Relaxation NOE Research Articles

Intra- and inter-ion-pair protonic-hydridic bonding in polyhydridobis(phosphine)rhenates

The hexahydridobis(phosphine)rhenate anions, [ReH6(PR3)2]- (PR3 = PCy3, P-i-Pr3, PPh3, PMe3) were generated by potassium hydride deprotonation of the neutral heptahydride conjugate acids (ReH7(PR3)2), isolated as their [K(18-crown-6)]+ and [K(1,10-diaza-18-crown-6)]+ salts, and characterized by NMR and IR spectroscopy and elemental analyses. Structures from single crystal X-ray diffraction were obtained for the [K(1,10-diaza-18-crown-6)]+salts and these indicate the presence of short protonic—hydridic bonds involving the hydrides of the anions and the proton donor NH moieties of the cations. The structure of [K(1,10-diaza-18-crown-6)][ReH6(P-i-Pr3)2] adopts a one-dimensional zigzag chain with alternating cations and anions connected and held together by inter-ion N-H···Hx-Re interactions (x = 1 or 2). Short distances between the NH protons of the cations and hydrides of the anion ranging from 1.6 to 1.9 Å are estimated for this complex. A different kind of chain structure is observed for [K(1,10-diaza-18-crown-6)][ReH6(PMe3)2] in which the combined effects of inter-ion protonic—hydridic bonding (N-H···Hx-Re) and inter-ion electrostatic interactions (ReH-x···K+···H-xRe), result in one-dimensional networks of alternating cations and anions, with the metals and hydrides occupying the interior and the organic moieties of the phosphine ligands and crown ether lining the exterior of cylindrical supramolecular assemblies. A combination of intra- and inter-ion protonic-hydridic and intra-ion-pair electrostatic interactions in [K(1,10-diaza-18-crown-6)][ReH6(PPh3)2] result in the formation of discrete two-dimensional {[K(1,10-diaza-18-crown-6)][ReH6(PPh3)2]}4 tetramers. The PCy3 salt is disordered but appears to consist of isolated 1:1 ion pairs containing strong intra-ion-pair NH···HRe bonding. The solid-state IR spectra of the [K(1,10-diaza-18-crown-6)]+ salts show low-frequency shifts for the NH bands relative to [K(1,10-diaza-18-crown-6)][BPh4], and perturbed Re-H bands relative to those in the [K(18-crown-6)]+ salts. The magnitude of ΔνNH is related to the basicity of the anion as indicated by the pKαTHF of the conjugate acid form (ReH7(PR3)2), which increases as PPh3 < < PMe3 < P-i-Pr3 < PCy3. Solution 1H NMR, NOE, and T1 relaxation measurements of [K(1,10-diaza-18-crown-6)][ReH6(PPh3)2] indicate that these interactions also persist in toluene solutions of this compound.Key words: rhenium, hydride, phosphine, hydrogen bonding, self-assembly.

NMR Detection of the Conformational Distortion Induced in Cyclodextrins by Introduction of Alkyl or Aromatic Substituents

The conformational features of alkylated, benzoylated, and benzylated cyclodextrins in solution were analysed by NOE and proton-selective relaxation methods and were compared to those of native compounds.

Backbone dynamics of the human CC chemokine eotaxin: fast motions, slow motions, and implications for receptor binding.

Eotaxin is a member of the chemokine family of about 40 proteins that induce cell migration. Eotaxin binds the CC chemokine receptor CCR3 that is highly expressed by eosinophils, and it is considered important in the pathology of chronic respiratory disorders such as asthma. The high resolution structure of eotaxin is known. The 74 amino acid protein has two disulfide bridges and shows a typical chemokine fold comprised of a core of three antiparallel beta-strands and an overlying alpha-helix. In this paper, we report the backbone dynamics of eotaxin determined through 15N-T1, T2, and [1H]-15N nuclear Overhauser effect heteronuclear multidimensional NMR experiments. This is the first extensive study of the dynamics of a chemokine derived from 600, 500, and 300 MHz NMR field strengths. From the T1, T2, and NOE relaxation data, parameters that describe the internal motions of eotaxin were derived using the Lipari-Szabo model free analysis. The most ordered regions of the protein correspond to the known secondary structure elements. However, surrounding the core, the regions known to be functionally important in chemokines show a range of motions on varying timescales. These include extensive subnanosecond to picosecond motions in the N-terminus, C-terminus, and the N-loop succeeding the disulfides. Analysis of rotational diffusion anisotropy of eotaxin and chemical exchange terms at multiple fields also allowed the confident identification of slow conformational exchange through the "30s" loop, disulfides, and adjacent residues. In addition, we show that these motions may be attenuated in the dimeric form of a synthetic eotaxin. The structure and dynamical basis for eotaxin receptor binding is discussed in light of the dynamics data.

