Nuclear Overhauser Enhancement Measurements Research Articles

Toward more reliable measurements of NOE effects in CEST spectra at around -1.6 ppm (NOE (-1.6)) in rat brain.

Recently, a new relayed nuclear Overhauser enhancement (NOE) saturation transfer effect at around -1.6 parts per million, termed NOE(-1.6), and its potential applications in tumor and stroke were reported by several institutes. However, there is a concern of the reproducibility of NOE(-1.6) measurements because it is not reported by many other publications. This paper aims to study the influence of typically overlooked experimental settings on the NOE(-1.6) signal and to build a framework for more reliable measurements of NOE(-1.6) at 9.4T. Z-spectra were obtained in rat brains. A fitting approach was performed to quantify all known saturation transfer effects except NOE(-1.6). Residual signals were obtained by removing these confounding effects from Z-spectra and were then used to quantify NOE(-1.6). Multislice imaging was performed to study the NOE(-1.6) dependence on brain regions. The influences of euthanasia, anesthesia, breathing gases, and RF irradiation power were also evaluated. Results demonstrate that the NOE(-1.6) signal contributions are often not clearly observable in raw Z-spectra at relatively high irradiation powers due to, for example, the direct water saturation effect, but they can be visualized after removing other nonspecific effects. In addition, the NOE(-1.6) effect depends on brain region, decreases postmortem, shifts after long-duration anesthesia, and may be enhanced by increasing O2 and N2 O breathing air concentrations. Because the NOE(-1.6) effect is more susceptible to the direct water saturation effect and more sensitive to physiological conditions than are other CEST effects, incorporating known sensitivities into the experimental design and data analysis is necessary to ensure more reliable NOE(-1.6) results.

High-resolution small RNA structures from exact nuclear Overhauser enhancement measurements without additional restraints

RNA not only translates the genetic code into proteins, but also carries out important cellular functions. Understanding such functions requires knowledge of the structure and dynamics at atomic resolution. Almost half of the published RNA structures have been solved by nuclear magnetic resonance (NMR). However, as a result of severe resonance overlap and low proton density, high-resolution RNA structures are rarely obtained from nuclear Overhauser enhancement (NOE) data alone. Instead, additional semi-empirical restraints and labor-intensive techniques are required for structural averages, while there are only a few experimentally derived ensembles representing dynamics. Here we show that our exact NOE (eNOE) based structure determination protocol is able to define a 14-mer UUCG tetraloop structure at high resolution without other restraints. Additionally, we use eNOEs to calculate a two-state structure, which samples its conformational space. The protocol may open an avenue to obtain high-resolution structures of small RNA of unprecedented accuracy with moderate experimental efforts.

On the origins of chemical exchange saturation transfer (CEST) contrast in tumors at 9.4 T.

Chemical exchange saturation transfer (CEST) provides an indirect means to detect exchangeable protons within tissues through their effects on the water signal. Previous studies have suggested that amide proton transfer (APT) imaging, a specific form of CEST, detects endogenous amide protons with a resonance frequency offset 3.5 ppm downfield from water, and thus may be sensitive to variations in mobile proteins/peptides in tumors. However, as CEST measurements are influenced by various confounding effects, such as spillover saturation, magnetization transfer (MT) and MT asymmetry, the mechanism or degree of increased APT signal in tumors is not certain. In addition to APT, nuclear Overhauser enhancement (NOE) effects upfield from water may also provide distinct information on tissue composition. In the current study, APT, NOE and several other MR parameters were measured and compared comprehensively in order to elucidate the origins of APT and NOE contrasts in tumors at 9.4 T. In addition to conventional CEST methods, a new intrinsic inverse metric was applied to correct for relaxation and other effects. After corrections for spillover, MT and T1 effects, corrected APT in tumors was found not to be significantly different from that in normal tissues, but corrected NOE effects in tumors showed significant decreases compared with those in normal tissues. Biochemical measurements verified that there was no significant enhancement of protein contents in the tumors studied, consistent with the corrected APT measurements and previous literature, whereas quantitative MT data showed decreases in the fractions of immobile macromolecules in tumors. Our results may assist in the better understanding of the contrast depicted by CEST imaging in tumors, and in the development of improved APT and NOE measurements for cancer imaging.

