Significant Chemical Shift Differences Research Articles

Three-dimensional solution NMR structure of Apo-L75F-TrpR, a temperature-sensitive mutant of the tryptophan repressor protein.

L75F-TrpR is a temperature-sensitive mutant of the tryptophan repressor protein of Escherichia coli in which surface-exposed residue leucine 75 in the DNA binding domain is replaced with phenylalanine. Biochemical and biophysical studies had suggested global alterations in dynamics for L75F-TrpR, although the structure was apparently similar to that of wild-type TrpR. Herein, we report the three-dimensional solution structure of apo-L75F-TrpR determined by multidimensional ((1)H, (15)N, and (13)C) solution NMR spectroscopy. An ensemble of structures was generated from 769 unique NOE-based distance restraints, 68 dihedral angle restraints, and 62 hydrogen bond distance restraints. Apo-L75F-TrpR exhibits a three-dimensional (3D) fold very similar to that of apo-WT-TrpR, with a dimeric core of four alpha-helices (A-C and F) from each subunit, and less well-defined D and E helical regions of the DNA binding domains. Despite their many similarities, wild-type and mutant proteins display significant chemical shift differences, one cluster of which is in the B-C turn, too distant to be ascribed solely to ring current effects from Phe75. Differences in NOE patterns and amide proton exchange rates are also observed in the B-C turn region. The data provide evidence that this point mutation exerts local effects on structure and stability in the DNA binding domain, and propagates long-range effects through the tertiary structure.

Characterization of the DNA-binding domains from the yeast cell-cycle transcription factors Mbp1 and Swi4.

The minimal DNA-binding domains of the Saccharomyces cerevisiae transcription factors Mbp1 and Swi4 have been identified and their DNA binding properties have been investigated by a combination of methods. An approximately 100 residue region of sequence homology at the N-termini of Mbp1 and Swi4 is necessary but not sufficient for full DNA binding activity. Unexpectedly, nonconserved residues C-terminal to the core domain are essential for DNA binding. Proteolysis of Mbp1 and Swi4 DNA-protein complexes has revealed the extent of these sequences, and C-terminally extended molecules with substantially enhanced DNA binding activity compared to the core domains alone have been produced. The extended Mbp1 and Swi4 proteins bind to their cognate sites with similar affinity [K(A) approximately (1-4) x 10(6) M(-)(1)] and with a 1:1 stoichiometry. However, alanine substitution of two lysine residues (116 and 122) within the C-terminal extension (tail) of Mbp1 considerably reduces the apparent affinity for an MCB (MluI cell-cycle box) containing oligonucleotide. Both Mbp1 and Swi4 are specific for their cognate sites with respect to nonspecific DNA but exhibit similar affinities for the SCB (Swi4/Swi6 cell-cycle box) and MCB consensus elements. Circular dichroism and (1)H NMR spectroscopy reveal that complex formation results in substantial perturbations of base stacking interactions upon DNA binding. These are localized to a central 5'-d(C-A/G-CG)-3' region common to both MCB and SCB sequences consistent with the observed pattern of specificity. Changes in the backbone amide proton and nitrogen chemical shifts upon DNA binding have enabled us to experimentally define a DNA-binding surface on the core N-terminal domain of Mbp1 that is associated with a putative winged helix-turn-helix motif. Furthermore, significant chemical shift differences occur within the C-terminal tail of Mbp1, supporting the notion of two structurally distinct DNA-binding regions within these proteins.

The acid-induced folded state of Sac7d is the native state.