Assessing potential bias in the determination of rotational correlation times of proteins by NMR relaxation.

The various factors that influence the reliable and efficient determination of the correlation time describing molecular reorientation of proteins by NMR relaxation methods are examined. Nuclear Overhauser effects, spin-lattice, and spin-spin relaxation parameters of 15N NMR relaxation in ubiquitin have been determined at 17.6, 14.1, 11.7 and 9.4 Tesla. This unusually broad set of relaxation parameters has allowed the examination of the influence of chemical shift anisotropy, the functional form of the model-free spectral density, and the reliability of determined spin-spin relaxation parameters on the characterization of global tumbling of the protein. Treating the 15N chemical shift anisotropy (CSA) as an adjustable parameter, a consensus value of -170 +/- 15 ppm for the breadth of the chemical shift tensor and a global isotropic correlation time of 4.1 ns are found when using the model-free spectral density to fit T1 and NOE data from all fields. The inclusion of T2 relaxation parameters in the determination of the global correlation time results in its increase to 4.6 ns. This apparent inconsistency may explain a large portion of the discrepancy often found between NMR- and fluorescence-derived tau m values for proteins. The near identity of observed T2 and T1 rho values suggests that contributions from slow motions are not the origin of the apparent inconsistency with obtained T1 and NOE data. Various considerations suggest that the origin of this apparent discrepancy may reside in a contribution to the spectral density at zero frequency that is not represented by the simple model-free formalism in addition to the usual experimental difficulties associated with the measurement of these relaxation parameters. Finally, an axially symmetric diffusion tensor for ubiquitin is obtained using exclusively T1 and NOE data. A recommendation is reached on the types and combinations of relaxation data that can be used to reliably determine tau m values. It is also noted that the reliable determination of tau m values from 15N T1 and NOE relaxation parameters will become increasingly difficult as tau m increases.

Dynamics of a bis-intercalator DNA complex by 1H-detected natural abundance 13C NMR spectroscopy.

The dynamics of the DNA oligomer d(CGCTAGCG)2 (CTSYM) and its complex with the dye 1,1-(4,4,8,8-tetramethyl-4, 8-diazaundecamethylene)-bis-4-(3-methyl-2,3-dihydro-(benzo-1, 3-thiazole)-2-methylidene)-quinolinium tetraiodide (TOTO) (CTSYMTOTO) bis-intercalated at the 5'-CT-3' sequence steps have been determined from NMR relaxation parameters. Longitudinal and transverse 13C relaxation rates and heteronuclear NOE relaxation data were acquired and have been analyzed in the context of the Lipari and Szabo model-free formalism. The overall rotational correlation time for the CTSYM is 3.44 ns and the CTSYMTOTO is 3.48 ns. The generalized order parameters (S2) for methine carbons in the CTSYM and CTSYMTOTO are relatively high but nonuniform for the molecules and show sequence context and conformation-dependent variations. Average values of S2 = 0.79 +/- 0.02 for the CTSYM, S2 = 0.80 +/- 0.04 for the CTSYMTOTO aromatic spins, S2 = 0.76 +/- 0.02 for the CTSYM, and S2 = 0.83 +/- 0.05 for the CTSYMTOTO deoxyribose spins were found. The S2 values for the 5'-terminal deoxyribose are lower than for the other residues. The DNA backbone in CTSYMTOTO is distorted and elongated at the site of intercalation, and the C3' atom of the C3 deoxyribose residue has a very low S2 = 0.57 +/- 0.06. The low order for this spin is interpreted in terms of exchange between the C2'-endo and O1'-endo conformations of the C3 deoxyribose. Significant chemical exchange processes were found for most of the aromatic spins in CTSYM that are interpreted in terms of microsecond to millisecond time scale dynamics. The microsecond to millisecond dynamics of the bases in CTSYM are quenched upon TOTO complex formation due to unwinding of the helix and an increase in the surface area of the bases in mutual contact and the large surface area in contact with the intercalated dye. The derived order parameters combined with the solution structure provide motional models for conformational changes induced in the backbone in response to the ligand binding.

Dynamics of the conformational ensemble of partially folded bovine pancreatic trypsin inhibitor.