Comprehensive description of NMR cross-correlated relaxation under anisotropic molecular tumbling and correlated local dynamics on all time scales

A simple general expression for the NMR cross-correlated relaxation rate under anisotropic molecular tumbling is presented for globular proteins. The derivation includes effects of fast and slow motion of the interaction tensors and correlation between them. Expressions suitable for practical analysis are tailored in dependence of standard order parameters of the individual interactions. It is shown that these order parameters must be sensitive to slow motion (slower than molecular tumbling) for detection of slow correlated motion. Such order parameters are those obtained from residual dipolar couplings but not those obtained from T1, T2, and heteronuclear Nuclear Overhauser Enhancement measurements.

Structure and Evolutionary Analysis of a Non-biological ATP-binding Protein

We present a structural and functional analysis of the evolutionary optimization of a non-biological protein derived from a library of random amino acid sequences. A series of previously described in vitro selection experiments transformed a low-affinity ancestral sequence into a stably folded, high affinity ATP binding protein structure. While the evolutionarily optimized protein differs from its ancestral sequence through the accumulation of 12 amino acid mutations, the means by which those mutations enhance the stability and functionality of the protein were not well understood. We used a combination of mutagenesis, biochemistry, and NMR spectroscopy to investigate the structural and functional significance of each mutation. We solved the three-dimensional structure of the folding optimized protein by solution NMR, which revealed a fourth strand of the β-sheet of the α/β-fold that was not observed in an earlier crystallographic analysis of a less stable version of the protein. The structural rigidity of the newly identified β-strand was confirmed by T 1, T 2, and heteronuclear nuclear Overhauser enhancement (NOE) measurements. Biochemical experiments were used to examine point mutations that revert the optimized protein back to the ancestral residue at each of the 12 sites. A combination of structural and functional data was then used to interpret the significance of each amino acid mutation. The enhanced ATP affinity was largely due to the emergence of a patch of positive charge density on the protein surface, while the increased solubility resulted from several mutations that increased the hydrophilicity of the protein surface, thereby decreasing protein aggregation. One mutation may stabilize the hydrophobic face of the β-sheet.

Investigation of local chain dynamics in poly(di-n-alkylitaconate)s

13C NMR in solution and molecular dynamics simulations were used to discuss the respective roles of intramolecular constraints and intermolecular contributions in poly(di-n-alkylitaconate)s (PDAI) with various side chain lengths. These polymers exhibit the unusual phenomenon of two distinct glass transitions when the alkyl side chains contain at least six carbon atoms. Nuclear Overhauser enhancement and spin-lattice relaxation time measurements in solution for PDAI’s with different side-chain lengths clearly indicated the existence of a competition between internal plasticization and organization of the alkyl parts of the side chains. This organisation decreases the flexibility of the long side chains and slows down the internal mobility of the main chain and carbonyl groups. Molecular modelling of the relative organization of two polymer chains demonstrates the strong tendency of the long alkyl parts to order and form discrete regions, which can be related to the existence of the second low-temperature glass transition in PDAI’s with long side groups.

Aggregation behavior of acrylamide/2-phenoxyethyl acrylate and its interaction with sodium dodecyl sulfate in aqueous solution studied by proton 1D and 2D NMR

