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1.
Chemical shift anisotropy (CSA) tensor parameters have been determined for the protonated carbons of the purine bases in an RNA kissing complex in solution by extending the model-independent approach [Fushman, D., Cowburn, D. (1998) J. Am. Chem. Soc. 120, 7109–7110]. A strategy for determining CSA tensor parameters of heteronuclei in isolated X–H two-spin systems (X = 13C or 15N) in molecules undergoing anisotropic rotational diffusion is presented. The original method relies on the fact that the ratio κ2=R2auto/R2cross of the transverse auto- and cross-correlated relaxation rates involving the X CSA and the X–H dipolar interaction is independent of parameters related to molecular motion, provided rotational diffusion is isotropic. However, if the overall motion is anisotropic κ2 depends on the anisotropy D||/D of rotational diffusion. In this paper, the field dependence of both κ2 and its longitudinal counterpart κ1=R1auto/R1cross are determined. For anisotropic rotational diffusion, our calculations show that the average κav = 1/2 (κ12), of the ratios is largely independent of the anisotropy parameter D||/D. The field dependence of the average ratio κav may thus be utilized to determine CSA tensor parameters by a generalized model-independent approach in the case of molecules with an overall motion described by an axially symmetric rotational diffusion tensor.  相似文献   

2.
A new approach is described for measuring chemical shift anisotropy (CSA)/dipolar cross-correlated relaxation (CCR) rates based on the selection of the individual 15N doublet components prior to the relaxation period. The method uses the spin-state-selective element (S3E) of Sørensen and co-authors [Meissner et al. (1997) J. Mag. Reson., 128, 92–97]. The main advantage of the new method compared to other J-resolved experiments is that it does not create problems of additional signal overlap encountered in coupled spectra. At the same time, this approach allows a simpler control of magnetization pathways than the indirect methods. The method is demonstrated for the B3 domain of protein G.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s10858-004-7562-8  相似文献   

3.
An approach to the determination of the orientation of the carbonyl chemical shift (CS) tensor in a 13C-15N-1H dipolar coupled spin network is proposed. The method involves the measurement of the Euler angles of the 13C-15N and 15N-1H dipolar vectors in the 13C CS tensor principal axes system, respectively, via a 13C-15N REDOR experiment and by a 2D relayed anisotropy correlation of the 13C CSA (2) and 15N-1H dipolar interaction (1). Via numerical simulations the sensitivity of the 1 cross sections of the 2D spectrum to the Euler angles of the 15N-1H bond vector in the 13C CSA frame is shown. Employing the procedure outlined in this work, we have determined the orientation of the 13C CS tensor in the peptide plane of the dipeptide AibAib-NH2 (Aib = -aminoisobutyric acid). The Euler angles are found to be (CN, CN) = (34° ± 2°, 88° ± 2° ) and (NH, NH) = (90° ± 10°, 80° ± 10° ). From the measured Euler angles it is seen that the 33 and 22 components of the 13C CS tensor approximately lie in the peptide plane.  相似文献   

4.
Carbonyl 13C′ relaxation is dominated by the contribution from the 13C′ chemical shift anisotropy (CSA). The relaxation rates provide useful and non-redundant structural information in addition to dynamic parameters. It is straightforward to acquire, and offers complimentary structural information to the 15N relaxation data. Furthermore, the non-axial nature of the 13C′ CSA tensor results in a T1/T2 value that depends on an additional angular variable even when the diffusion tensor of the protein molecule is axially symmetric. This dependence on an extra degree of freedom provides new geometrical information that is not available from the NH dipolar relaxation. A protocol that incorporates such structural restraints into NMR structure calculation was developed within the program Xplor-NIH. Its application was illustrated with the yeast Fis1 NMR structure. Refinement against the 13C′ T1/T2 improved the overall quality of the structure, as evaluated by cross-validation against the residual dipolar coupling as well as the 15N relaxation data. In addition, possible variations of the CSA tensor were addressed. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.  相似文献   

