Transferred Nuclear Overhauser Effect Research Articles

Dynamic NMR studies of ligand-receptor interactions: design and analysis of a rapidly exchanging complex of FKBP-12/FK506 with a 24 kDa calcineurin fragment.

Dynamic NMR methods, such as differential line broadening and transferred NOE spectroscopy, are normally reserved for the study of small molecule ligand interactions with large protein receptors. Using a combination of isotope labeling and isotope edited NMR, we have extended these techniques to characterize interactions of a much larger protein/drug complex, FKBP-12/ FK506 with its receptor protein, calcineurin. In order to examine this multicomponent system by dynamic NMR methods, the 93 kDa, tightly bound FKBP-12/FK506/Cn complex was replaced with a lower affinity, rapidly exchanging system consisting of FKBP-12/FK506 (13 kDa), recombinant calcineurin subunit B (CnB) (20 kDa), and a synthetic peptide (4 kDa) corresponding to the B binding domain (BBD) of calcineurin catalytic subunit A (CnA). Analysis of 1H-13C HSQC data acquired for the FKBP-12/ 13C-FK506 and FKBP-12/13C-FK506/CnB/BBD complexes indicates that FKBP-12/FK506 and CnB/BBD are in fast exchange in the quaternary complex. Comparison of proton line widths shows significant broadening of resonances along the macrocycle backbone at 13-CH, 13-OMe, 15-OMe, 18-CH2, 20-CH, 21-CH, and 25-Me, as well as moderate broadening on the macrocycle backbone at 17-Me, 24-CH, and the pyranose 12-CH2 protons. The tri-substituted olefin and cyclohexyl groups also show moderate broadening at the 27-Me, 28-CH, and 30-CH2 positions, respectively. Unexpectedly, little line broadening was observed for the allyl resonances of FK506 in the quaternary complex, although 13C longitudinal relaxation measurements suggest this group also makes contacts with calcineurin. In addition, intermolecular transfer NOE peaks were observed for the allyl 37-CH2, 21-CH, 30-CH2, 13-OMe, 15-OMe, 17-Me, 25-Me, and 27-Me groups, indicating that these are potential sites on the FK506 molecule that interact with calcineurin.

Conformation of surface exposed N-terminus part of bacteriorhodopsin studied by transferred NOE technique

Interaction of the monoclonal antibody A5 raised against native bacteriorhodopsin (BR) with the synthetic peptide pGlu 1-Ala-Gln-Ile-Thr-Gly-Arg 7-NH 2, corresponding to the amino acid sequence 1–7 was studied by transferred nuclear Overhauser effect (TRNOE) spectroscopy. The denaturing reagents and the specially designed pulse sequences which eliminate broad signals from the TRNOE spectra were used to favour evaluation of the TRNOE peaks. On the basis of the data obtained, the conformation of peptide bound with A5 was calculated. A model of the mutual arrangement of bacteriorhodopsin N-terminus and the first transmembrane α-helical segment 8–32 was proposed.

Kinetic, Binding, and NMR Studies of Perdeuterated Yeast Phosphoglycerate Kinase and Its Interactions with Substrates

Perdeuterated yeast phosphoglycerate kinase (2HPGK) was prepared from yeast cells grown in 99.9% 2H2O and an acid hydrolysate from deuterated algal cells. Kinetic and binding studies suggested that perdeuterated enzyme was similar to the isotopically normal PGK. The use of 2HPGK not only eliminated the spectral overlap between the enzyme and substrate nuclear Overhauser effect (NOE) cross-peaks, but also permitted observation of weak transfer NOE cross-peaks between the substrate protons that are greater than 4 Å apart. Intensity of NOE cross-peaks was used to determine the interproton distances of enzyme bound Mg(II)dATP. These distances were then used in model building studies to determine the conformation of Mg(II)dATP. The average conformation of enzyme bound dATP is anti with O4′/C2′ endo ribose pucker and trans, gauche about the C4′-C5′ bond. Although many spin diffusion pathways were eliminated by protein deuteration, spin diffusion was still observed between the protons of the substrate at mixing times longer than 25 ms.