H-1 NMR self-diffusion coefficient, spin-lattice relaxation time, spin-spin relaxation time, and two-dimensional nuclear Overhauser enhancement (2D NOESY) measurements have been used to study the association behavior of a novel hydrophobically associating copolymer composed of acrylamide ( AM) and a small amount of 2-phenoxyethyl acrylate ( POEA), and its interaction with the anionic surfactant sodium dodecyl sulfate (SDS). Three sets of copolymers with approximately the same composition but with different hydrophobic POEA contents were investigated. The POEA contents for these copolymers were about 1.41, 1.03, and 0.56 mol% respectively, as validated by H-1 NMR spectra. Self-diffusion coefficient measurements show that the aggregation process occurs in a relative narrow concentration range and the critical association concentrations (cacs), of these copolymers are within this narrow concentration range, which are in agreement with those measured by viscosity measurements (6 g L-1). Above this concentration, the hydrophobic POEA moieties are found to associate and possibly build a transitory three-dimensional network along the polyacrylamide (PAM) backbones, which induces a strong decrease in NMR parameters including self-diffusion coefficients and relaxation times. The surfactant SDS showed a significant interaction with the copolymer in the dilute solution. Addition of SDS resulted in the binding of SDS on copolymer POEA-PAM segments and reinforced the interchain transient network formation of copolymer at a concentration below its cac. In the SDS/POEA-PAM mixed systems, the hydrophobic methylene groups of the SDS molecules were preferentially located in the vicinity of the phenoxy groups of the POEA hydrophobes.

13C NMR relaxation time measurements of macrocyclic ether complexes. Part XII: the association constants of benzo[15]crown-5 and benzo[12]crown-4 with NaClO4·H2O

Abstract In order to investigate the cation binding power of macrocyclic ethers the 13 C NMR relaxation times, T 1 , of free benzo[15]crown-5 and benzo[12]crown-4 as well as their 1/1 Na + complexes were determined at different temperatures in acetonitrile. The dipolar contributions to NMR relaxation were separated by nuclear Overhauser enhancement measurements. The dipolar relaxation times of free and complexed crown ethers were explained on the basis of intramolecular [ 1 H]– 13 C interactions of the molecules CH 2 –O–CH 2 backbone. From 13 C NMR relaxation times observed at extreme narrowing conditions the association constants, K a , of the sodium-crown ether complexes were obtained as a function of temperature; the Na + associations were shown to depend on the carbon–oxygen sites reducing directly the O–C–H relaxation times, T 1 . Benzo[15]crown-5 displayed different T 1 for different carbon segments on the ring, while benzo[12]crown-4 showed less T 1 changes among the macrocyclic backbone carbons. Complexed macrocyclic ethers exhibited higher barrier energies for internal motion than the uncomplexed macrocycles because the oxygen charges of the former are attracted by the cation in a complex.

Characterization of the [Ag(dmb)(2)(+)](n) oligomers (dmb = 1,8-diisocyano-p-menthane) in solution.

The crystallographically characterized polymers [[Ag(dmb)(2)]Y](n) (Y = BF(4)(-), NO(3)(-), ClO(4)(-)) extensively dissociate in solution, contrarily to the Cu analogue, and common molecular weight determination techniques such as light scattering, osmometric, and intrinsic viscosity measurements fail to provide data allowing full characterization. Using pulsed NMR experiments, notably (13)C NMR T(1) (spin lattice relaxation time) and NOE (nuclear Overhauser enhancement) measurements on various ionic [[Ag(dmb)(2)]Y](n) materials (Y = BF(4)(-), NO(3)(-), ClO(4)(-)) and their related mononuclear [Ag (CN-t-Bu)(4)]Y salts in acetonitrile-d(3) (as comparative standards), the dipole-dipole spin lattice relaxation times (T(1)(DD)) of a selected quaternary (13)C probe are measured. These data allow us to extract the correlation times (tau(c)), which in turn permit us to estimate the volume of the tumbling species in solution. The comparison of the data between the [Ag(dmb)(2)(+)](n) and Ag(CN-t-Bu)(4)(+) species indicates the oligomeric nature of the former species, where the average number of Ag(dmb)(2)(+) approximately 8 (M(n) approximately 4000-5000).