5.
13C‐nmr chemical shift tensor components are reported for a 13C‐labeled Gly1 amide carbonyl carbon of a glycylglycine (Gly1Gly2) single crystal, a GlyGly · HNO3 single crystal and a GlyGly · HCl · H2O single crystal, for which the three‐dimensional crystal structures have already been determined by x‐ray diffraction. The tensor components were measured by changing the angle between the crystal plane and the applied magnetic field by using a goniometer designed in this work for use in conventional 13C cross‐polarization/magic angle spinning nmr probe. From these experimental data, the principal values of the 13C chemical shift tensor and its directions for the Gly1 amide carbonyl carbon were determined. It was found that the 13C chemical shift tensor components (δ11, δ22, and δ33) for the Gly1 amide carbonyl carbon in GlyGly and GlyGly · HNO3 with a >NH · · · OC< type of hydrogen bond depend on the hydrogen‐bond length and the directions of the δ22 components of these peptides are along the hydrogen‐bonded >CO bond axis. In addition, the magnitude of the deviation from the bond axis depends on the hydrogen‐bond angle. Further, the experimental result for GlyGly · HCl · H2O with a  O H · · ·OC< type of hydrogen bond was discussed. © 1999 John Wiley & Sons, Inc. Biopoly 50: 61–69, 1999  相似文献   

6.
Dynamics and structure of (1–36)bacteriorhodopsin solubilized in chloroform/methanol mixture (1:1) were investigated by 1H-15N NMR spectroscopy under a hydrostatic pressure of 2000 bar. It was shown that the peptide retains its spatial structure at high pressure. 15N transverse and longitudinal relaxation times, 15N{1H} nuclear Overhauser effects, chemical shifts and the translation diffusion rate of the peptide at 2000 bar were compared with the respective data at ambient pressure [Orekhov et al. (1999) J. Biomol. NMR, 14, 345–356]. The model free analysis of the relaxation data for the helical 9–31 fragment revealed that the high pressure decreases the overall rotation and translation diffusion, as well as apparent order parameters of fast picosecond internal motions (S2 f) but has no effect on internal nanosecond motions (S2 s and s) of the peptide. The decrease of translation and overall rotation diffusion was attributed to the increase in solvent viscosity and the decrease of apparent order parameters S2 f to a compression of hydrogen bonds. It is suggested that this compression causes an elongation of H-N bonds and a decrease of absolute values of chemical shift anisotropy (CSA). In particular, the observed decrease of S2 f at 2000 bar can be explained by 0.001 nm increase of N-H bond lengths and 10 ppm decrease of 15N CSA values.  相似文献   

7.
A detailed analysis of peptide backbone amide (HN) and Hα chemical shifts reveals a consistent pattern for β hairpins and three-stranded β sheets. The Hα’s at non-hydrogen-bonded strand positions are inwardly directed and shifted downfield ~1 ppm due largely to an anisotropy contribution from the cross-strand amide function. The secondary structure associated Hα shift deviations for the H-bonded strand positions are also positive but much smaller (0.1–0.3 ppm) and the turn residues display negative Hα chemical shift deviations (CSDs). The pattern of (HN) shift deviations is an even better indicator of both hairpin formation and register, with the cross-strand H-bonded sites shifted downfield (also by ~1 ppm) and with diagnostic values for the first turn residue and the first strand position following the turn. These empirical observations, initially made for [2:2]/[2:4]-type-I' and -II' hairpins, are rationalized and can be extended to the analysis of other turns, hairpin classes ([3:5], [4:4]/[4:6]), and three-stranded peptide β-sheet models. The Hα’s at non-hydrogen-bonded sites and (HN)’s in the intervening H-bonded sites provide the largest and most dependable measures of hairpin structuring and can be used for melting studies; however the intrinsic temperature dependence of (HN) shifts deviations needs to reflect the extent of solvent sequestration in the folded state. Several observations made in the course of this study provide insights into β-sheet folding mechanisms: (1) The magnitude of the (HN) shifts suggests that the cross-strand H-bonds in peptide hairpins are as short as those in protein β sheets. (2) Even L-Pro-Gly turns, which are frequently used in unfolded controls for hairpin peptides, can support hairpin populations in aqueous fluoroalcohol media. (3) The good correlation between hairpin population estimates from cross-strand H-bonded (HN) shift deviations, Hα shift deviations, and structuring shifts at the turn locus implies that hairpin folding transitions approximate two-state behavior. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.  相似文献   