Restricted Rotation and NOE Transfer: A Conformational Study of Some Substituted (9-Anthryl)carbinol Derivatives

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTRestricted Rotation and NOE Transfer: A Conformational Study of Some Substituted (9-Anthryl)carbinol DerivativesInnocenzo de Riggi, Albert Virgili, Maria de Moragas, and Carlos JaimeCite this: J. Org. Chem. 1995, 60, 1, 27–31Publication Date (Print):January 1, 1995Publication History Published online1 May 2002Published inissue 1 January 1995https://doi.org/10.1021/jo00106a010RIGHTS & PERMISSIONSArticle Views220Altmetric-Citations34LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (627 KB) Get e-AlertsSupporting Info (2)»Supporting Information Supporting Information Get e-Alerts

Solution conformation of a cyclophilin-bound proline isomerase substrate.

Cyclophilin (CyP) is the 17.8-kDa cytosolic receptor of the immunosuppressant cyclosporin A (CsA) and also a peptidyl prolyl cis-trans isomerase (PPIase). In order to gain insights into the PPIase mechanism, transferred nuclear Overhauser effect (TRNOE) measurements by two-dimensional 1H NMR were used to determine the conformation of the isomerase-bound standard model substrate suc-AAPF-pNA. Results indicate a cis-like conformation for the CyP-bound substrate with the A-P peptide bond being no more than 40 degrees out of planarity.

Comparison of the membrane-bound states of two structurally similar delta-selective opioid peptides by transferred nuclear Overhauser effect spectroscopy and molecular modeling.

NMR spectroscopic, peptide-membrane conformational studies on [D-Pen2,D-Pen5]-enkephalin (DPDPE), an opioid receptor selective peptide, and an acyclic analog of DPDPE (DPDPE reduced at the disulfide bond) were conducted. The NMR method of transferred nuclear Overhauser effect (TRNOE) was used to obtain NOE profiles of the free and membrane bound forms of DPDPE and acyclic DPDPE. After comparison of the profiles of both peptides in the free and membrane-bound states, we hypothesize that the cyclic DPDPE undergoes little if any conformational change upon interaction with the membrane. However, for the acyclic analog, large changes in the NOE profile associated with backbone and side-chain groups were observed after interaction with the membrane. Results of computerized molecular modeling studies also were consistent with our theory that the free and membrane-bound forms of cyclic DPDPE have very similar free and membrane-bound states. The free acyclic DPDPE has a reverse turn conformation with sidechains situated so that hydrophobic surface exposure to aqueous solution is minimized. After membrane interaction, the acyclic DPDPE has an extended conformation near the carboxy terminus with aromatic sidechains widely separated. We propose that the interaction of the acyclic DPDPE with the membrane surface is mediated by the amino terminus. We further propose that the interaction of the cyclic DPDPE with the membrane surface is limited because the D-Pen2 side chain is covalently bonded and the aromatic side chains and backbone are only slightly altered after membrane contact. Permeability studies by Ramaswami et al. [(1992) Biochim. Biophys. Acta 1109(2), 195-202] demonstrated that the acyclic DPDPE permeated through membranes at a rate 4 times greater than cyclic DPDPE. We conclude that conformational and topographical flexibility may be critical factors in peptide-membrane interactions and permeability of bilayer membranes to opioid peptides.

Transfer nuclear Overhauser effect study of the conformation of oxytocin bound to bovine neurophysin I.