A single-disulfide variant of bovine pancreatic trypsin inhibitor (BPTI), [14-38]Abu, is a partially folded ensemble which includes two, and in one case three, conformations that interconvert slowly enough to exhibit separate cross-peaks in the amide region of homonuclear and heteronuclear NMR spectra. Each conformation is itself composed of many subconformations in rapid equilibrium. Partially folded BPTI undergoes local motions that are slow, noncooperative, independent fluctuations of short segments within the chain. Cooperative global unfolding of the ensemble is also observed. Heteronuclear NMR has been used to measure interconversion rate constants of partially folded conformational substates; the rate constants differ for each residue and vary over an order of magnitude. For local fluctuation, the forward rate constants for amide protons of the antiparallel beta-sheet are significantly smaller than the rest of the molecule, consistent with other indications that this is the most stable part of the partially folded protein. The reverse rate constants also vary; they are the highest for Ala 27 in the turn between the strands in the sheet and for Phe 33 in the antiparallel beta-sheet. Global unfolding interconversion rate constants vary over a 3-fold range, consistent with previously observed deviations from two-state behavior. Fast backbone dynamics, from T1, T2, and NOE relaxation parameters, are obtained for the slowly interchanging conformations in the partially folded ensemble. Clear differences are observed between the two conformations; one is more flexible and less compact than the other. In the more flexible and disordered partially folded conformation, intermediate exchange is detected for some backbone amides, namely, those in the central beta-sheet and the turn. These same sheet and turn residues are more ordered in the globally denatured ensemble as well. Our results suggest that the turn initiates formation of a partially folded ensemble in which the slow-exchange core is the most stable region and in which segmental fluctuations reflect multiple nuclei for folding of the rest of the molecule.

Dynamics in psoralen-damaged DNA by 1H-detected natural abundance 13C NMR spectroscopy.

The dynamics of the DNA oligomer d(GCGTACGC)2 and the 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen-DNA furanside monoadduct (MAf) of this oligomer have been determined from NMR relaxation parameters. Longitudinal and transverse 13C relaxation rates and heteronuclear NOE relaxation data have been measured at natural abundance and have been analyzed in the context of the Lipari and Szabo model-free formalism. The generalized order parameters for methine carbons in the octamer sequence d(GCGTACGC)2 (UM) are relatively and uniformly high for the entire molecule. The generalized order parameters for methine carbons in the MAf are significantly lower for the deoxyribose bearing the damaged thymidine base and for the bases flanking the lesion on the undamaged strand, indicating additional conformational flexibility due to the lesion. The order parameters for the bases on the damaged strand flanking the lesion remain high. Analysis of the relaxation data indicates substantial chemical exchange for the adenosine residues in the UM TpA site, and this chemical exchange is quenched upon MAf formation. These data are discussed in terms of a model for DNA damage recognition by the nucleotide excision repair system.

Internal dynamics of human ubiquitin revealed by 13C-relaxation studies of randomly fractionally labeled protein

The use of random, fractional 13C-enrichment combined with low pass filtration has allowed the determination of NMR relaxation parameters at an unprecedented number of sites within recombinant human ubiquitin. Essentially complete 1H, 13C, and 15N resonance assignments for the protein are reported. Carbon spin lattice and heteronuclear NOE relaxation data have been analyzed in the context of the Lipari-Szabo model free formalism. The generalized order parameters for 56 main chain alpha C-H vectors have been determined and are found to correspond to the highly restricted motion seen in previous studies of the motion of amide N-H vectors. In distinct contrast, the analysis presented here indicates an unexpected range of dynamics within the interior of the protein. The generalized order parameters of 45 methyl groups of human ubiquitin have been determined. The methyl groups of Thr and Ala residues show generalized order parameters ranging from the Woessner limit (0.111) to below 0.01. Generalized order parameters for all methyl groups of the seven isoleucine residues were determined. With one exception, the generalized order parameters of the gamma methyls were equal to or greater than the corresponding delta methyls, indicating higher mobility away from the main chain. Generalized order parameters for 11 methyl groups of leucine residues were also determined. In six of the seven cases where the generalized order parameters of both prochiral methyl groups were determined, the pro-R methyl consistently shows a higher value than the pro-S methyl group. Generalized order parameters for seven methyl groups of four valines were also determined. There is no apparent correlation of methyl group prochirality with the value of the generalized order parameter. These data have several implications and generally indicate that the interior of the protein is heterogeneously dynamic.