8.
The effects of chemical shift anisotropy (CSA) are evident in line-shapes or side-band analysis in solid-state NMR, in the observed line positions in partially oriented samples, and in relaxation effects in liquid-state studies. In all of these cases, the effective shielding tensor is influenced by fast vibrational averaging in addition to larger-amplitude internal motions and to overall libration or rotation. Here we compute the contributions of vibrational averaging (including zero-point motions) to the CSA relaxation strengths for the nitrogen and carbonyl carbon in two simple peptide models, and for snapshots taken from a path-integral simulation of a small protein. Because the 15N shielding tensor is determined by all the atoms of the peptide group, it is less influenced by vibrational motion than (for example) the N–H dipolar interaction, which is more sensitive to the motion of the light hydrogen atom. Computed order parameters for CSA averaging are hence much closer to unity than are N–H dipolar order parameters. This leads to a reduction by about 9% in the magnitude of the amide nitrogen CSA that is needed to fit liquid-state relaxation data. Similar considerations apply to the carbonyl carbon shielding tensor, but in this case the differences between dipolar and CSA averaging are smaller. These considerations will be important for making comparisons between CSA tensors extracted from various NMR experiments, and for comparisons to quantum chemical calculations carried out on static conformers.  相似文献   

9.
Here we propose a method for the measurement of the 15N CSA/dipolar relaxation interference based on direct comparison of the 15N doublet components observed in a 1H-coupled 1H-15N HSQC-type spectrum. This allows the determination of the cross-correlation rates with no need for correction factors associated with other methods. The signal overlap problem of coupled HSQC spectra is addressed here by using the IPAP scheme (Ottiger et al., 1998). The approach is applied to the B3 domain of protein G to show that the method provides accurate measurements of the 15N CSA/dipolar cross-correlation rates.  相似文献   

10.
A set of TROSY-HNCO (tHNCO)-based 3D experiments is presented for measuring 15N relaxation parameters in large, membrane-associated proteins, characterized by slow tumbling times and significant spectral overlap. Measurement of backbone 15N R 1, R , 15N–{1H} NOE, and 15N CSA/dipolar cross correlation is demonstrated and applied to study the dynamic behavior of the homotetrameric KcsA potassium channel in SDS micelles under conditions where this channel is in the closed state. The micelle-encapsulated transmembrane domain, KcsATM, exhibits a high degree of order, tumbling as an oblate ellipsoid with a global rotational correlation time, τc = 38 ± 2.5 ns, at 50 °C and a diffusion anisotropy, , corresponding to an aspect ratio a/b ≥ 1.4. The N- and C-terminal intracellular segments of KcsA exhibit considerable internal dynamics (S 2 values in the 0.2–0.45 range), but are distinctly more ordered than what has been observed for unstructured random coils. Relaxation behavior in these domains confirms the position of the C-terminal helix, and indicates that in SDS micelles, this amphiphilic helix does not associate into a stable homotetrameric helical bundle. The relaxation data indicate the absence of elevated backbone dynamics on the ps–ns time scale for the 5-residue selectivity filter, which selects K+ ions to enter the channel. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at . An erratum to this article can be found at  相似文献   

