Intermolecular Nuclear Overhauser Effects Research Articles

Chemical Tagging with tert-Butyl and Trimethylsilyl Groups for Measuring Intermolecular Nuclear Overhauser Effects in a Large Protein-Ligand Complex.

Intermolecular 1 H-1 H nuclear Overhauser effects (NOE) present a powerful tool to assess contacts between proteins and binding partners, but are difficult to identify for complexes of high molecular weight. This report shows that intermolecular NOEs can readily be observed following chemical labeling with tert-butyl or trimethylsilyl (TMS) groups. Proteins can be furnished with tert-butyl or TMS groups site-specifically using genetically encoded unnatural amino acids or by chemical modification of single cysteine residues. No isotope labeling is required. The approach is demonstrated with the 95 kDa complex between tetrameric E. coli single-stranded DNA binding protein (SSB) and single-stranded DNA.

Revival of the Intermolecular Nuclear Overhauser Effect for Mapping Local Protein Hydration Dynamics.

The highly heterogeneous hydration dynamics of protein-water interfaces is considered important for protein stability and dynamics, protein folding, enzymatic activity, and even drug design. The nuclear Overhauser effect (NOE) between protein and water protons is the only experimental observable which, in principle, can provide a map of locally resolved hydration dynamics. However, its utility was questioned in various theoretical studies that emphasized the contributions of long-range NOE interactions. We show by a detailed analysis based on molecular dynamics simulations that, contrary to recent claims, the protein-water NOE is an excellent observable to map local hydration dynamics at the protein surface.

Separation of enilconazole enantiomers in capillary electrophoresis with cyclodextrin-type chiral selectors and investigation of structure of selector-selectand complexes by using nuclear magnetic resonance spectroscopy.

In the present study, the enantiomer migration order (EMO) of enilconazole in the presence of various cyclodextrins (CDs) was investigated by capillary electrophoresis (CE). Opposite EMO of enilconazole were observed when β-CD or the sulfated heptakis(2-O-methyl-3,6-di-O-sulfo)-β-CD (HMDS-β-CD) was used as the chiral selectors. Nuclear magnetic resonance (NMR) spectroscopy was used to study the mechanism of chiral recognition between enilconazole enantiomers and those two cyclodextrins. On the basis of rotating frame nuclear Overhauser (ROESY) experiments, the structure of an inclusion complex between enilconazole and β-CD was derived, in which (+)-enilconazole seemed to form a tighter complex than the (-)-enantiomer. This correlates well with the migration order of enilconazole enantiomers observed in CE. No evidence of complexation between enilconazole and HMDS-β-CD could be gathered due to lack of intermolecular nuclear Overhauser effect (NOE). Most likely the interaction between enilconazole and HMDS-β-CD leads to formation of a shallow external complex that is sufficient for separation of enantiomers in CE but cannot be evidenced based on ROESY experiment. Thus, in this particular case CE documents the presence of intermolecular interactions which are at least very difficult to be evidenced by other instrumental techniques.

Detection of intermolecular transferred‐NOE interactions in small and medium size protein complexes: RANTES complexed with a CCR5 N‐terminal peptide

NMR is a powerful tool for studying structural details of protein/peptide complexes exhibiting weak to medium binding (KD > 10 μm). However, it has been assumed that intermolecular nuclear Overhauser effect (NOE) interactions are difficult to observe in such complexes. We demonstrate that intermolecular NOEs can be revealed by combining the 13 C-edited/13 C-filtered experiment with the transferred NOE effect (TRNOE). Due to the TRNOE phenomenon, intermolecular NOE cross peaks are characterized by both the chemical shifts (CSs) of the protein protons and the average CSs of the peptide protons, which are dominated by the CSs of the protons of the free peptide. Previously, the TRNOE phenomenon was used almost exclusively to investigate the conformation of small ligands bound to large biomolecules. Here, we demonstrate that TRNOE can be extended to enable the study of intermolecular interactions in small- and medium-sized protein complexes. We used the 13 C-edited/13 C-filtered TRNOE experiment to study the interactions of the chemokine regulated upon activation, normal T cell, expressed and secreted (RANTES) with a 27-residue peptide, containing two sulfotyrosine residues, representing the N-terminal segment of the CCR5 receptor ((Nt-CCR5(1-27). The TRNOE phenomenon led to more than doubling of the signal-to-noise ratios (SNRs) for the intermolecular NOEs observed in the 13 C-edited/13 C-filtered experiment for the 11.5-kDa monomeric RANTES/Nt-CCR5(1-27) complex. An even better improvement in the SNR was achieved with dimeric Nt-CCR5(1-27)/RANTES (23 kDa), especially in comparison with the spectra measured with a 1 : 1 protein to peptide ratio. In principle, the isotope-edited/isotope-filtered TRNOE spectrum can discern all intermolecular interactions involving nonexchangeable protons in the complex.