Study of protein dynamics in solution by measurement of 13C?-13CO NOE and 13CO longitudinal relaxation

(13)C(α)-(13)CO homonuclear NOE and (13)CO T(1) relaxation were measured for a 20 kDa protein using tripleresonance pulse sequences. The experiments were sufficiently sensitive to obtain statistically significant differences in relaxation parameters over the molecule. The (13)C(α)-(13)CO cross-relaxation rate, obtained from these data, is directly proportional to an order parameter describing local motion and it is largely independent of the local correlation time. It is therefore a relatively straightforward observable for the identification of local dynamics.

Internal dynamics of human ubiquitin revealed by 13C-relaxation studies of randomly fractionally labeled protein.

The use of random, fractional 13C-enrichment combined with low pass filtration has allowed the determination of NMR relaxation parameters at an unprecedented number of sites within recombinant human ubiquitin. Essentially complete 1H, 13C, and 15N resonance assignments for the protein are reported. Carbon spin lattice and heteronuclear NOE relaxation data have been analyzed in the context of the Lipari-Szabo "model free" formalism. The generalized order parameters for 56 main chain alpha C-H vectors have been determined and are found to correspond to the highly restricted motion seen in previous studies of the motion of amide N-H vectors. In distinct contrast, the analysis presented here indicates an unexpected range of dynamics within the interior of the protein. The generalized order parameters of 45 methyl groups of human ubiquitin have been determined. The methyl groups of Thr and Ala residues show generalized order parameters ranging from the Woessner limit (0.111) to below 0.01. Generalized order parameters for all methyl groups of the seven isoleucine residues were determined. With one exception, the generalized order parameters of the gamma methyls were equal to or greater than the corresponding delta methyls, indicating higher mobility away from the main chain. Generalized order parameters for 11 methyl groups of leucine residues were also determined. In six of the seven cases where the generalized order parameters of both prochiral methyl groups were determined, the pro-R methyl consistently shows a higher value than the pro-S methyl group. Generalized order parameters for seven methyl groups of four valines were also determined. There is no apparent correlation of methyl group prochirality with the value of the generalized order parameter. These data have several implications and generally indicate that the interior of the protein is heterogeneously dynamic.

Composite spectral density functions for the interpretation of the 13C NMR relaxation behavior of polymers containing hydrocarbon side chains. Free and restricted side chain motion

Abstract13C NMR NT1 and NOE have been calculated by using composite spectral density functions describing polymer chain segmental motion and internal rotation of a hydrocarbon side chain attached to the polymer backbone. Numerical results at two magnetic fields are presented as a function of the various motional parameters characterizing the various models. NT1 and NOE relaxation parameters are well behaved and appear to have practical value for describing the dynamics of these systems. The models have been applied to the relaxation data of poly(n‐butyl methacrylate) and poly(n‐hexyl methacrylate) in toluene solutions. The dynamics of the two polymers are characterized by a very localized backbone motion and restricted internal rotation about successive CC bonds of the side chains. © 1995 John Wiley & Sons, Inc.

Backbone Dynamics of a Highly Disordered 131 Residue Fragment of Staphylococcal Nuclease

In order to characterize the dynamic properties of the denatured state of staphylococcal nuclease, R1, R2, and NOE relaxation parameters have been measured for the backbone 15N nuclei of a 131 residue fragment that serves as a model of the denatured state under non-denaturing conditions. The relaxation data indicate a wide range of amplitudes for segmental motion and are inconsistent with a random coil conformation. An optimal value of 7·8 ns was obtained for the molecular rotational correlation time τm based on the analysis of the 79 residues for which R1, R2, and NOE relaxation data could be obtained. This value corresponds roughly to the slowest detectable motion on the nanosecond time scale and is of a magnitude consistent with global tumbling of a large portion of the molecule. For the majority of residues, experimental data could be described most adequately in terms of a modified "model-free" formalism which includes contributions from internal motions on both an intermediate (τe) and a fast time scale (τf) in the context of slow overall tumbling (τm). The generalized order parameter S2, which gives the amplitude of motions on time scales faster than τm, correlates with sequence hydrophobicity and suggests a relationship between chain flexibility and sequence propensity for hydrophobic collapse. The fractional populations of three α-helices in the protein show a stronger correlation with S2 values and hydrophobicities than with intrinsic helix propensities. These observations suggest that secondary structure may be preferentially stabilized in hydrophobic segments of the sequence.

The solution structure of a 3'-phenazinium (Pzn) tethered DNA-RNA duplex with a dangling adenosine: r(5'G-AUUGAA3'):d(5'TCAATC3'-Pzn).