11.
Density functional theory was employed to study the influence of O-phosphorylation of serine, threonine, and tyrosine on the amidic 15N chemical shielding anisotropy (CSA) tensor in the context of the complex chemical environments of protein structures. Our results indicate that the amidic 15N CSA tensor has sensitive responses to the introduction of the phosphate group and the phosphorylation-promoted rearrangement of solvent molecules and hydrogen bonding networks in the vicinity of the phosphorylated site. Yet, the calculated 15N CSA tensors in phosphorylated model peptides were in range of values experimentally observed for non-phosphorylated proteins. The extent of the phosphorylation induced changes suggests that the amidic 15N CSA tensor in phosphorylated proteins could be reasonably well approximated with averaged CSA tensor values experimentally determined for non-phosphorylated amino acids in practical NMR applications, where chemical surrounding of the phosphorylated site is not known a priori in majority of cases. Our calculations provide estimates of relative errors to be associated with the averaged CSA tensor values in interpretations of NMR data from phosphorylated proteins.  相似文献   

12.
The dependence of the 13C chemical shift on side-chain orientation was investigated at the density functional level for a two-strand antiparallel β-sheet model peptide represented by the amino acid sequence Ac-(Ala)3-X-(Ala)12-NH2 where X represents any of the 17 naturally occurring amino acids, i.e., not including alanine, glycine and proline. The dihedral angles adopted for the backbone were taken from, and fixed at, observed experimental values of an antiparallel β-sheet. We carried out a cluster analysis of the ensembles of conformations generated by considering the side-chain dihedral angles for each residue X as variables, and use them to compute the 13C chemical shifts at the density functional theory level. It is shown that the adoption of the locally-dense basis set approach for the quantum chemical calculations enabled us to reduce the length of the chemical-shift calculations while maintaining good accuracy of the results. For the 17 naturally occurring amino acids in an antiparallel β-sheet, there is (i) good agreement between computed and observed 13Cα and 13Cβ chemical shifts, with correlation coefficients of 0.95 and 0.99, respectively; (ii) significant variability of the computed 13Cα and 13Cβ chemical shifts as a function of χ1 for all amino acid residues except Ser; and (iii) a smaller, although significant, dependence of the computed 13Cα chemical shifts on χξ (with ξ ≥ 2) compared to χ1 for eleven out of seventeen residues. Our results suggest that predicted 13Cα and 13Cβ chemical shifts, based only on backbone (φ,ψ) dihedral angles from high-resolution X-ray structure data or from NMR-derived models, may differ significantly from those observed in solution if the dihedral-angle preferences for the side chains are not taken into account. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.  相似文献   

13.
The dynamics of the nucleobase and the ribose moieties in a 14-nt RNA cUUCGg hairpin-loop uniformly labeled with 13C and 15N were studied by 13C spin relaxation experiments. R1, R and the 13C-{1H} steady-state NOE of C6 and C1′ in pyrimidine and C8 and C1′ in purine residues were obtained at 298 K. The relaxation data were analyzed by the model-free formalism to yield dynamic information on timescales of pico-, nano- and milli-seconds. An axially symmetric diffusion tensor with an overall rotational correlation time τc of 2.31±0.13 ns and an axial ratio of 1.35±0.02 were determined. Both findings are in agreement with hydrodynamic calculations. For the nucleobase carbons, the validity of different reported 13C chemical shift anisotropy values (Stueber, D. and Grant, D. M., 2002 J. Am. Chem. Soc. 124, 10539–10551; Fiala et al., 2000 J. Biomol. NMR 16, 291–302; Sitkoff, D. and Case, D. A., 1998 Prog. NMR Spectroscopy 32, 165–190) is discussed. The resulting dynamics are in agreement with the structural features of the cUUCGg motif in that all residues are mostly rigid (0.82 < S2 < 0.96) in both the nucleobase and the ribose moiety except for the nucleobase of U7, which is protruding into solution (S2 = 0.76). In general, ribose mobility follows nucleobase dynamics, but is less pronounced. Nucleobase dynamics resulting from the analysis of 13C relaxation rates were found to be in agreement with 15N relaxation data derived dynamic information (Akke et al., 1997 RNA 3, 702–709). Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.  相似文献   