This study reports the structure of the peptide hormone oxytocin bound to its carrier protein, neurophysin I, obtained by nuclear magnetic resonance techniques. At the pH value of 2.1 in our experiments, the ligand is in fast exchange with its carrier protein, allowing the use of transfer-NOE methods. The number of distance constraints for the peptide being limited, considerable attention has been paid to an accurate distance determination. The resulting accurate distance limits were used as input for a distance geometry calculation followed by a restrained molecular dynamics run. Convergence to a well-defined family of structures for oxytocin in its bound state was reached. Both the backbone and the side-chain conformations differ between the bound form and the crystal structure of free oxytocin [Wood, S. P., et al. (1986) Science 232, 633]. These differences, as well as other structural features of the bound form, are discussed in terms of interactions made with the carrier protein. Transfer-NOE experiments at low peptide protein ratios provide direct experimental evidence for contacts between the oxytocin Tyr2 residue and an aromatic residue of neurophysin. The resonance assignments of the aromatic groups [Whittaker, B. A., et al. (1985) Biochemistry 24, 2782] together with the recently published X-ray structure of the neurophysin II protein complexed with a dipeptide [Chen et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4240] allow us to assign the aromatic signal on the protein to the neurophysin Phe22 residue.

A T1 rho-filtered two-dimensional transferred NOE spectrum for studying antibody interactions with peptide antigens

Transferred nuclear Overhauser effect (TRNOE) spectroscopy can be used to study intra- and intermolecular interactions of bound ligands complexed with large proteins. However, the 2D NOE (NOESY) spectra of large proteins are very poorly resolved and it is very difficult to discriminate the TRNOE cross peaks, especially those due to intermolecular interactions, from the numerous cross peaks due to intramolecular interactions in the protein. In previous studies we measured two-dimensional difference spectra that show exclusively TRNOE and exchange cross-peaks (Anglister, J., 1990. Quart. Rev. Biophys. 23:175-203). Here we show that a filtering method based on the difference between the T1rho values of the ligand and the protein protons can be used to directly obtain a two-dimensional transferred NOE spectrum in which the background cross-peaks due to intramolecular interactions in the protein are very effectively removed. The usefulness of this technique to study protein ligand interactions is demonstrated for two different antibodies complexed with a peptide of cholera toxin (CTP3). It is shown that the T1 rho-filtering alleviates t problems encountered in our previous measurements of TRNOE by the difference method. These problems were due to imperfections in the subtraction of two spectra measured for two different samples.

Conformation of beta-methylmelibiose bound to the ricin B-chain as determined from transferred nuclear Overhauser effects.

Transferred nuclear Overhauser effect (TRNOE) experiments have revealed a change in the torsion angles about the alpha-1-6 glycosidic bond of methyl beta-melibioside upon binding of the melibioside to the ricin B-chain (Rb). A full relaxation rate matrix simulation of experimental buildup curves aided in quantitative interpretation of 1D selective inversion recovery TRNOE experiments. The data are consistent with a model in which both major (omega approximately 170 degrees) and minor (omega approximately -60 degrees) conformers for methyl beta-melibioside are significantly populated in solution while the Rb/methyl beta-melibioside complex has little of the minor conformer populated. The results indicate that the ricin B-chain excludes binding of certain ligand conformations on the basis of unfavorable interactions between the protein surface and remote portions of the disaccharide system.

Theory and experimental results of transfer NOE experiments. II. The influence of residual mobility and relaxation centers inside the protein on the size of transfer NOEs

