Two-dimensional Nuclear Overhauser Spectroscopy Research Articles

Investigating lignin from Canna edulis ker residues induced activation of α-amylase: Kinetics, interaction, and molecular docking

The lignin isolated from C. edulis ker residues showed a significant activating effect on α-amylase. Further studies revealed that the isolated lignin formed a 1:1 complex with α-amylase through hydrogen bonding and quenched fluorescence of α-amylase with a static quenching procedure. Binding with lignin led to conformational and granular size changes of α-amylase. Two-dimensional nuclear Overhauser spectroscopy (2D-NOESY) spectra suggested that OH in G units and β-O-4 structure were the major binding sites of lignin on the α-amylase molecule. Molecular docking studies indicated that the binding residue on α-amylase for lignin was not the same as for chloride ions, and the major binding force was hydrogen bonding. Furthermore, the docking results also showed the structural change of lignin induced by α-amylase. Thus, this work provided a new insight into the interaction between lignin from Canna edulis ker residues and α-amylase, which may be beneficial to apply lignin in the food industry.

Solution NMR characterization of magnetic/electronic properties of azide and cyanide-inhibited substrate complexes of human heme oxygenase: Implications for steric ligand tilt

Solution 2D 1H NMR was carried out on the azide-ligated substrate complex of human heme oxygenase, hHO, to provide information on the active site molecular structure, chromophore electronic/magnetic properties, and the distal H-bond network linked to the exogenous ligand by catalytically relevant oriented water molecules. While 2D NMR exhibited very similar patterns of two-dimensional nuclear Overhauser spectroscopy cross peaks of residues with substrate and among residues as the previously characterized cyanide complex, significant, broadly distributed chemical shift differences were observed for both labile and non-labile protons. The anisotropy and orientation of the paramagnetic susceptibility tensor, χ, were determined for both the azide and cyanide complexes. The most significant difference observed is the tilt of the major magnetic axes from the heme normal, which is only half as large for the azide than cyanide ligand, with each ligand tilted toward the catalytically cleaved α-meso position. The difference in chemical shifts is quantitatively correlated with differences in dipolar shifts in the respective complexes for all but the distal helix. The necessity of considering dipolar shifts, and hence determination of the orientation/anisotropy of χ, in comparing chemical shifts involving paramagnetic complexes, is emphasized. The analysis shows that the H-bond network cannot detect significant differences in H-bond acceptor properties of cyanide versus azide ligands. Lastly, significant retardation of distal helix labile proton exchange upon replacing cyanide with azide indicates that the dynamic stability of the distal helix is increased upon decreasing the steric interaction of the ligand with the distal helix.

Evaluating spin diffusion in MAS-NOESY spectra of phospholipid multibilayers

Biological semisolids such as aqueous phospholipid dispersions are amenable to experiments that combine magic-angle spinning (MAS) to yield high-resolution 1H NMR spectra and two-dimensional nuclear Overhauser spectroscopy (NOESY) to estimate proton-proton distances. Using several selectively deuterated dimyristoylphosphatidylcholines, a systematic investigation has been made into the origin of an anomalous NOESY crosspeak between methyl groups of the lipid headgroup and the acyl chains. Although the spin-relaxation and line-narrowing behavior in these multilamellar systems argue against efficient spin diffusion, interdigitated or chain-bendback structural models that bring these 1H nuclei into close proximity are not implicated by the MAS-NOESY results for this phospholipid system. A mechanism has been proposed for spin communication between the two types of methyl protons within a conventional lipid bilayer structure: through-space interactions link the headgroup and backbone protons on adjacent molecules, and then spin diffusion occurs along the acyl chains.

Structural study of a DNA.RNA hybrid duplex with a chiral phosphorothioate moiety by NMR: extraction of distance and torsion angle constraints and imino proton exchange rates.