14.
The 31P CP-MAS NMR spectra of trans-square-planar complexes of dihalonickel(II) complexes with tribenzyl-, tricyclohexyl- and tricyclohexylmethylphosphines have been examined and the chemical shift tensors determined. The spans, δ11-δ33, of the tensor components decrease with change in the halide, Cl > Br > I, for all the tertiary phosphines due principally to the deshielding of the δ33 component.  相似文献   

15.
NOEs between the β-protons of cysteine residues across disulfide bonds in proteins provide direct information on the connectivities and conformations of these important cross-links, which are otherwise difficult to investigate. With conventional [U-13C, 15N]-proteins, however, fast spin diffusion processes mediated by strong dipolar interactions between geminal β-protons prohibit the quantitative measurements and thus the analyses of long-range NOEs across disulfide bonds. We describe a robust approach for alleviating such difficulties, by using proteins selectively labeled with an equimolar mixture of (2R, 3S)-[β-13C; α,β-2H2] Cys and (2R, 3R)-[β-13C; α,β-2H2] Cys, but otherwise fully deuterated. Since either one of the prochiral methylene protons, namely β2 (proS) or β3 (proR), is always replaced with a deuteron and no other protons remain in proteins prepared by this labeling scheme, all four of the expected NOEs for the β-protons across disulfide bonds could be measured without any spin diffusion interference, even with long mixing times. Therefore, the NOEs for the β2 and β3 pairs across each of the disulfide bonds could be observed at high sensitivity, even though they are 25% of the theoretical maximum for each pair. With the NOE information, the disulfide bond connectivities can be unambiguously established for proteins with multiple disulfide bonds. In addition, the conformations around disulfide bonds, namely χ2 and χ3, can be determined based on the precise proton distances of the four β-proton pairs, by quantitative measurements of the NOEs across the disulfide bonds. The feasibility of this method is demonstrated for bovine pancreatic trypsin inhibitor, which has three disulfide bonds.  相似文献   

16.
Major urinary protein (MUP) is a pheromone-carrying protein of the lipocalin family. Previous studies by isothermal titration calorimetry (ITC) show that the affinity of MUP for the pheromone 2-methoxy-3-isobutylpyrazine (IBMP) is mainly driven by enthalpy, with a small unfavourable entropic contribution. Entropic terms can be attributed in part to changes in internal motions of the protein upon binding. Slow internal motions can lead to correlated or anti-correlated modulations of the isotropic chemical shifts of carbonyl C′ and amide N nuclei. Correlated chemical shift modulations (CSM/CSM) in MUP have been determined by measuring differences of the transverse relaxation rates of zero- and double-quantum coherences ZQC{C′N} and DQC{C′N}, and by accounting for the effects of correlated fluctuations of dipole–dipole couplings (DD/DD) and chemical shift anisotropies (CSA/CSA). The latter can be predicted from tensor parameters of C′ and N nuclei that have been determined in earlier work. The effects of complexation on slow time-scale protein dynamics can be determined by comparing the temperature dependence of the relaxation rates of APO-MUP (i.e., without ligand) and HOLO-MUP (i.e., with IBMP as a ligand). Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.  相似文献   

17.
Two novel HSQC-IPAP approaches are proposed to achieve α/β spin-state editing simultaneously for 13C and 15N in a single NMR experiment. The pulse schemes are based on a time-shared (TS) 2D 1H,13C/1H,15N-HSQC correlation experiment that combines concatenated echo elements for simultaneous J(CH) and J(NH) coupling constants evolution, TS evolution of 13C and 15N chemical shifts in the indirect dimension and heteronuclear α/β-spin-state selection by means of the IPAP principle. Heteronuclear α/β-editing for all CH n (n = 1–3) and NH n (1–2) multiplicities can be achieved in the detected F2 dimension of a single TS-HSQC-F2-IPAP experiment. On the other hand, an alternative TS-HSQC-F1-IPAP experiment is also proposed to achieve α/β-editing in the indirect F1 dimension. Experimental and simulated data is provided to evaluate these principles in terms of sensitivity and performance simultaneously on backbone and side-chain CH, CH2, CH3, NH, and NH2 spin systems in uniformly 13C/15N-labeled proteins and in small natural-abundance peptides.  相似文献   