Experimental evidence is presented for a strong dependence of transfer NOE values on the Larmor frequency for certain residues of a peptide in its complex with a protein. This dependency has been explained by residual mobility in these residues of the peptide when bound. As a consequence, effective correlation times exist for some proton-proton pairs that are at least one order of magnitude smaller than expected for a rigid complex. This is shown to be the main reason for the failure to observe certain transfer NOEs in such a complex when studied at proton frequencies of 400 MHz or less. Another factor that can reduce the size of transfer NOES is the possibility of leakage of magnetization toward relaxation centers inside the protein. We have observed several intermolecular NOE cross peaks demonstrating such an intermolecular exchange of magnetization. The influence of cross relaxation to relaxation centers in the protein on the buildup and magnitude of proton transfer NOES for the bound and the free ligand is described in a model of the ligand-protein system that consists of a set of Bloch equations in which chemical exchange, transfer of magnetization, and relaxation sinks in the protein have been included. Simulations have been done for the cases of fast and intermediate exchange on the spin-lattice relaxation time scale while in all cases bound and free ligand signals were assumed to be in fast exchange on the chemical-shift scale. It is found that the magnitude of intramolecular transfer NOEs is reduced by the presence of cross relaxation from the peptide into the protein, in particular when the protein has relaxation centers such as methyl groups and aromatic rings. It is shown that these effects can reduce the size of transfer NOE effects by an order of magnitude for those ligand protons that are directly in contact with the protein. The simulations for ligand protein concentration ratios of 2, 10, and 100 with various numbers of relaxation sinks in the protein show that concentration ratios and mixing times must be optimized in order to observe sizeable transfer NOES in 2D NMR experiments. The influence of these effects on the accuracy of distances determined via transfer NOEs is also discussed.

Conformation of 6,7-dimethyl-8-ribityllumazine bound to β-subunits of heavy riboflavin synthase: Transferred nuclear overhauser effect (TrNOE) studies employing ω1-13C-filtered NOESY including a novel technique for zero quantum suppression

The β-subunits of heavy riboflavin synthase catalyze the formation of 6,7-dimethyl-8-ribityllumazine. The interaction of the13C-labelled enzyme product with isolated β-subunits was studied by transfer NOE measurements using ω1=13C-filtered NOESY and13C-1H-relayed NOESY. The advantages of these techniques are the removal of residual enzyme signals, the simplification of zero quantum suppression, and the improvement of water suppression which enabled the semiquantitative study of1H-1H distances of the ligand in the bound state. The preferred conformation of the ribityl side chain was calculated on the basis of the measured distances.

EPSP synthase inhibitor design I. Conformations of enzyme bound shikimate-3-phosphate and 5-enolpyruvoylshikimate-3-phosphate using TRNOE

The conformations of S3P, 1 and EPSP, 2 bound to E. Coli EPSP synthase have been determined using two dimensional transfer NOE (2D TRNOE) measurements. Both S3P and EPSP bound to the enzyme retain the half-chair conformation with the phosphate group in an axial position similar to “free” S3P in solution. 4 This cis enol proton of bound EPSP is in close proximity to H 4 on the ring. The conformations of S3P, 1 and EPSP, 2 bound to E. Coli EPSP synthase have been determined using 2d transfer NOE experiments.

NMR Study of the Dynamic Stereochemistry of Substituted Bicyclo[4.2.2]decanes and Bicyclo[5.2.2]undecane

AbstractVarious NMR methods have been applied to investigate the ring‐reversal process in the title compounds. In order to measure barriers an exact assignment of proton‐exchange connectivities was required. This assignment was made using phase‐sensitive NOESY and difference NOE and saturation transfer spectroscopies. The barriers for ring reversal were found to differ substantially between compounds 1–3 on one hand (9–10.5 kcal mol−1), and compound 4 on the other (20.2 kcal mol−1). This was rationalized in terms of the different geometries of the ring systems.

Statistical analysis of double NOE transfer pathways in proteins as measured in 3D NOE-NOE spectroscopy

The recent development of three-dimensional NMR spectroscopy has alleviated the problem of overlap of resonances. However, also for the 3D experiments resonance assignment strategies have usually relied upon knowledge about spin systems, combined with information about short (sequential) distances. For doubly (15N/13C)-labelled molecules, a novel assignment strategy has been developed. In this paper we address the possibilities of an assignment strategy for proteins, based solely upon the use of NOE data. For this, the 3D NOE-NOE experiment seems most suitable. Therefore, we have made a theoretical evaluation of double NOE transfer pathways in 28 protein crystal structures. We identify 95 connectivities which are most likely to be observed as cross peaks in a 3D NOE-NOE spectrum of a protein. Given the occurrence of one of these 95 connectivities, we evaluate the chances of occurrence for the others. Analysis of these conditional probabilities allowed the construction of five patterns of related, highly correlated cross peaks which resemble the conventional idea of spin systems to some extent and may provide a basis for assignment and secondary structure analysis from 3D NOE-NOE data alone.