Sac7d unfolds at low pH in the absence of salt, with the greatest extent of unfolding obtained at pH 2. We have previously shown that the acid unfolded protein is induced to refold by decreasing the pH to 0 or by addition of salt (McCrary BS, Bedell J. Edmondson SP, Shriver JW, 1998, J Mol Biol 276:203-224). Both near-ultraviolet circular dichroism spectra and ANS fluorescence enhancements indicate that the acid- and salt-induced folded states have a native fold and are not molten globular. 1H,15N heteronuclear single quantum coherence NMR spectra confirm that the native, acid-, and salt-induced folded states are essentially identical. The most significant differences in amide 1H and 15N chemical shifts are attributed to hydrogen bonding to titrating carboxyl side chains and through-bond inductive effects. The 1H NMR chemical shifts of protons affected by ring currents in the hydrophobic core of the acid- and salt-induced folded states are identical to those observed in the native. The radius of gyration of the acid-induced folded state at pH 0 is shown to be identical to that of the native state at pH 7 by small angle X-ray scattering. We conclude that acid-induced collapse of Sac7d does not lead to a molten globule but proceeds directly to the native state. The folding of Sac7d as a function of pH and anion concentration is summarized with a phase diagram that is similar to those observed for other proteins that undergo acid-induced folding except that the A-state is encompassed by the native state. These results demonstrate that formation of a molten globule is not a general property of proteins that are refolded by acid.

NMR analysis of the binding of a rhodanese peptide to a minichaperone in solution

A detailed structural analysis of interactions between denatured proteins and GroEL is essential for an understanding of its mechanism. Minichaperones constitute an excellent paradigm for obtaining high-resolution structural information about the binding site and conformation of substrates bound to GroEL, and are particularly suitable for NMR studies. Here, we used transferred nuclear Overhauser effects to study the interaction in solution between minichaperone GroEL(193-335) and a synthetic peptide (Rho), corresponding to the N-terminal α-helix (residues 11 to 23) of the mitochondrial rhodanese, a protein whose in vitro refolding is mediated by minichaperones. Using a 60 kDa maltose-binding protein (MBP)-GroEL(193-335) fusion protein to increase the sensitivity of the transferred NOEs, we observed characteristic sequential and mid-range transferred nuclear Overhauser effects. The peptide adopts an α-helical conformation upon binding to the minichaperone. Thus the binding site of GroEL is compatible with binding of α-helices as well as extended β-strands. To locate the peptide-binding site on GroEL(193-335), we analysed changes in its chemical shifts on adding an excess of Rho peptide. All residues with significant chemical shift differences are localised in helices H8 and H9. Non-specific interactions were not observed. This indicates that the peptide Rho binds specifically to minichaperone GroEL(193-335). The binding region identified by NMR in solution agrees with crystallographic studies with small peptides and with fluorescence quenching studies with denatured proteins.

Assignment of Relative Configuration to Acyclic Compounds Based on (13)C NMR Shifts. A Density Functional and Molecular Mechanics Study.

1,3-Dimethylated hydrocarbon segments occur frequently as structural elements in polyketide natural products. The (13)C NMR chemical shifts of a series of model compounds containing such segments can be well reproduced by a combination of molecular mechanics and SOS-DFPT/IGLO calculations. (13)C NMR chemical shifts are calculated on MM3 geometries and are Boltzmann weighted according to the MM3 energies. On the basis of the resulting thermally averaged chemical shifts, all diastereomers of the model compounds can be unequivocally distinguished. Significant differences in chemical shifts occur at methyl groups and methylene groups that are adjacent to a single stereogenic center. The method is applied to predict the relative configuration of two stereocenters in the side chains of two natural products, sambutoxin and the bradykinin inhibitor L-755,897.

Solution structure of a human cystatin A variant, cystatin A2-98 M65L, by NMR spectroscopy. A possible role of the interactions between the N- and C-termini to maintain the inhibitory active form of cystatin A.