The 3'-Pzn group tethered to an oligo-DNA stabilizes a DNA-RNA hybrid duplex structure by 13 degrees C compared to the natural counterpart. This report constitutes the first full study of the conformational features of a hybrid DNA-RNA duplex, which has been possible because of the unique stabilization of this rather small duplex by the tethered 3'-Pzn moiety (Tm approximately 40 degrees C from NMR). In this study, a total of 252 inter- and intra-strand torsional and distance constraints along with the full NOE relaxation matrix, taking into account the exchange process of imino and amino protons with water, have been used. The 3'-Pzn-promoted stabilization of the DNA-RNA hybrid duplex results in detailed local conformational characteristics such as the torsion angles of the backbone and sugar moieties that are close to the features of the other natural DNA-RNA hybrids (i.e. sugars of the RNA strand are 3'-endo, but the sugars of the DNA strand are intermediate between A- and B-forms of DNA, 72 degrees < P < 180 degrees; note however, that the sugars of our DNA strand have a C1-exo conformation: 131 degrees < P < 154 degrees). This study suggests that 3'-Pzn-tethered smaller oligo-DNA should serve the same purpose as a larger oligo-DNA as a antisense inhibitor of the viral mRNA. Additionally, these types of tethered oligos have been found to be relatively more resistant to the cellular nuclease. Moreover, they are taken up quite readily through the cellular membrane (14) compared to the natural counterparts.

A structural study of the naringin-?-cyclodextrin complex

The structure of the inclusion complex formed between naringin (naringenin-7-O-β-neohesperidoside) andβ-cyclodextrin (BCD) was studied in detail by UV and NMR spectroscopic techniques and potentiometry. A binding constant value of 1016±150M−1 was arrived at from UV studies. Potentiometric studies showed that pK values of 4′-OH and 5-OH were affected byα andβ-cyclodextrins. One-dimensional difference NOE and spin-lattice relaxation time (T1) measurements indicated that the aglycone protion was affected more than the neohesperidoside portion. TheT1 values analysed for local motions indicated thatτ c values of complexed naringin was higher than that of free naringin. The internal rotation calculated for different groups showedτ i values for the phenolic and dihydrobenzopyran portion decrease by a factor of 2. Also a ξ value of 0.12–0.17 observed for the aglycone portion indicated that the coupling between guest and host is weak. All the studies have shown that the disposition in which the phenol group at 2 is inside the BCD cavity with 4-keto and 5-OH hydrogen bonded to the secondary hydroxyl groups at the rim of the wider end of the BCD cavity is the most probable one.

13C NMR study of 2‐iodoso‐ and 2‐iodoxy‐benzoic acids and their sodium salts

AbstractThe 13C NMR spectra for selected 2‐iodosobenzoic acids, their sodium salts and the sodium salts of the corresponding iodoxybenzoic acids were measured and carbon assignments made using 2D and NOE experiments and relaxation times. Iodoso and iodoxy groups deshielded the ipso‐carbon in these compounds by approximately 25 and 55 ppm, respectively, relative to the corresponding iodo compounds. The 13C NMR spectra of the 2‐iodosobenzoate anions were almost identical with those of the corresponding free acids, both possessing cyclic structures. The iodoxybenzoic acids are either insoluble in, or oxidize, suitable NMR solvents (i.e. DMSO‐d6, DMF‐d7). The cyclic structure postulated for iodoxybenzoate anions is supported by their 13C chemical shifts.

Influence of molecular geometry on uncertainty in distances determined from NOE

The initial-slope and full-relaxation-matrix methods of extracting internuclear distances from NOE data are examined with special emphasis on the effect of the geometrical arrangement of the spins on the accuracy of the ratios of distances determined. The source of geometric error may be understood in terms of the relationship of molecular conformation to the relative sizes of the eigenvalues of the NOE relaxation matrix. If these eigenvalues differ greatly in magnitude, large distance errors may result. It is shown tha for certain molecular geometries the distance between spins may be accurately measured by either method, while some geometries require the full-matrix analysis for accuracy. There are a few structures which cannot be resolved by either procedure. These distinctions are revealed by an analytical solution for the homonuclear NOE in an isotropically tumbling three-spin system in the limit of long correlation time. This solution was utilized to obtain computer simulation of the NOE for a variety of geometrical arrangements. Contour maps are presented to depict the errors in the distances determined from NOE and to indicate exactly which structures are resistant to NOE analysis.