The solution structure of the thiophosphate-modified DNA.RNA hybrid duplex d(GCTATAApsTGG).r(CCAUUAUAGC) has been studied by NMR. Two samples with pure stereochemistry in the modified phosphate have been investigated. Two-dimensional NMR (2D NMR) methods have been applied to assign nearly all the resonances in both duplexes. Scalar coupling constants have been determined by comparing quantitative simulations with experimental double-quantum filtered COSY (DQF-COSY) cross-peaks. More than 300 distance constraints have been obtained from two-dimensional nuclear Overhauser spectroscopy (2D NOE) spectra recorded in D2O and H2O by using a complete relaxation matrix analysis as implemented in the program MARDIGRAS. This hybrid duplex presents a heteronomous structure. Riboses in the RNA strand are found in a N-type conformation typical of the A-form family as shown by the lack of H1'-H2' cross-peaks in DQF-COSY spectra and confirmed by the measured interproton distances. In contrast, the DNA strand adopts a different conformation with sugar puckers partially in the S-type domain, which is not in agreement with the A-family of structures. Coupling constants in deoxyriboses are not consistent with any single sugar conformation. Therefore, sugar pucker pseudorotation parameters are calculated according to a two-state dynamic equilibrium between N- and S-type conformers. In general, the population of major S conformer is lower than in double-stranded DNA duplexes, indicating that hybrid duplexes may be more flexible than pure DNA or RNA. The only differences observed in the spectra between the two stereoisomers studied originate from resonances of protons located near the modified phosphate. No significant differences in interproton distance have been detected, and only a slight difference of sugar pucker in the 5' neighbor has been found. The sulfur atom appears to be well-accommodated without further changes in the structure of the hybrid.

NMR studies of peptides derived from the putative binding regions of cartilage proteins. No evidence for binding to hyaluronan.

Previous work has implicated sequences within the tandem repeats of cartilage link protein in the interaction of link protein with hyaluronan. This conclusion was based on competitive inhibition experiments using synthetic peptides (Goetinck, P. F., Stirpe, N. S., Tsonis, P. A., and Carlone, D. (1987) J. Cell Biol. 105, 2403-2407). Further investigation of this system using high resolution nuclear magnetic resonance, circular dichroism, and competitive inhibition with other peptides indicates that the previously observed inhibition of link protein-hyaluronan binding was not caused by peptide-hyaluronan interactions. Instead, nonspecific aggregation of the peptides with link protein is proposed to account for all of the experimental data. Consequently, there is no direct experimental evidence to support the conclusion that these sequences in the tandem repeats of link protein are responsible for the link protein-hyaluronan interaction. If these peptides do represent the hyaluronan binding regions of link protein, these results imply a highly structure-dependent interaction between link protein and hyaluronan. Conformational analysis of the peptides using two-dimensional nuclear Overhauser spectroscopy indicates that the linear peptides do not adopt any stable secondary structure. However, several residues in the disulfide-looped peptides exhibit connectivities, suggesting a relatively long-lived extended chain conformation, consistent with predictions of secondary structure based on sequence analysis.

Nuclear Magnetic Resonance Comparison of the Binding Sites of Mithramycin and Chromomycin on the Self-complementary Oligonucleotide d(ACCCGGGT) 2: Evidence that the Saccharide Chains have a role in Sequence Specificity

A comprehensive two-dimensional 1H nuclear magnetic resonance spectral analysis of the ternary 4:2:1 mithramycin-Mg 2+-d(A 1C 2C 3C 4G 5G 6G 7T 8) 2 complex and the ternary 2:1:1 chromomycin-Mg 2+-d(A 21C 2C 3C 4C 4G 5G 6G 7T 8) 2 complex is presented. The self-complementary oligonucleotide is found to bind two dimers of mithramycin in two identical off-center binding sites such that the twofold symmetry of the oligonucleotide is retained. In contrast, the same oligonucleotide binds only one dimer of chromomycin in a single but distinct of-center binding site. Two-dimensional nuclear Overhauser spectroscopy experiments show that the aglycone binding site of the drug dimer in each complex extends over almost four base-pairs and is similar in length to other complexes between chromomycin or mithramycin and oligonucleotides. The data demonstrate that the chromomycin dimer binding site is offset by one base-pair step from the dimer binding site in the mithramycin complex. This preferred binding site prevents two dimers of chromomycin binding to d(ACCCGGGT) 2 for steric reasons and lends further support to previous work that showed the 5′-CG base-pair site is less favored by these drugs compared to the 5′GC and 5′-GG, 5′-CC sites. Evidence is presented that suggests mithramycin may occupy either of two distinct binding sites on d(ACCCGGGT) 2 when the drug concentration is not saturating. The nuclear magnetic resonance data demonstrate that the saccharide chains of this family of drugs do have a role in determining the binding site on nucleotides and as a consequence the CDE trisaccharide chain may alter its conformation to fulfil this role. Titration of mithramycin up to a drug-duplex ratio of 7:1 reveals further association of mithramycin with the complex but no new drug-oligonucleotide nuclear Overhauser enhancement contacts.