Uncovering Key Structural Features of an Enantioselective Peptide-Catalyzed Acylation Utilizing Advanced NMR Techniques.

We report on a detailed NMR spectroscopic study of the catalyst-substrate interaction of a highly enantioselective oligopeptide catalyst that is used for the kinetic resolution of trans-cycloalkane-1,2-diols via monoacylation. The extraordinary selectivity has been rationalized by molecular dynamics as well as density functional theory (DFT) computations. Herein we describe the conformational analysis of the organocatalyst studied by a combination of nuclear Overhauser effect (NOE) and residual dipolar coupling (RDC)-based methods that resulted in an ensemble of four final conformers. To corroborate the proposed mechanism, we also investigated the catalyst in mixtures with both trans-cyclohexane-1,2-diol enantiomers separately, using advanced NMR methods such as T1 relaxation time and diffusion-ordered spectroscopy (DOSY) measurements to probe molecular aggregation. We determined intramolecular distance changes within the catalyst after diol addition from quantitative NOE data. Finally, we developed a pure shift EASY ROESY experiment using PSYCHE homodecoupling to directly observe intermolecular NOE contacts between the trans-1,2-diol and the cyclohexyl moiety of the catalyst hidden by spectral overlap in conventional spectra. All experimental NMR data support the results proposed by earlier computations including the proposed key role of dispersion interaction.

Examination of Trifluoroethanol Interactions with Trp-Cage through MD Simulations and Intermolecular Nuclear Overhauser Effects.

Molecular dynamics simulations of the protein model Trp-cage in 42% 2,2,2-trifluoroethanol (TFE)-water at 318 K have been carried out with the goal of exploring solvent fluorine-peptide hydrogen nuclear spin cross-relaxation. TFE5 and TFE6 models of TFE developed in previous work from this laboratory were used with the TIP5PE model of water. System densities and component translational diffusion coefficients were well predicted by the simulations, as were many of the cross-relaxation parameters (ΣHF) for which experimental values are available. However, the calculated ΣHF values were too small for some hydrogens of the Trp6 indole ring and for amino acid hydrogens near this residue in the native structure. Simulations carried out with unfolded versions of Trp-cage suggest that underestimates of ΣHF are the result of insufficient sampling of conformational motions that expose these hydrogens to interactions with solvent molecules during simulations of the native peptide. Consideration of the relative amounts of TFE and water surrounding the Trp-cage structure indicates that the composition of the solvent mixture at distances beyond ∼1.5 nm from the surface of the peptide is close to the composition of the bulk solvent, but, as observed by others, TFE tends to accumulate preferentially near the surface of the peptide. Both TFE and water molecules make contacts with the peptide surface; water molecules predominate in contacts with the peptide backbone atoms, and TFE molecules generally preferentially interact with side chains. Translational diffusion of solvent molecules appears to slow down near the surface of the peptide.

Site specific polarization transfer from a hyperpolarized ligand of dihydrofolate reductase.