The solution structure of a human cystatin A variant, cystatin A2-98 M65L, which maintains the full inhibitory activity of the wild-type protein, was determined at pH 3.8 by sD/3D heteronuclear double- and triple-resonance NMR spectroscopy. The structure is based on a total of 1343 experimental restraints, comprising 1139 distance, 154 phi and chi 1 torsion angle restraints, and 50 distance constraints for 25 backbone hydrogen bonds. A total of 15 structures was calculated using the YASAP protocol with X-PLOR, and the atomic rms distribution about the mean coordinate positions for residues 8-93 was 0.55 +/- 0.10 A for the backbone atoms and 1.05 +/- 0.11 A for all heavy atoms. The structure consists of five antiparallel beta-sheets and two short alpha-helices. Comparison with the X-ray structure of cystatin B in the papain complex shows that the conformation of the first binding loop is quite similar to that of cystatin A, with an rms deviation of 0.78 A for the backbone atoms in the 43-53 region (cystatin A numbering). The second binding loop, however, is significantly different in the two structures, with an rms deviation greater than 2 A. There are some other significant differences, especially for the N-terminal and alpha-helix regions. The overall structure of cystatin A is also compared with the recently reported NMR structure of the wild-type cystatin A (stefin A) at pH 5.5 (Martin et al., 1995) and reveals the following features. that differ in our structure from the previous one: (1) the N-terminal segment, which was unstructured in the previous report, folds over in close vicinity to the C-terminus, as revealed by the distinctive NOEs between those segments; (2) two discrete short alpha-helices linked by a type II reverse turn were found, instead of the continuous single alpha-helix with a slight kink shown in the previous structure; (3) the second binding loop, which was not well converged in the previous study at pH 5.5, is determined very well in our structure. The effect of the N-terminal truncation on the cystatin A structure was examined by comparing the 1H-15N HSQC spectrum of cystatin A2-98 with that of the cystatin A5-98 variant, which lacks the anti-papain activity, revealing significant chemical shift differences in the residual N-terminal segment and the first binding loop, together with small shifts in the other parts.(ABSTRACT TRUNCATED AT 400 WORDS)

Heteronuclear NMR studies of cobalt corrinoids—17. characterization of neopentylcobinamide and neopentyl-13-epicobinamide by 1H and 13C NMR spectroscopy: Inferred corrin ring conformations from chemical shift differentials

While the 1H and 13C NMR spectra of neopentylcobalamin (NpCbl) and its epimer at corrin ring C(13), neopentyl-13-epiCbl (Np-13-epiCbl), are extremely broad, apparently due to chemical exchange between the base-on and base-off species, the cobinamide derivatives, neopentyl cobinamide (NpCbi +) and neopentyl-13-epicobinamide (Np-13-epiCbi +), have sharp, well-resolved NMR spectra. The 1H and 13C NMR spectra of NpCbi + and Np-13-epiCbi + have now been completely assigned by modern two-dimensional NMR methodologies. Comparison of the 13C spectra of these two complexes shows that significant chemical shift differences occur at a variety of corrin ring and peripheral carbon atoms and are not localized near the site of epimerization. Similarly, comparison of the 13C NMR spectra of NpCbi + and 5′-deoxyadenosylcobinamide (AdoCbi +) shows differences at many corrin ring and peripheral carbons. A first attempt at discerning differences in corrin ring conformation from such differences in 13C chemical shift has been made by comparing the X-ray crystal structures and 13C NMR spectra of 5′-deoxyadenosylcobalamin (coenzyme B 12, AdoCbl) and cyanocobalamin (vitamin B 12, CNCbl). After elimination of carbon atoms whose chemical shifts are likely to be significantly affected by differences in the inductive effect of the Ado and CN ligands, and after consideration of differential anisotropic shielding effects in the two complexes due to the presence or absence of the Ado ligand, the difference in magnetic anisotropy of the central cobalt atom, the change in position of the axial nucleotide and differences in the magnetic anisotropy of the corrin ligand, 15 peripheral carbon atoms [C(2), C(18), C(20), C(25), C(26), C(30), C(36), C(37), C(41), C(46), C(47), C(48), C(54), C(55), C(60)] emerge as candidate “reporter” carbons whose 13C chemical shifts may be useful in deducing conformational differences in cobalt corrinoids. Application of this method to adeninylpropylcobalamin (AdePrCbl), for which the X-ray crystal structure and absolute NMR assignments are known, correctly predicts the gross conformational differences between the corrin ring of AdePrCbl and that of CNCbl. Use of these reporter carbon chemical shifts suggests that in NpCbi +, the fold angle, defined as the angle between the “northern” and “southern” planes of the corrin ring, is reduced relative to AdoCbi +. Comparison of the chemical shifts of the reporter carbon atoms in NpCbi + and Np-13-epiCbi + suggests that the fold angle in the former is larger than that for the latter.