Theoretical evaluation of the two-dimensional transferred nuclear overhauser effect

A theoretical evaluation of the two-dimensional intramolecular transferred nuclear Overhauser effect (TRNOE) is presented using a full relaxation matrix approach. The application of this analysis to the study of 6-24 residue peptides binding to proteins ranging in size from 18,000 to 80,000 Da is considered. The effects of variables such as mixing time, fraction of bound peptide, free and bound correlation times, and other contributions to spin-lattice relaxation rates are examined in an attempt to offer practical experimental guidelines for the design of a TRNOE experiment.

A diffusion equation approach to spin diffusion in biomolecules

A theoretical description of 1H- 1H dipolar nuclear spin relaxation in a multispin system has been worked out by forming a diffusion equation for a one-dimensional chain of equidistant spins. The spin-diffusion equation is formed from first principles by assuming nearest neighbor interactions for a molecule undergoing isotropic random reorientation. This equation describes diffusion only in the long correlation limit (for ( ωτ c > 1.118) and is solved for driven NOE experiments, for spins in a chain of infinite length (0 < × < ∞), or for spins in a chain of finite length (0 < × < L). The solutions are obtained using the method of the Laplace transform for specified initial and boundary conditions. The observed selectivity of the NOE transfer in driven NOE experiments on a biomolecule which has a correlation factor ωτ c ∼ 3 is indeed in conformity with the predictions obtained from the spin-diffusion equation.

Conformation of NAD+ Bound to Allosteric L-Lactate Dehydrogenase Activated by Chemical Modification

On modification of arginine residues with 2,3-butanedione, the Thermus caldophilus L-lactate dehydrogenase is converted to an activated form that is independent of an allosteric effector, fructose 1,6-bisphosphate (Fru-1,6-P2). The conformation of NAD+ bound to the modified enzyme in the absence of Fru-1,6-P2 was investigated by means of proton NMR, analyzing the time dependence of the transferred nuclear Overhauser effect (TRNOE) and TRNOE action spectra. The inter-proton distances determined on TRNOE analysis indicated that both the nicotinamide riboside moiety and the adenosine moiety of NAD+ were in the anti conformation, the ribose rings being in the C3'-endo form. This conformation was almost the same as that of NAD+ bound to the native enzyme-Fru-1,6-P2 complex, rather than that of NAD+ bound to the free native enzyme. These results suggest that the C3'-endo-anti form of the enzyme-bound NAD+ is essential for the activation of the T. caldophilus L-lactate dehydrogenase.

Relayed-NOE experiments in the rotating frame for sequence analysis of peptides

Proton spectra of peptides consist of a set of spin systems for the individual amino acid residues, which are usually identified via their characteristic J-coupling patterns (I) in 2D correlation spectra. However, the examination of a peptide with unknown amino acid sequence or the presence of several amino acids with identical spin systems requires an assignment of each spin system to its position in the peptide chain. The sequence analysis of a peptide makes use of correlations across the peptide bonds between a-protons and the amide protons of the following amino acid, either via heteronuclear long-range couplings to carbonyl carbons (favorably by COLOC (2) or its inverse variant (3)) or directly via NOE effects (I) (NOESY (4)). However, especially in linear peptides the H, resonances are often not sufficiently resolved for an unambiguous assignment. This serious problem can be circumvented by the use of the Relayed-NOESY experiment (5), advantageously with double-quantum filter (6), where the magnetization transfer via NOE between NH’+’ and HL is followed by a transfer through J coupling from HL to NH’ within the same amino acid residue. This results in a cross peak between NH’ and NH’+’ which appears only on one side of the diagonal due to the asymmetry of the mixing sequence (7) (cf. Fig. 1). Unfortunately, many oligopeptides with molecular weights of about 1000 show only very weak NOE effects in high-field spectrometers due to their unfavorable correlation times. Instead of conventional NOE spectra, ROESY (8, 9) experiments can be used to measure NOE effects in the rotating frame (ROE effects), which are almost independent of the correlation time. We describe here the recently proposed (IO) possibility of using spin-lock sequences in Relayed-NOESY experiments for both the NOE and the Jtransfer part of the mixing time. Figure 2 shows the pulse sequences which are discussed in this paper. The DQF-Relayed-NOESY experiment exists in two versions, which differ in the order of NOE transfer and J trandkr. Since the desired cross peaks always show antiphaae s&uctaue in one dime i$ seems to be favoraMe to have antiphase in w2 because of’tlte bd%cs resduhta in that dimension. This can be achieved by placing ~JVrans~&z&eNOEtrans&ass&owninF~ Z?a.Tbecorrespondingsequence wi~theJ~r~~eNOE~(givingrise~bo~inwl)is~f~ omitted in Fig. 2.