Mixed Micelles of Polyethylene Glycol (23) Lauryl Ether with Ionic Surfactants Studied by Proton 1D and 2D NMR

1H NMR chemical shift, spin–lattice relaxation time, spin–spin relaxation time, self-diffusion coefficient, and two-dimensional nuclear Overhauser enhancement (2D NOESY) measurements have been used to study the nonionic–ionic surfactant mixed micelles. Cetyl trimethyl ammonium bromide (CTAB) and sodium dodecyl sulfate (SDS) were used as the ionic surfactants and polyethylene glycol (23) lauryl ether (Brij-35) as the nonionic surfactant. The two systems are both with varying molar ratios of CTAB/Brij-35 (C/B) and SDS/Brij-35 (S/B) ranging from 0.5 to 2, respectively, at a constant concentration of 6 mM for Brij-35 in aqueous solutions. Results give information about the relative arrangement of the surfactant molecules in the mixed micelles. In the former system, the trimethyl groups attached to the polar heads of the CTAB molecules are located between the first oxy-ethylene groups next to the hydrophobic chains of Brij-35 molecules. These oxy-ethylene groups gradually move outward from the hydrophobic core of the mixed micelle with an increase in C/B in the mixed solution. In contrast to the case of the CTAB/Triton X-100 system, the long flexible hydrophilic poly oxy-ethylene chains, which are in the exterior part of the mixed micelles, remain coiled, but looser, surrounding the hydrophobic core. There is almost no variation in conformation of the hydrophilic chains of Brij-35 molecules in the mixed micelles of the SDS/Brij-35 system as the S/B increases. The hydrophobic chains of both CTAB and SDS are co-aggregated with Brij-35, respectively, in their mixed micellar cores.

Mixed Micelles of Triton X-100 and Cetyl Trimethylammonium Bromide in Aqueous Solution Studied by 1H NMR

1H NMR chemical shift, spin−lattice relaxation time, spin−spin relaxation time, and two-dimensional nuclear Overhauser enhancement (2D NOESY) measurements show that Triton X-100 (TX-100) molecules coaggregate with cetyl trimethylammonium bromide (CTAB) molecules in aqueous solutions. The concentration of TX-100 in the mixed solutions of this study is 3 mM, with varying molar ratios of CTAB/TX-100 (C/T) ranging from 0.5 to 2.9. The results give information about the structure of the mixed micelles. The α-methylene group of CTAB is in the near vicinity of the phenoxy ring of TX-100. The trimethyl group attached to the polar head of CTAB locates between the first oxyethylene group next to the phenoxy ring of TX-100, and the end methyl group of CTAB is close to those of TX-100. The closely packed (coiled) hydrophilic polyoxyethylene chains in the exterior part of the mixed micelles gradually extend with an increase in C/T in the mixed solution. CTAB and TX-100 molecules are uniformly mixed in the micelles in ...

Backbone dynamics of receptor binding and antigenic regions of a Pseudomonas aeruginosa pilin monomer.

Pilin is the major structural protein that forms type IV pili of various pathogenic bacteria, including Pseudomonas aeruginosa. Pilin is involved in attachment of the bacterium to host cells during infection, in the initiation of immune response, and serves as a receptor for a variety of bacteriophage. We have used (15)N nuclear magnetic resonance relaxation measurements to probe the backbone dynamics of an N-terminally truncated monomeric pilin from P. aeruginosa strain K122-4. (15)N-T(1), -T(2), and [(1)H]-(15)N nuclear Overhauser enhancement measurements were carried out at three magnetic field strengths. The measurements were interpreted using the Lipari-Szabo model-free analysis, which reveals the amplitude of spatial restriction for backbone N-NH bond vectors with respect to nano- to picosecond time-scale motions. Regions of well-defined secondary structure exhibited consistently low-amplitude spatial fluctuations, while the terminal and loop regions showed larger amplitude motions in the subnano- to picosecond time-scale. Interestingly, the C-terminal disulfide loop region that contains the receptor binding domain was found to be relatively rigid on the pico- to nanosecond time-scale but exhibited motion in the micro- to millisecond time-scale. It is notable that this disulfide loop displays a conserved antigenic epitope and mediates binding to the asialo-GM(1) cell surface receptor. The present study suggests that a rigid backbone scaffold mediates attachment to the host cell receptor, and also maintains the conformation of the conserved antigenic epitope for antibody recognition. In addition, slower millisecond time-scale motions are likely to be crucial for conferring a range of specificity for these interactions. Characterization of pilin dynamics will aid in developing a detailed understanding of infection, and will facilitate the design of more efficient anti-adhesin synthetic vaccines and therapeutics against pathogenic bacteria containing type IV pili.

