Nuclear Overhauser Effect Cross Research Articles

Insights into dynamic properties of water in lipidic cubic phases by 2D nuclear Overhauser effect (NOE) NMR spectroscopy

Two-dimensional NOE (nuclear Overhauser effect) NMR spectroscopy was employed to investigate the dynamic properties of water within lyotropic bicontinuous lipidic cubic phases (LCPs) formed by monoolein (MO). Experiments observed categorically different effective residence times of water molecules: (i) in proximity to the glycerol moiety of MO, and (ii) adjacent to the hydrophobic chain towards the hydrocarbon tail of MO, as evidenced by the opposite signs of intermolecular NOE cross peaks between protons of water and those of MO in 2D 1H–1H NOESY spectra. Spectroscopic data delineating the different effective residence times of water molecules within both the gyroid (QIIG) and diamond (QIID) phase groups corresponding to hydration levels of 35 and 40 wt%, respectively, are presented. Additionally, an increase in effective residence time of water molecules in proximity to the glycerol moiety of MO in LCPs was observed upon storage at ambient temperature and in the presence of an additive lipid, cholesterol. Atom-specific NOE build-up curves for protons of water and those of MO are also given. The results presented herein provide new insight into the physicochemical properties and behaviour of water in LCPs, and demonstrate an additional avenue for experimental study of water–lipid interactions and hydration dynamics in model membranes and nanomaterials using 2D NOE NMR spectroscopy.

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.

Interaction of naphthalene derivatives with lipids in membranes studied by the 1H-nuclear Overhauser effect and molecular dynamics simulation

The location, orientation, and dynamics of hydrophobic small molecules in lipid membranes are studied through combined use of solution-state (1)H-NMR and MD simulation. 1-Naphthol and 1-methylnaphthalene were adopted as the small molecules with or without hydrophilic groups. The nuclear Overhauser effect (NOE) measurement was performed for large unilamellar vesicles (100 nm in diameter) composed of dimyristoylphosphatidylcholine (DMPC) and the naphthalene derivative. The transient NOE-SE (spin-echo) scheme previously reported (J. Phys. Chem. B, 2011, 115, 9106-9115) was employed to quantitatively determine the NOE cross relaxation rate constant between DMPC and the naphthalene derivative. The observed NOE shows that both the naphthalene derivatives distribute over a wide domain across the normal of the essentially planar membrane ranging from the hydrophobic core to the hydrophilic headgroup. The experimental NOE information was further refined in combination with the analysis of time correlation functions in MD simulation. It was found that 1-naphthol exhibits a slight preference for pointing its OH group toward the hydrophilic domain of the membrane and that no definite preference can be concluded for the orientation of 1-methylnaphthalene. When 1-naphthol and 1-methylnaphthalene are compared, the NOE is stronger for 1-naphthol due to the restricted motion of the OH group. The slowdown of the 1-naphthol motion is also evidenced by the (1)H spectral line width.

A helix-turn motif in the C-terminal domain of histone H1.

The structural study of peptides belonging to the terminal domains of histone H1 can be considered as a step toward the understanding of the function of H1 in chromatin. The conformational properties of the peptide Ac-EPKRSVAFKKTKKEVKKVATPKK (CH-1), which belongs to the C-terminal domain of histone H1(o) (residues 99-121) and is adjacent to the central globular domain of the protein, were examined by means of 1H-NMR and circular dichroism. In aqueous solution, CH-1 behaved as a mainly unstructured peptide, although turn-like conformations in rapid equilibrium with the unfolded state could be present. Addition of trifluoroethanol resulted in a substantial increase of the helical content. The helical limits, as indicated by (i,i + 3) nuclear Overhauser effect (NOE) cross correlations and significant up-field conformational shifts of the C(alpha) protons, span from Pro100 to Val116, with Glu99 and Ala117 as N- and C-caps. A structure calculation performed on the basis of distance constraints derived from NOE cross peaks in 90% trifluoroethanol confirmed the helical structure of this region. The helical region has a marked amphipathic character, due to the location of all positively charged residues on one face of the helix and all the hydrophobic residues on the opposite face. The peptide has a TPKK motif at the C-terminus, following the alpha-helical region. The observed NOE connectivities suggest that the TPKK sequence adopts a type (I) beta-turn conformation, a sigma-turn conformation or a combination of both, in fast equilibrium with unfolded states. Sequences of the kind (S/T)P(K/R)(K/R) have been proposed as DNA binding motifs. The CH-1 peptide, thus, combines a positively charged amphipathic helix and a turn as potential DNA-binding motifs.

NMR solution structure of the C-terminal fragment 255-316 of thermolysin: a dimer formed by subunits having the native structure.