Protein-ligand interaction is often characterized using polarization transfer by the intermolecular nuclear Overhauser effect (NOE). For such NOE experiments, hyperpolarization of nuclear spins presents the opportunity to increase the spin magnetization, which is transferred, by several orders of magnitude. Here, folic acid, a ligand of dihydrofolate reductase (DHFR), was hyperpolarized on (1)H spins using dissolution dynamic nuclear polarization (D-DNP). Mixing hyperpolarized ligand with protein resulted in observable increases in protein (1)H signal predominantly in the methyl group region of the spectra. Using (13)C single quantum selection in a series of one-dimensional spectra, the carbon chemical shift ranges of the corresponding methyl groups can be elucidated. Signals observed in these hyperpolarized spectra could be confirmed using 3D isotope filtered NOESY spectra, although the hyperpolarized spectra were obtained in single scans. By further correlating the signal intensities observed in the D-DNP experiments with the occurrence of short distances in the crystal structure of the protein-ligand complex, the observed methyl proton signals could be matched to the chemical shifts of six amino acids in the active site of DHFR-folic acid binary complex. These data demonstrate that (13)C chemical shift selection of protein resonances, combined with the intrinsic selectivity towards magnetization originating from the initially hyperpolarized spins, can be used for site specific characterization of protein-ligand interactions.

Solution Structure of Molecular Associations Investigated Using NMR for Polysaccharides: Xanthan/Galactomannan Mixtures.

Although the intermolecular nuclear Overhauser effect (NOE) signal was valuable to elucidate molecular association structure, it could not always be observed for associated molecules due to the short spin-spin relaxation time T2 in NMR measurements, especially for high molar mass systems. While almost no study has been reported for high molar mass polymers (>1 × 10(6)), especially for polysaccharide-polysaccharide interactions, NOE signals were observed for the first time between two different types of polysaccharides, xanthan and galactomannan (locust bean gum), forming a synergistic gel, as a direct evidence of intermolecular binding of polysaccharides. The NOE peak was found between pyruvic acid in xanthan and anomeric proton of mannose of galactomannan. This NOE signal was observed only when mixing time >0.5 s, indicating indirect NOEs caused by spin diffusion. Therefore, this NOE could not be used to construct the molecular models. However, it is a direct evidence for the binding between two different types of polysaccharide to elucidate the synergistic gelation. This NOE signal was observed only for low molar mass galactomannans (1.4 × 10(4)). T2 of pyruvate methyl drastically decreased at low temperatures in the presence of synergistic interaction, suggesting that pyruvate group at terminal end of side chain in xanthan plays an essential role in synergistic interaction.

Interactions of the Chemokine CCL5/RANTES with Medium-Sized Chondroitin Sulfate Ligands

Interactions of the chemokine CCL5 (RANTES) with glycosaminoglycans (GAGs) are crucial to the CCL5-mediated inflammation process. However, structural information on interactions between CCL5 and longer GAG fragments is lacking. In this study, the interactions between oligosaccharides derived from chondroitin sulfate and a dimeric variant of CCL5 were investigated using solution nuclear magnetic resonance. The data indicate that, in addition to the BBXB motif in the 40s loop, GAGs also contact residues in the N loop in a manner similar to interactions between chemokine and the receptor N terminus, leading to possible stabilization of the dimer. Using 2,2,6,6-tetramethylpiperidin-1-yl)oxidanyl-tagged hexasaccharides, the binding orientation of the hexasaccharides was shown to be highly dependent on thesulfation pattern of the N-acetyl galactosamine groups. Finally, a model of the CCL5 dimer complexed to chondroitin sulfate hexasaccharides was constructed using paramagnetic relaxation enhancement and intra- and intermolecular nuclear Overhauser effect constraints.

Pair dynamics and the intermolecular nuclear Overhauser effect (NOE) in liquids analysed by simulation and model theories: application to an ionic liquid.

Combining simulation and model theories, this paper analyses the impact of pair dynamics on the intermolecular nuclear Overhauser effect (NOE) in liquids. For the first time, we give a distance resolved NOE. When applied to the ionic liquid 1-ethyl-3-methyl-imidazolium tetrafluoroborate the NOE turns out to be of long-range nature. This behaviour translates to the experimentally measured cross- and longitudinal relaxation rates. We were able to calculate the heteronuclear NOE from simulation data, despite the high computational effort. Model theories are computationally less demanding and cover the complete frequency range of the respective spectral density function, they are usually based on a very simple pair distribution function and the solution of the diffusion equation. In order to model the simulated data sufficiently, these simplifications in structure and dynamics have to be generalised considerably.