A pH-dependent Conformational Change of Staphylococcal Nuclease: Hydrogen-1 Two-Dimensional Nuclear Overhauser Spectroscopy Studies.

Two-dimensional nuclear magnetic resonance (2d-NMR) dipolar correlation spectroscopy (NOESY) for the calcium binding protein, staphylococcal nuclease, was used to study the conformations in solution as a function of pH. A series of 2d-NOESY spectra of the nuclease H124L-Ca(++)-thymidine 3'5'-bisphosphate ternary complex demonstrated significant pH dependent changes: NOE cross peaks between 46His delta H and an aliphatic proton at 2.66 ppm appeared at pH 10.2, but not at pH 5.5. Some other NOE cross peaks between aromatic and aliphatic protons displayed chemical shift changes in omega 2 or both dimensions (omega 1, omega 2). These results indicated that a local rearrangement accompanies the ionization of the functional groups and has a role in enzyme function.

Magic-angle spinning NMR studies of molecular organization in multibilayers formed by 1-octadecanoyl-2-decanoyl-sn-glycero-3-phosphocholine

Magic-angle spinning 1H and 13C nuclear magnetic resonance (NMR) have been employed to study 50%-by-weight aqueous dispersions of 1-octadecanoyl-2-decanoyl-sn-glycero-3-phosphocholine (C[18]:C[10]PC) and 1-octadecanoyl-2-d19-decanoyl-PC (C[18]:C[10]PC-d19), mixed-chain phospholipids which can form interdigitated multibilayers. The 1H NMR linewidth for methyl protons of the choline headgroup has been used to monitor the liquid crystalline-to-gel (LC-to-G) phase transition and confirm variations between freezing and melting temperatures. Both 1H and 13C spin-lattice relaxation times indicate unusual restrictions on segmental reorientation at megahertz frequencies for C(18):C(10)PC as compared with symmetric-chain species in the LC state; nevertheless each chemical moiety of the mixed-chain phospholipid exhibits motional behavior that may be classified as liquidlike. Two-dimensional nuclear Overhauser spectroscopy (NOESY) on C(18):C(10)PC and C(18):C(10)PC-d19 reveals cross-peaks between the omega-methyl protons of the C18 chain and the N-methyl protons of the phosphocholine headgroup, and several experimental and theoretical considerations argue against an interpretation based on spin diffusion. Using NMR relaxation times and NOESY connectivities along with a computational formalism for four-spin systems (Keepers, J. W., and T. L. James. 1984. J. Magn. Reson. 57:404-426), an estimate of 3.5 A is obtained for the average distance between the omega-methyl protons of the C18 chain and the N-methyl protons of the phosphocholine headgroup. This finding is consistent with a degree of interdigitation similar to that proposed for organized assemblies of gel-state phosphatidylcholine molecules with widely disparate acyl-chain lengths (Hui, S. W., and C.-H. Huang. 1986. Biochemistry. 25:1330-1335); however, acyl-chain bendback or other intermolecular interactions may also contribute to the NOESY results. For multibilayers of C(18):C(10)PC in the gel phase, 13C chemical-shift measurements indicate that trans conformers predominate along both acyl chains. 13C Spin-lattice relaxation times confirm the unusual motional restrictions noted in the LC state; nevertheless, 13C and 1H rotating-frame relaxation times indicate that the interdigitated arrangement enhances chain or bilayer motions which occur at mid-kilohertz frequencies.