Conformational analysis of diribosylribitol phosphate by NMR spectroscopy and molecular dynamics

The conformation of diribosylribitol phosphate was studied by means of NMR spectroscopy and molecular dynamics (MD). Starting from 3J(1H,1H), 3J(31P,1H) and 3J(31P,13C) couplings, measured from 1H NMR, 13C NMR and COSY spectra, the conformations of the ribose residues and the phosphate linkage were determined. The measurement of nuclear Overhauser enhancements (NOE) from NOESY and NOEDIFF experiments allowed the determination of constraints around the glycosidic bonds. The results of the simulation by MD (GROMOS) in water at 300 K during 2 ns agree in general with those obtained by NMR and suggest a high degree of conformational freedom. Copyright © 2000 John Wiley & Sons, Ltd.

Conformational analysis of diribosylribitol phosphate by NMR spectroscopy and molecular dynamics

The conformation of diribosylribitol phosphate was studied by means of NMR spectroscopy and molecular dynamics (MD). Starting from 3J(1H,1H), 3J(31P,1H) and 3J(31P,13C) couplings, measured from 1H NMR, 13C NMR and COSY spectra, the conformations of the ribose residues and the phosphate linkage were determined. The measurement of nuclear Overhauser enhancements (NOE) from NOESY and NOEDIFF experiments allowed the determination of constraints around the glycosidic bonds. The results of the simulation by MD (GROMOS) in water at 300 K during 2 ns agree in general with those obtained by NMR and suggest a high degree of conformational freedom. Copyright © 2000 John Wiley & Sons, Ltd.

Tumor suppressor INK4: comparisons of conformational properties between p16INK4A and p18INK4C

The INK4 (inhibitor of cyclin-dependent kinase 4) family consists of four tumor-suppressor proteins: p15INK4B, p16INK4A, p18INK4C, and p19INK4D. While their sequences and structures are highly homologous, they show appreciable differences in conformational flexibility, stability, and aggregation tendency. Here, p16 and p18 were first compared directly by NMR for line broadening and disappearance, then investigated by three different approaches in search of the causes of these differences. From denaturation experiments it was found that both proteins are marginally stable with low denaturation stability (1.94 and 2.98 kcal/mol, respectively). Heteronuclear 1H-15N nuclear Overhauser enhancement measurements revealed very limited conformational flexibility on the pico- to nanosecond time-scale for both p16 and p18. H/2H exchange of amide protons monitored by NMR on three proteins (p16, p18 as well as p15), however, revealed markedly different rates in the order p18<p16⩽p15. A subset of very slowly exchanging residues (about 19 in total) was identified in p18, including 16 residues in the region of the fourth ankyrin repeat, probably as a result of a stabilizing effect by the extra ankyrin repeat. Thus, while INK4 proteins may have similar low thermodynamic stability as well as limited flexibility on the pico- to nanosecond time-scale, they display pronounced differences in the conformational flexibility on the time-scale of minutes to hours. Further analyses suggested that differences in H/2H exchange rates reflect differences in the kinetic stability of the INK4 proteins, which in turn is related to differences in the aggregation tendency.

Structure and conformation of complex carbohydrates of glycoproteins, glycolipids, and bacterial polysaccharides.

For nuclear magnetic resonance determinations of the conformation of oligosaccharides in solution, simple molecular mechanics calculations and nuclear Overhauser enhancement measurements are adequate for small oligosaccharides that adopt single, relatively rigid conformations. Polysaccharides and larger or more flexible oligosaccharides generally require additional types of data, such as scalar and dipolar coupling constants, which are most conveniently measured in 13C-enriched samples. Nuclear magnetic resonance relaxation data provide information on the dynamics of oligosaccharides, which involves several different types of internal motion. Oligosaccharides complexed with lectins and antibodies have been successfully studied both by X-ray crystallography and by nuclear magnetic resonance spectroscopy. The complexes have been shown to be stabilized by a combination of polar hydrogen bonding interactions and van der Waals attractions. Although theoretical calculations of the conformation and stability of free oligosaccharides and of complexes with proteins can be carried out by molecular mechanics methods, the role of solvent water for these highly polar molecules continues to present computational problems.