Conformational properties of the -35 region of the trp promoter in solution: comparison of the wild-type sequence with an AT transversion.

The majority of the 1H NMR resonances of the protons in a tetradecamer containing the -35 region of the trp promoter d(GCTGTTGACAATTA): d(TAATTGTCAACAGC) and in the TA transversion have been assigned. The conformational properties of the nucleotides have been determined and compared in the two duplexes. Analysis of spin-spin coupling and NOEs shows that all sugar puckers are in the south domain (i.e. near C2' endo) and the glycosidic torsion angles are anti (chi approximately 110 degrees). The NMR data are consistent with the duplex being in the B family of conformations. Significant differences in chemical shifts between the two molecules were observed only for nearest neighbours to the transversion site, suggesting the absence of long range conformational effects. This was confirmed by the similarity of coupling constants and NOEs. Other properties are also not greatly affected at positions more than two base pairs from the mutation site. These results are consistent with the hypothesis that unconstrained oligonucleotides are highly flexible, and can readily accommodate significant perturbations of the local structure, such as a transversion.

Conformation and dynamics of an Fab'-bound peptide by isotope-edited NMR spectroscopy

The dynamics and conformation of the peptide antigen MHKDFLEKIGGL bound to the Fab' fragment of the monoclonal antipeptide antibody B13A2, raised against a peptide from myohemerythrin, have been investigated by isotope-edited NMR techniques. The peptides were labeled with 15N (98%) or 13C (99%) at the backbone of individual amino acid residues. Well-resolved amide proton and nitrogen backbone resonances were obtained and assigned for eight of the 12 residues of this bound peptide. Significant resonance line width and chemical shift differences were observed. The 15N and 1H line width variations are attributed to differential backbone mobilities among the bound peptide residues which are consistent with the previously mapped epitope of this peptide antigen. Local structural information was obtained from isotope-directed NOE studies. The approximate distances associated with the experimental NOEs were estimated on the basis of a theoretical NOE analysis involving the relative integrated intensities of the NOE and source peaks. In this way, the sequential NH-NH NOEs obtained for seven of the Fab'-bound peptide residues were shown to correspond to interproton separations of approximately 3 A or less. Such short distances indicate that the backbone dihedral angles of these residues are in the alpha rather than the beta region of phi,psi conformational space; the peptide most likely adopts a helical conformation from F5 to G11 within the antibody combining site. The significance of these results with respect to the type and extent of conformational information obtainable from studies of high molecular weight systems is discussed.

Conformation of MeAla6‐cyclosporin A by NMR

MeAla6-cyclosporin A (MeAla6-CsA) is a unique CsA analog that shows weak immunosuppressive activity and yet binds strongly to the proposed cytosolic protein receptor, cyclophilin (CyP). Preliminary 1H NMR data showed significant chemical shift differences between spectra of MeAla6-CsA and CsA, suggesting different preferred conformations. A more detailed study, however, revealed that the backbone conformations of the two molecules are essentially identical, and that the differences can be accounted for, principally, by the sidechain motions of the MeBmt-1, MeLeu-9, and -10 residues. ROE and coupling constant data show that in MeAla6-CsA, the preferred chi 1 rotamers for MeLeu-9 and -10 are + 180 degrees (T), whereas in CsA there is a more even distribution of rotamer populations for MeLeu-10, and a preferred -60 degrees (G-) chi 1 rotamer for MeLeu-9. Similar data argue that the sidechain of MeBmt-1 is more restricted in its motion in MeAla-CsA than in CsA. Temperature studies suggest that these preferred rotamers for MeAla6-CsA may increase the stability of the hydrogen bond between NH(7) and CO(11), but prevent particular residues, especially the essential MeBmt-1 sidechain, from adopting orientations required to elicit immunosuppressive activity. The significant changes observed in the preferred orientations for the sidechains of the MeBmt-1, MeLeu-9, and MeLeu-10 residues in MeAla6-CsA argue that the particular orientations which they assume in CsA are not essential for cyclophilin binding.