The solution structure of the C-terminal fragment 255-316 of thermolysin has been determined by two-dimensional proton NMR methods. For this disulfide-free fragment there was a previous proposal according to which it would fold into a stable helical structure forming a dimer at concentrations above 0.06 mM. A complete assignment of the proton NMR resonances of the backbone and amino acid side chains of the fragment was first performed using standard sequential assignment methods. On the basis of 729 distance constraints derived from unambiguously assigned nuclear Overhauser effect (NOE) proton connectivities, the three-dimensional structure of a monomeric unit was then determined by using distance geometry and restrained molecular dynamic methods. The globular structure of fragment 255-316 of thermolysin in solution, composed of three helices, is largely coincident with that of the corresponding region in the crystallographic structure of intact thermolysin [Holmes, M. A., & Matthews, B. W. (1982) J. Mol. Biol. 160, 623-639]. This fact allowed identification as intersubunit of up to 52 NOE cross correlations, which were used to dock the two subunits into a symmetric dimer structure. The obtained dimeric structure served as the starting structure in a final restrained molecular dynamic calculation subjected to a total of 1562 distance constraints. In the resulting dimeric structure, the interface between the two subunits, of a marked hydrophobic character, coincides topologically with the one between the 255-316 fragment and the rest of the protein in the intact enzyme. The present work decisively shows that the thermolysin fragment 255-316 can attain a stable and nativelike structure independently of the rest of the polypeptide chain. Considering that the thermolysin molecule is constituted of two structural domains of equal size (residues 1-157 and 158-316), the results of this study show that autonomously folding units can be substantially smaller than entire domains.

Models of the cytochromes b. 8. Two-dimensional nuclear Overhauser and exchange spectroscopy studies of paramagnetic "cavity" type (tetraphenylporphinato)iron(III) complexes of planar ligands

The paramagnetic bis-2-methylimidazole complex of (tetramesitylporphinato)iron(III) chloride, with short TI and T, relaxation times, was investigated in CD2C12 by NOESY (nuclear Overhauser and exchange spectroscopy) experiments as a function of temperature (-29, -54, -74 C). The two-dimensional (2-D) maps obtained at -29 and -54 C revealed cross peaks due to both chemical exchange and the nuclear Overhauser effect (NOE), while the map obtained at -74 C showed only NOE cross peaks. The NOE cross correlations are consistent with the 2-MeImH ligands being positioned over or near the meso carbon atoms, rather than over the porphyrin nitrogens, as previously suggested by Nakamura and Groves (Tetrahedron 1988, 44, 3225). The nonbonded interactions between the methyl groups of the 2-methylimidazole (2-MeImH) ligands and the mesityl groups of the porphyrin phenyl rings for this orientation of the axial ligands produce a strongly ruffled porphyrin core having two oblong cavities at right angles to each other, one above and one below the plane of the porphyrin ring, which hold the hindered axial ligands in perpendicular planes over the meso positions. The chemical exchange cross peaks are indicative of interconversion of all resonances of a particular type, due to rotation of the axial ligands rather than axial ligand exchange. This interpretation is confirmed by the fact that there are no cross peaks observed between free and coordinated 2-MelmH resonances at any of the temperatures investigated. Rotation of the 2-MeImH ligands requires that alternate phenyl rings flap up and down as the ligands rotate. The coupling pattern between the pyrrole-H protons is consistent with that expected on the basis of the extrapolated intercepts of the Curie plot.

Two-dimensional magnetization exchange spectroscopy of Anabaena 7120 ferredoxin. Nuclear Overhauser effect and electron self-exchange cross peaks from amino acid residues surrounding the iron-sulfur (2Fe-2S) cluster

Hyperfine 1H NMR signals of the 2Fe-2S* vegetative ferredoxin from Anabaena 7120 have been studied by two-dimensional (2D) magnetization exchange spectroscopy. The rapid longitudinal relaxation rates of these signals required the use of very short nuclear Overhauser effect (NOE) mixing times (0.5-20 ms). The resulting pattern of NOE cross-relaxation peaks when combined with previous 1D NOE results [Dugad, L. B., La Mar, G. N., Banci, L., & Bertini, I. (1990) Biochemistry 29, 2263-2271] led to elucidation of the carbon-bound proton spin systems from each of the four cysteines ligated to the 2Fe-2S* cluster in the reduced ferredoxin. Additional NOE cross peaks were observed that provide information about other amino acid residues that interact with the iron-sulfur cluster. NOE cross peaks were assigned tentatively to Leu27, Arg42, and Ala43 on the basis of the X-ray coordinates of oxidized Anabaena 7120 ferredoxin [Rypniewski, W.R., Breiter, D.R., Benning, M.M., Wesenberg, G., Oh, B.-H., Markley, J.L., Rayment, I., & Holden, H. M. (1991) Biochemistry 30, 4126-4131]. Three chemical exchange cross peaks were detected in magnetization exchange spectra of half-reduced ferredoxin and assigned to the 1H alpha protons of Cys49 and Cys79 [both of whose sulfur atoms are ligated to Fe(III)] and Arg42 (whose amide nitrogen is hydrogen-bonded to one of the inorganic sulfurs of the 2Fe-2S* cluster). The chemical exchange cross peaks provide a means of extending assignments in the spectrum of reduced ferredoxin to assignments in the spectrum of the oxidized protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Determining local conformational variations in DNA: Nuclear magnetic resonance structures of the DNA duplexes d(CGCCTAATCG) and d(CGTCACGCGC) generated using back-calculation of the nuclear overhauser effect spectra, a distance geometry algorithm and constrained molecular dynamics

