatic ligands BASA, benzylamine, and benzamidine exhibit Ka values of approximately 4.0, approximately 0.04, and approximately 0.03 mM-1, respectively. The relative stability of these interactions indicates a strict requirement for both cationic and anionic polar groups in the ligand, whereas the presence of a lipophilic aromatic group seems to be of lesser consequence. Ligand-induced shifts of r-K2 (1)H-NMR signals and two-dimensional nuclear Overhauser effect (NOESY) experiments in the presence of 6-AHA reveal direct involvement of residues Tyr36, Trp62, Phe64, and Trp72 (kringle residue numbering convention) in ligand binding. Starting from the X-ray crystallographic structure of HPg K4 and the intermolecular 1H-NMR NOE data, two models of the K2 lysine binding site complexed to 6-AHA have been derived which differ mainly in the extent of electrostatic pairing between the K2 Arg56 and Glu57 side chains. Competition between these two conformations in equilibrium may account for the relatively lesser affinity of the K2 domain for zwitterionic lysine-type ligands.

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.

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. Proteins 28:268–284, 1997 © 1997 Wiley-Liss Inc.

Polymyxin B nonapeptide: Conformations in water and in the lipopolysaccharide-bound state determined by two-dimensional NMR and molecular dynamics

Polymyxin B nonapeptide (PMN) is a derivative of polymyxin B, an alpha, gamma-diaminobutyric acid-rich decapeptide from Bacillus polymyxa that displays antimicrobial and lipopolysaccharide (LPS)-antagonistic activities. The conformations of PMN in aqueous solution as well as in the LPS-bound state have been studied by 1H two-dimensional nmr methods in conjunction with molecular dynamics techniques. The aqueous structure of the free peptide is characterized by a type II beta-turn centered around D-Phe5 and Leu6, and an inverse -turn at Thr9. The LPS-bound conformation of PMN was studied by transferred nuclear Overhauser effect experiments. The essential features of the cyclic portion of free aqueous PMN are mostly preserved when the peptide is bound to LPS; however, the linear dipeptide fragment as well as the side chains of the heptatpeptide ring show a conformational change and a reduction in mobility. The LPS-bound PMN structure was used to construct a model of the lipid A-polymyxin B (decapeptide) complex, which allows the rationalization of several experimental observations concerning the binding of polymyxin to, and consequent neutralization of, the toxicity of LPS, and may be of value in the rational therapeutic targeting of endotoxin.