Epigallocatechin Gallate Decreases the Micellar Solubility of Cholesterol via Specific Interaction with Phosphatidylcholine

The mechanisms underlying the effect of epigallocatechin gallate (EGCG) on the micellar solubility of cholesterol were examined. EGCG eliminated both cholesterol and phosphatidylcholine (PC) from bile salt micelles in a dose-dependent manner in vitro. When the bile salt micelles contained a phospholipid other than PC, neither cholesterol nor the phospholipid was eliminated following the addition of EGCG. When vesicles comprised of various phospholipids were prepared and, EGCG was added to the vesicles, EGCG effectively and exclusively eliminated only PC. An intermolecular nuclear Overhauser effect (NOE) was observed between PC and EGCG in bile salt micelles with EGCG added, but not between cholesterol and EGCG, by using a NOE-correlated spectroscopy nuclear magnetic resonance method. The results of binding analyses using surface plasmon resonance (SPR) showed that EGCG did not bind to cholesterol. These observations strongly suggest that EGCG decreases the micellar solubility of cholesterol via specific interaction with PC.

Investigation of Ethanol–Peptide and Water–Peptide Interactions through Intermolecular Nuclear Overhauser Effects and Molecular Dynamics Simulations

Molecular dynamics simulations have been used to explore solvent-solute intermolecular nuclear Overhauser effects (NOEs) on NMR (nuclear magnetic resonance) signals of [val5]angiotensin dissolved in 35% ethanol-water (v/v). Consideration of chemical shift, coupling constant and intramolecular NOE data suggest that conformations of the peptide are adequately sampled by simulations of up to 0.6 μs duration. Calculated cross relaxation terms at 0 and 25 °C are compared to experimental values and to terms predicted using a particulate model of the solvent. Many calculated solvent NOEs are in agreement with experimental results; disagreements are particularly striking for hydrogens of the Phe8 residue of the peptide. Calculations show that individual molecules of either solvent component can spend many ns in association with the peptide but dipolar interactions within such a complex account for only a few percent of an observed cross relaxation rate. Most parts of the peptide interact selectively with ethanol. Diffusion of both solvent components is slowed when they are close to the peptide. Solvent-solute cross relaxation terms for acetic acid in the same solvent obtained from simulations agree with experiment. Preferential interactions of solvent molecules with acetic acid are largely absent, as are effects of this solute on solvent diffusion rates.

Solubilization Mechanism and Characterization of the Structural Change of Bacterial Cellulose in Regenerated States through Ionic Liquid Treatment

A statistical approach was used to characterize the heterogeneous structures of bacterial cellulose samples pretreated with four kinds of ionic liquids (ILs). The structural heterogeneity of these samples was measured by Fourier transform infrared spectroscopy as well as solid-state NMR methods such as cross-polarization magic-angle spinning and dipolar-assisted rotational resonance. The obtained data matrices were then evaluated by principal components analysis. The measured 1-D data clearly revealed the modification of crystalline cellulose; in addition, the statistical approach revealed subtle structural changes that occurred upon pretreatment with different kinds of ILs. To investigate whether such regenerated structural changes occurred because of solubilization, we examined the intermolecular nuclear Overhauser effect between cellulose and an IL. Our results clarify how the nucleophilic imidazole is attacked and suggest that the cation of the IL is associated with the collapse of hydrogen bonds in cellulose.