Water Mobility and Crystallization Action of Lactose‐water Systems by Oxygen‐17 and Carbon‐13 NMR

ABSTRACTSystems ranging in concentration from 10‐60g lactose/100g water were investigated by fully proton decoupled 17O and fully proton decoupled 13C Nuclear Magnetic Resonance (NMR). The relation between 17O NMR transverse relaxation rate measurements and lactose concentration showed water mobility decreased linearly with increasing lactose concentration up to 25g lactose/100g water and decreased rapidly and nonlinearly above that level. Water mobility was used to monitor lactose crystallization in supersaturated solutions with time. The 13C NMR showed no significant differences in chemical shifts of carbon atom peaks in lactose molecules during crystallization. However, depending on the method of sample preparation, mutarotation could be observed.

Conformational analysis of the tumor-associated carbohydrate antigen 19-9 and its Lea blood group antigen component as related to the specificity of monoclonal antibody CO19-9.

A structural comparison between the synthetic, tumor-associated 19-9 tetrasaccharide, NeuAc alpha 2----3Gal beta 1----3GlcNAc(4----1 alpha Fuc)-O(CH2)8CO2CH3 and its Lea blood group antigen component, Gal beta 1----3GlcNAc(4----1 alpha Fuc)-O(CH2)8CO2CH3 was carried out by two-dimensional 1H NMR spectroscopy and hard-sphere energy calculations. Significant chemical shift differences between the two molecules were detected only for protons at or near the linkage site of NeuAc to the Lea trisaccharide core. Coupling constants for the ring protons of both molecules did not suggest major deviation from the 4C1 chair conformation for Gal and GlcNAc, the 1C4 conformation for Fuc, or the 2C5 conformation for NeuAc. Two-dimensional nuclear Overhauser enhancement experiments revealed through-space, inter-proton interactions that corresponded to some extent with those predicted by diffraction data and hard-sphere energy minimization programs for both saccharides. However, a significant number of interactions did not obey the distance dependence predicted from a rigid structure model. These data suggest that, while the average conformation of the 19-9 antigen's Lea core may be invariant to NeuAc alpha 2----3Gal linkage, the dynamics of the Lea trisaccharide are altered upon sialylation. Data also indicate that the terminal NeuAc linkage is more flexible than the inter-residue bonds of the core trisacharide. This analysis, in combination with the fact that the monoclonal anti-19-9 antibody CO 19-9 does not cross-react with the Lea antigen, provides evidence in favor of NeuAc as an epitope-creating unit involved directly at the antibody binding site. However, given the possible role of variable dynamics in epitope formation, these results do not preclude crucial roles in antibody recognition for regions on the 19-9 antigen that are distanced from NeuAc.

ChemInform Abstract: Some Selenium Analogues of the Anionic Tridentate Tripodal Sulfur Ligand, Tris(diphenylthiophosphinyl)methanide.

Abstract A new potential tridentate tripodal ligand, bis (diphenylselenophosphinyl)diphenylthiophosphinylmethanide, and its neutral protonated parent have been synthesized. The stable conformations of HTrisSSe2 and its trichalcogen analogues can be determined by variable temperature 31p NMR spectroscopy because of significant differences in chemical shifts for a particular chalcogenophosphinyl group in different stereochemical orientations within the sterically hindered molecules.