A transfer nuclear Overhauser effect study of coenzyme binding to distinct sites in binary and ternary complexes in glutamate dehydrogenase.

The oxidized coenzyme NAD binds to two sites per subunit of bovine liver glutamate dehydrogenase with equal affinity in the absence of dicarboxylic acid coligands. In the presence of glutarate or 2-oxoglutarate, the affinity to one site is unchanged, but the affinity to the other (presumed to be the active site) is considerably increased and now requires two dissociation constants to describe its saturation. A combination of transfer nuclear Overhauser effects (TRNOE) together with an examination of the slopes of TRNOE time dependence indicates that while NAD is bound in a syn conformation at both binding sites, NADP (which binds only to the active site) is bound in a syn-anti mixture. The existence of N6 to N3' and N6 and N2' and N1' to N3' NOE's with NAD suggests that the two coenzyme binding sites are located near enough to allow intermolecular NOE's. In the presence of 2-oxoglutarate where only binding to the active site is effectively observed, the conformation of either coenzyme is syn. Modeling studies using the distance estimates from the TRNOE results suggest that the nicotinamide ribose approximates a 3'-endo conformation. The absence of evidence for intermolecular NOE's under these conditions indicates that while the active and regulatory NAD sites per subunit are in close proximity, the six active sites per hexamer are located greater than 5 A apart.

NMR analyses of the conformations of L-isoleucine and L-valine bound to Escherichia coli isoleucyl-tRNA synthetase.

The 400-MHz 1H NMR spectra of L-isoleucine and L-valine were measured in the presence of Escherichia coli isoleucyl-tRNA synthetase (IleRS). Because of chemical exchange of L-isoleucine or L-valine between the free state and the IleRS-bound state, a transferred nuclear Overhauser effect (TRNOE) was observed among proton resonances of L-isoleucine or L-valine. However, in the presence of isoleucyl adenylate tightly bound to the amino acid activation site of IleRS, no TRNOE for L-isoleucine or L-valine was observed. This indicates that the observed TRNOE is due to the interaction of L-isoleucine or L-valine with the amino acid activation site of IleRS. The conformations of these amino acids in the amino acid activation site of IleRS were determined by the analyses of time dependences of TRNOEs and TRNOE action spectra. The IleRS-bound L-isoleucine takes the gauche+ form about the C alpha-C beta bond and the trans form about the C beta-C gamma 1 bond. The IleRS-bound L-valine takes the gauche- form about the C alpha-C beta bond. Thus, the conformation of IleRS-bound L-valine is the same as that of IleRS-bound L-isoleucine except for the delta-methyl group. The side chain of L-isoleucine or L-valine lies in an aliphatic hydrophobic pocket of the active site of IleRS. Such hydrophobic interaction with IleRS is more significant for L-isoleucine than for L-valine. The TRNOE analysis is useful for studying the amino acid discrimination mechanism of aminoacyl-tRNA synthetases.