Investigation of Methanol–Peptide Nuclear Overhauser Effects through Molecular Dynamics Simulations

Intermolecular nuclear Overhauser effects (NOEs) produced by interactions of methanol with [val(5)]angiotensin in 25% methanol-water at 0 °C were examined through molecular dynamics (MD) simulations and compared to experimental results. Calculated average (3)J(NHCαH) spin coupling constants, conformation-sensitive chemical shift changes, and intramolecular (1)H-(1)H NOEs indicated that peptide conformations present over the course of simulation trajectories of 100-300 ns are likely similar to those present in the experimental system. Calculated cross-relaxation terms for the methanol-peptide interactions showed the same trends as corresponding experimental data but were about a factor of 3 too large. The lack of agreement between observed and calculated cross-relaxation terms probably has origins in characteristics of the simulations that lead to overestimation of translational diffusion coefficients of the system components. Simulations confirmed the heterogeneity of the methanol-water solvent at the molecular level, with clusters of methanol and water molecules changing their size and composition on a subpicosecond time scale. Most peptide hydrogens are preferentially solvated by interactions with methanol molecules. Simulations suggest that diffusion of water and methanol molecules near the peptide is slowed as these species approach the peptide backbone.

Peptide-Protein Interactions Suggest That Acetylation of Lysines 381 and 382 of p53 Is Important for Positive Coactivator 4-p53 Interaction

The human transcriptional positive coactivator 4 (PC4) activates several p53-dependent genes. It has been demonstrated that this is a consequence of direct interaction with p53. Previously, we have concluded that PC4 interacts mainly with the C-terminal negative regulatory domain of p53 through its DNA binding C-terminal half. NMR chemical shift perturbation studies with peptide fragments indicated that amino acids 380-386 of p53 are crucial for interaction with PC4. This was verified by fluorescence anisotropy and sedimentation velocity studies. A peptide consisting of p53-(380-386) sequence, when attached to a cell penetration tag and nuclear localization signal, localizes to the nucleus and inhibits luciferase gene expression from a transfected plasmid carrying a Luc gene under a p53-dependent promoter. Acetylation of lysine 382/381 enhanced the binding of this peptide to PC4 by about an order of magnitude. NMR and mutagenesis studies indicated that serine 73 of PC4 is an important residue for recognition of p53. Intermolecular nuclear Overhauser effect placed aspartate 76 in the vicinity of lysine 381, indicating that the region around residues 73-76 of PC4 is important for p53 recognition. We conclude that the 380-386 region of p53 interacts with the region around residues 73-76 of PC4, and acetylation of lysine 382/381 of p53 may play an important role in modulating p53-PC4 interaction and as a consequence PC4 mediated activation of p53 target genes.

Interactions of Nonprotic Organic Solvents with [Val5]angiotensin in Water

Intermolecular solvent-solute nuclear Overhauser effects have been used to explore interactions of the organic component of acetonitrile-water, acetone-water, and dimethyl sulfoxide-water mixtures with the peptide hormone [val(5)]angiotensin. As reported by the NOEs, many cross relaxation terms for interactions of these organic cosolvents are adequately accounted for using a hard spheres interaction model in which encounters of peptide and cosolvent molecules take place by mutual diffusion. However, there are indications of localized solvent-peptide interactions that are not well described by this model. In dimethyl sulfoxide-water at 0 °C, organic solvent near the C-terminal Phe8 residue and the Val3 residue produce strongly enhanced cross-relaxation terms. NOEs for all peptide N-H protons and the protons of the Tyr4 aromatic ring were significantly more positive than expected in 33% acetone-water (v/v) at 0 °C, while those for most side-chain protons were close to predictions of the hard sphere model. All peptide-organic solvent NOEs in 35% acetonitrile water (v/v) at 0 °C are consistent with the hard spheres interaction model.

Coupling liquid phase electrochemistry with nuclear magnetic resonance spectroscopy and its applications

The combination of liquid nuclear magnetic resonance spectroscopy (NMR) and electrochemistry (EC) is a new technique with promising prospect which provides novel, exciting, and crucial insights into the processes near or on the electrode surface at a molecular or atomic level. The development of in situ EC-NMR spectroscopy with flow or static electrolysis was summarized, the structures of the electrolysis cell and the characteristics of the NMR spectra were analyzed, and the feature of the in situ EC-NMR spectroscopy in practice was described in detail. The electrolysis electrode located at radio-frequency detection area of the NMR spectrometer reduced homogeneity of the magnetic field. Furthermore, the electrolysis current results in broadened spectral lines and loss of resolution. The working electrode consisting of a thin metal film and the electrolysis current parallel to the static magnetic field can overcome such shortcomings. Besides, several high-resolution methods in inhomogeneous fields were discussed and compared, such as intramolecular multiple quantum coherences, nutation echos, intermolecular nuclear Overhauser effects, and intermolecular multiple quantum coherences.