SOME SELENIUM ANALOGUES OF THE ANIONIC TRIDENTATE TRIPODAL SULFUR LIGAND, TRIS(DIPHENYLTHIOPHOSPHINYL) METHANIDE

Abstract A new potential tridentate tripodal ligand, bis (diphenylselenophosphinyl)diphenylthiophosphinylmethanide, and its neutral protonated parent have been synthesized. The stable conformations of HTrisSSe2 and its trichalcogen analogues can be determined by variable temperature 31p NMR spectroscopy because of significant differences in chemical shifts for a particular chalcogenophosphinyl group in different stereochemical orientations within the sterically hindered molecules.

13C and pulse nuclear magnetic resonance spectroscopy of wheat proteins

High resolution 13C nuclear magnetic resonance experiments were carried out on dry and hydrated gluten, glutenin and gliadin. The spectra of hydrated materials displayed narrower line widths than the cross-polarisation magic angle spinning spectra of the solids, but there were no significant differences in chemical shifts. Gluten, glutenin and gliadin spectra were similar and did not change when the hydrated proteins were heated at 80 °C/30 min, 1H spin-spin relaxation spectra (T2) of the proteins in D2O revealed a high degree (~ 65%) of segmental mobility. This increased with temperature but returned to a value close to the original on cooling. These data indicate that wheat proteins have open structures and that these are retained after heating, even though the formation of new disulphide bonds dramatically changes their rheological properties.

Chemical properties and stereoisomerism of heterogeneous long chain bases in lysosphingolipids by positive ion fast atom bombardment mass spectrometry and carbon-13 NMR spectroscopy.

Positive ion fast atom bombardment (FAB) mass spectrometry of galactopsychosine and glucopsychosine was capable of showing not only the pseudo molecular ion peaks, but also various fragment ion peaks such as protonated sphingosine and its fragment ions. The percent distribution of sphingosine and dihydrosphingosine in each lysosphingolipid was determined by GLC of the trimethyl-silylated derivatives of long chain bases after methanolysis and was comparable to the relative intensities of ion peaks derived from the sphingosine and dihydrosphingosine groups. The FAB mass spectra showed that during the fast atom bombardment the sphingosine more preferentially gave rise to one and/or two fragment ions by loss of one and/or two molecules of water than the dihydrosphingosine did. The stereoisomerism of sphingosylphosphorylcholine containing mainly L-threo-sphingosine could be reconfirmed by carbon-13 NMR spectroscopy. Furthermore, although the carbon-13 NMR signals of sphingosine C-1, C-2, C-3, C-4, and C-5 showed significant chemical shift differences between D-erythro and L-threo-sphingosines of lysosphingolipids, it was concluded that the signal position of sphingosine C-3 was the most important for the determination of D-erythro and L-threo configuration in the long chain base moieties of lysosphingolipids.

Synthesis and physico‐chemical properties of (R, S)‐6‐ditrideuteriomethylamino‐4,4‐diphenylheptan‐3‐one hydrochloride (methadone‐d6)

AbstractA reaction sequence is described to synthesize (R, S)‐6‐ditrideuteriomethylamino‐4,4‐diphenylheptan‐3‐one hydrochloride ( methadone‐d6.HCl ) in 10.5% overall yield starting from dimethylamine‐d6 hydrochloride. While methadone‐d6.HCl had rather similar physicochemical properties compared to methadone.HCl in the solid phase, a divergent behaviour was observed in solution ( pKa, chromatographic and 13C‐NMR data ). A higher basicity along with a more important hydrophilicity were observed for the d6‐derivative. A 13C‐NMR study showed significant differences in the 13C chemical shifts, which were attributed in part to the intrinsic nature of D itself and in part to a conformational perturbation. The longer duration of antinociceptive action, along with higher t1/2 and clearance, and the depression of the metabolic N‐demethylation process were the only biological properties modified by the deuteration.

13C nuclear magnetic resonance of organophosphorus compounds: triphenyl phosphite and triphenyl phosphate

The 13C n.m.r. spectra of triphenyl phosphite and triphenyl phosphate show significant differences in chemical shifts and C–P coupling constants.