18.
Chemical shifts of amino acids in proteins are the most sensitive and easily obtainable NMR parameters that reflect the primary, secondary, and tertiary structures of the protein. In recent years, chemical shifts have been used to identify secondary structure in peptides and proteins, and it has been confirmed that 1Hα, 13Cα, 13Cβ, and 13C′ NMR chemical shifts for all 20 amino acids are sensitive to their secondary structure. Currently, most of the methods are purely based on one-dimensional statistical analyses of various chemical shifts for each residue to identify protein secondary structure. However, it is possible to achieve an increased accuracy from the two-dimensional analyses of these chemical shifts. The 2DCSi approach performs two-dimension cluster analyses of 1Hα, 1HN, 13Cα, 13Cβ, 13C′, and 15NH chemical shifts to identify protein secondary structure and the redox state of cysteine residue. For the analysis of paired chemical shifts of 6 data sets, each of the 20 amino acids has its own 15 two-dimension cluster scattering diagrams. Accordingly, the probabilities for identifying helix and extended structure were calculated by using our scoring matrix. Compared with existing the chemical shift-based methods, it appears to improve the prediction accuracy of secondary structure identification, particularly in the extended structure. In addition, the probability of the given residue to be helix or extended structure is displayed, allows the users to make decisions by themselves. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Grant sponsor: National Science Council of ROC; Grant numbers: NSC-94-2323-B006- 001, NSC-93-2212-E-006.  相似文献   

19.
We have studied the influence of hydrogenation on the relative stability of the low-lying isomers of the anionic B7 cluster, computationally. It is known that the pure-boron B7 cluster has a doubly (σ- and π-) aromatic C6v (3A1) quasi-planar wheel-type triplet global minimum (structure 1), a low-lying σ-aromatic and π-antiaromatic quasi-planar singlet C2v (1A1) isomer 2 (0.7 kcal mol−1 above the global minimum), and a planar doubly (σ- and π-) antiaromatic C2v (1A1) isomer 3 (7.8 kcal mol−1 above the global minimum). However, upon hydrogenation, an inversion in the stability of the species occurs. The planar B7H2 (C2v, 1A1) isomer 4, originated from the addition of two hydrogen atoms to the doubly antiaromatic B7 isomer 3, becomes the global minimum structure. The second most stable B7H2 isomer 5, originated from the quasi-planar triplet wheel isomer 1 of B7, was found to be 27 kcal mol−1 higher in energy. The inversion in stability occurs due to the loss of the doubly aromatic character in the wheel-type global minimum isomer (C6v, 3A1) of B7 upon H2−addition. In contrast, the planar isomer of B7 (C2v, 1A1) gains aromatic character upon addition of two hydrogen atoms, which makes it more stable. Figure The B7H2-global minimum structure and its σ-aromatic and π-antiaromatic MOs Dedicated to Professor Dr. Paul von Ragué Schleyer on the occasion of his 75th birthday.  相似文献   

20.
The catalytic center of the [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F in the oxidized states was investigated by electron paramagnetic resonance and electron–nuclear double resonance spectroscopy applied to single crystals of the enzyme. The experimental results were compared with density functional theory (DFT) calculations. For the Ni-B state, three hyperfine tensors could be determined. Two tensors have large isotropic hyperfine coupling constants and are assigned to the β-CH2 protons of the Cys-549 that provides one of the bridging sulfur ligands between Ni and Fe in the active center. From a comparison of the orientation of the third hyperfine tensor with the tensor obtained from DFT calculations an OH bridging ligand has been identified in the Ni-B state. For the Ni-A state broader signals were observed. The signals of the third proton, as observed for the “ready” state Ni-B, were not observed at the same spectral position for Ni-A, confirming a structural difference involving the bridging ligand in the “unready” state of the enzyme. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. Maurice van Gastel and Matthias Stein contributed equally to this work.  相似文献   

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