Two-dimensional nuclear magnetic resonance (n.m.r.) spectroscopy and a variety of computational techniques have been used to generate three-dimensional structures of the two DNA duplexes d(CGCCTAATCG) and d(CGTCACGCGC). The central six base-pairs in these two decamers contain all ten dinucleotide pairs in DNA and thus, represent a model system for investigating how the local structure of DNA varies with base sequence. Resonance assignments were made for the non-exchangeable base protons and most of the C-1′-C-4′ sugar protons in both decamers. Three-dimensional structures were generated using a distance geometry algorithm and these initial structures were refined by optimizing the fit of back-calculated spectra against the experimental two-dimensional nuclear Overhauser effect (NOE) spectra. This back-calculation procedure consists of calculating NOE cross relaxation rates for a given structure by solution of the Bloch equations, and directly accounts for spin diffusion effects. Use of this refinement procedure eliminates some assumptions that have been invoked when generating structures of DNA oligomers from n.m.r. data. Constrained energy minimization and constrained quenched molecular dynamics calculations were also performed on both decamers to help generate energetically favorable structures consistent with the experimental data. Analysis of the local conformational parameters of helical twist, helical rise, propeller twist, displacement and the α, β, γ,ε and ζ backbone torsion angles in these structures shows that these parameters span a large range of values relative to the X-ray data of nucleic acids. However, the glycosidic and pseudorotation angles are quite well defined in these structures. The implications that these results have for determination of local structural variations of DNA in solution, such as those predicted by Callidine's rules, are discussed. Our results differ significantly from some previous studies on determining local conformations of nucleic acids and comparisons with these studies are made.

Suppression of HOHAHA and “false” NOE cross peaks in CAMELSPIN spectra

The CAMELSPIN experiment, introduced by Bothner-By et al. (1, 2), makes it possible to study nuclear Overhauser effect (NOE) enhancements in molecules where the motional correlation time is such that there are no enhancements in conventional NOE spectra. In conventional NOE studies positive enhancements are expected for small rapidly tumbling molecules, while negative enhancements are expected for large slowly tumbling molecules. A significant number of medium-sized molecules have correlation times that result in NOE enhancements near to the zero-crossing point between these two extremes and hence show either no or only very small enhancements. The CAMELSPIN experiment detects cross relaxation between spinlocked transverse magnetization, in contrast to conventional NOE experiments which utilize longitudinal magnetization. The precise dynamics of cross relaxation between transverse magnetizations are such that positive NOE enhancements are seen for all correlation times (I, 3), thus making it possible to study molecules for which conventional NOE experiments yield no enhancements. The conditions of spin locking used in the CAMELSPIN experiment can also bring about coherence transfer between scalar-coupled spins (4-7). This type of transfer can be utilized for correlation spectroscopy of various kinds (such as the TOCSY experiment (6)), and has recently been dubbed HOHAHA by Bax et al. ( 7). Such transfer between scalar-coupled spins is very undesirable in CAMELSPIN spectra since the resulting cross peaks may obscure the desired NOE cross peaks or, more seriously, may give rise to “false” NOE cross peaks (8, 9). These false cross peaks arise from an NOE transfer followed by a HOHAHA transfer (or vice versa) and they are indistinguishable in a single spectrum from genuine NOE cross peaks. The presence of these false NOE cross peaks can thus cause substantial errors or ambiguities of interpretation. The purpose of this communication is to describe a modified method of recording CAMELSPIN spectra in which the HOHAHA mechanism is effectively suppressed, thus allowing such spectra to be interpreted without ambiguity. Differentiation of NOE and HOHAHA transfer is impossible using the usual methods of phase cycling or randomization of the mixing time. However, the two processes vary greatly in their sensitivity to the offset and field strength of the spin-locking field and have different time dependences. The method described here utilizes these

Comparative study of conformational behaviour of leucine and methionine enkephalinamides by1H-nuclear magnetic resonance spectroscopy

The conformational proclivity of leucine and methionine enkephalinamides in deuterated dimethyl sulphoxide has been investigated using proton magnetic resonance at 500 MHz. The resonances from the spin system of the various amino acid residues have been assigned from the 2-dimensional correlated spectroscopy spectra. The temperature variation of the amide proton shifts indicates that none of the amide proton is intramolecularly hydrogen-bonded or solvent-shielded. The analysis of vicinal coupling constants,3JHN.C2H,along with temperature coefficients and the absence of characteristic nuclear Overhauser effect cross peaks between the NH protons reveal that there is no evidence of the chain folding in these molecules. However, the observation of nuclear Overhauser effect cross peaks between the NH and the CαH of the preceding residue indicates preference for extended backbone conformation with preferred side chain orientations particularly of Tyr and Phe in both [Leu5]- and [Met5]-enkephalinamides.