13C NMR relaxation time measurements of macrocyclic ether complexes. The association of benzo[18]crown-6 with NaClO4·H2O in acetonitrile

Abstract Association constants for the complex Na+/benzo[18]crown-6 in acetonitrile solution were determined from 13C-NMR relaxation time measurements at different temperatures. The dipolar contributions to relaxation were separated by nuclear Overhauser enhancement measurements. The contributions are split into parts originating from uncomplexed and from complexed macrocycles assuming the 1:1 complex formation between Na+ and crown ether and by measuring solutions of different concentrations. The measured data were analysed to yield the respective relaxation times and the association constants as a function of the temperature. The results depend on the carbon site in the macrocycle thus showing preferences for binding of Na+. The dipolar relaxation times of the complex were explained on the basis of pseudorotational motion of the macrocycle which was proved with the MM+ geometry optimisation.

Synthesis of cyclic dipeptide templates, their incorporation into peptides and studies on their conformational and biological properties.

This study investigated the diastereoselective synthesis of three dipeptide templates 1, 2 and 3, which may be regarded as conformationally restricted analogs of H-Gly-Xaa-OH, in which Xaa constitutes an aromatic amino acid. Bond formation between alpha-C of Gly and the aromatic moiety was achieved by proton-catalyzed intramolecular electrophilic aromatic substitution. The absolute configuration of the dipeptide templates was determined by single-crystal X-ray crystallography or by nuclear Overhauser enhancement measurements. A protective group strategy was elaborated to allow their incorporation into peptide sequences by liquid phase as well as by solid-phase peptide synthesis. The templates were used to generate an enkephalin analog 15, a modified peptidic neurokinin antagonist 20 and two dermorphin derivatives (24 and 33). Molecular dynamic simulations with 15 and 20 revealed the preference for a turn-like motif for 15. The biological activity, as investigated by respective receptor binding and functional assays, was strongly diminished with all four derivatives, indicating that their receptor-relevant molecular geometries lie outside the examined conformational space.

Assigning the NMR spectra of aromatic amino acids in proteins: analysis of two Ets pointed domains

The measurement of interproton nuclear Overhauser enhancements (NOEs) and dihedral angle restraints of aromatic amino acids is a critical step towards determining the structure of a protein. The complete assignment of the resonances from aromatic rings and the subsequent resolution and identification of their associated NOEs, however, can be a difficult task. Shown here is a strategy for assigning the 1H, 13C, and 15N signals from the aromatic side chains of histidine, tryptophan, tyrosine, and phenylalanine using a suite of homo- and hetero-nuclear scalar and NOE correlation experiments, as well as selective deuterium isotope labelling. In addition, a comparison of NOE information obtained from homonuclear NOE spectroscopy (NOESY) and 13C-edited NOESY - heteronuclear single quantum correlation experiments indicates that high-resolution homonuclear two-dimensional NOESY spectra of selectively deuterated proteins are invaluable for obtaining distance restraints to the aromatic residues.Key words: NMR assignment, aromatic residue, transcription factor, NOE, dihedral angle.

Assigning the NMR spectra of aromatic amino acids in proteins: analysis of two Ets pointed domains.

The measurement of interproton nuclear Overhauser enhancements (NOEs) and dihedral angle restraints of aromatic amino acids is a critical step towards determining the structure of a protein. The complete assignment of the resonances from aromatic rings and the subsequent resolution and identification of their associated NOEs, however, can be a difficult task. Shown here is a strategy for assigning the 1H, 13C, and 15N signals from the aromatic side chains of histidine, tryptophan, tyrosine, and phenylalanine using a suite of homo- and hetero-nuclear scalar and NOE correlation experiments, as well as selective deuterium isotope labelling. In addition, a comparison of NOE information obtained from homonuclear NOE spectroscopy (NOESY) and 13C-edited NOESY-heteronuclear single quantum correlation experiments indicates that high-resolution homonuclear two-dimensional NOESY spectra of selectively deuterated proteins are invaluable for obtaining distance restraints to the aromatic residues.