Conformations of Betanova in aqueous trifluoroethanol

Conformations of the designed peptide Betanova in 42% trifluoroethanol/water (v/v) were explored. Circular dichroism (CD) observations provided no evidence for the presence of significant amounts of beta-structures in water, in TFE/water, or in ethanol/water. Nuclear magnetic resonance (NMR) diffusion experiments showed no significant difference in the hydrodynamic radius of the peptide in water and in 42% TFE/water. However, calculations indicated that the hydrodynamic radii of the triple-stranded beta-sheet, originally proposed for Betanova by Kortemme et al. (Science 1998, 281, 253-256), and a variety of partially folded forms of Betanova would be similar and likely could not be convincingly distinguished by diffusion experiments. Temperature coefficients (Deltadelta/DeltaT) of the peptide N--H chemical shifts are similar in water and 42% TFE/water, implying that most of these protons are highly solvent exposed in both solvents and likely do not participate in intramolecular hydrogen bonding interactions. Possible exceptions to this conclusion are the Lys9 and Lys15 residues, where a more positive coefficient may indicate that these residues are involved to some extent in local turn structures. Peptide proton-solvent fluorine intermolecular nuclear Overhauser effect (NOE)s at 25 degrees C were consistent with the presence of a mixture of conformations, which could include the triple-stranded beta-sheet structure as a minor component. At 0 degrees C, peptide-TFE NOEs indicated that TFE interacts strongly enough with many protons of Betanova that alcohol-peptide interactions persist for times of the order of nanoseconds, appreciably longer than the encounter time characteristic of mutual diffusion of TFE and the solute.

The Heterohexameric Complex Structure, a Component in the Non-classical Pathway for Fibroblast Growth Factor 1 (FGF1) Secretion

Fibroblast growth factors (FGFs) are key regulators of cell proliferation, tumor-induced angiogenesis, and migration. FGFs are essential for early embryonic development, organ formation, and angiogenesis. FGF1 also plays an important role in inflammation, wound healing, and restenosis. The biological effects of FGF1 are mediated through the activation of the four transmembrane phosphotyrosine kinase fibroblast growth factor receptors in the presence of heparin sulfate proteoglycans and, therefore, require the release of the protein into the extracellular space. FGF1 is exported through a non-classical release pathway involving the formation of a specific multiprotein complex. The protein constituents of this complex include FGF1, S100A13, and the p40 form of synaptotagmin 1 (Syt1). Because FGF1 plays an important role in tumor formation, it is clear that preventing the formation of the multiprotein complex would be an effective strategy to inhibit a wide range of cancers. To understand the molecular events in the FGF1 release pathway, we studied the FGF1-S100A13 tetrameric and FGF1-S100A13-C2A hexameric complex structures, which are both complexes possibly formed during the non-classical pathway of FGF1 release.

Structural Convergence between Cryo-EM and NMR Reveals Intersubunit Interactions Critical for HIV-1 Capsid Function

Mature HIV-1 particles contain conical-shaped capsids that enclose the viral RNA genome and perform essential functions in the virus life cycle. Previous structural analysis of two- and three-dimensional arrays of the capsid protein (CA) hexamer revealed three interfaces. Here, we present a cryoEM study of a tubular assembly of CA and a high-resolution NMR structure of the CA C-terminal domain (CTD) dimer. In the solution dimer structure, the monomers exhibit different relative orientations compared to previous X-ray structures. The solution structure fits well into the EM density map, suggesting that the dimer interface is retained in the assembled CA. We also identified a CTD-CTD interface at the local three-fold axis in the cryoEM map and confirmed its functional importance by mutagenesis. In the tubular assembly, CA intermolecular interfaces vary slightly, accommodating the asymmetry present in tubes. This provides the necessary plasticity to allow for controlled virus capsid dis/assembly.