Sequence-specific proton NMR assignments and secondary structure in solution of Escherichia coli trp repressor

Sequence-specific 1H NMR assignments are reported for the active L-tryptophan-bound form of Escherichia coli trp repressor. The repressor is a symmetric dimer of 107 residues per monomer; thus at 25 kDa, this is the largest protein for which such detailed sequence-specific assignments have been made. At this molecular mass the broad line widths of the NMR resonances preclude the use of assignment methods based on 1H-1H scalar coupling. Our assignment strategy centers on two-dimensional nuclear Overhauser spectroscopy (NOESY) of a series of selectively deuterated repressor analogues. A new methodology was developed for analysis of the spectra on the basis of the effects of selective deuteration on cross-peak intensities in the NOESY spectra. A total of 90% of the backbone amide protons have been assigned, and 70% of the alpha and side-chain proton resonances are assigned. The local secondary structure was calculated from sequential and medium-range backbone NOEs with the double-iterated Kalman filter method [Altman, R. B., & Jardetzky, O. (1989) Methods Enzymol. 177, 218-246]. The secondary structure agrees with that of the crystal structure [Schevitz, R., Otwinowski, Z., Joachimiak, A., Lawson, C. L., & Sigler, P. B. (1985) Nature 317, 782], except that the solution state is somewhat more disordered in the DNA binding region and in the N-terminal region of the first alpha-helix. Since the repressor is a symmetric dimer, long-range intersubunit NOEs were distinguished from intrasubunit interactions by formation of heterodimers between two appropriate selectively deuterated proteins and comparison of the resulting NOESY spectrum with that of each selectively deuterated homodimer. Thus, from spectra of three heterodimers, long-range NOEs between eight pairs of residues were identified as intersubunit NOEs, and two additional long-range intrasubunits NOEs were assigned.

400 MHz two-dimensional nuclear Overhauser spectroscopy on anesthetic interaction with lipid bilayer

Interaction between a volatile anesthetic, methoxyflurane, and dipalmitoylphosphatidylcholine (DPPC) vesicle membrane was analyzed by nuclear Overhauser effect (NOE) difference spectroscopy and two-dimensional nuclear Overhauser spectroscopy (NOESY). The NOE difference spectra were obtained by selectively irradiating methoxy protons (hydrophobic end) of the anesthetic: a negative nuclear Overhauser effect of -2.94% was observed with the choline methyl protons of DPPC. The NOESY spectra revealed a cross-peak between the anesthetic methoxy protons and the choline methyl protons. A dipole-dipole interaction exists between the hydrophobic end of the anesthetic and the hydrophilic head group of DPPC. No other cross-peaks were observed. The anesthetic orients itself at the membrane/water interface by interacting with the hydrophilic surface of the DPPC membrane, leaving the hydrophilic end of the anesthetic molecule in the aqueous phase. The preferred residence site of dipolar volatile anesthetics is the membrane/water interface.

Conformational analysis of the octamer d(G-G-C-C-G-G-C-C) in aqueous solution. A one-dimensional and two-dimensional proton NMR study at 300 MHz and 500 MHz.

Proton NMR studies in H2O and 2H2O were carried out on the self-complementary DNA octamer d(G-G-C-C-G-G-C-C) and compared with similar studies on the dimethylated analogue d(G-G-m5C-m5C-G-G-C-C) [Sanderson, M. R., Mellema, J.-R., van der Marel, G. A., Wille, G., van Boom, J. H. & Altona, C. (1983) Nucleic Acids Res. 11, 3333-3346]. Base, H1', H2' and H2" resonances were assigned by means of two-dimensional nuclear Overhauser spectroscopy (NOESY) and correlated spectroscopy (COSY) experiments. Chemical shift vs temperature profiles were used to analyze the temperature-dependent conformational behaviour and to extract thermodynamic parameters for the duplex-to-coil transition. Analysis of proton-proton couplings were used to discriminate between J1'2' and J1'2" and between the H2' and H2" resonances as well as to obtain conformational parameters of the sugar rings. From the NOESY and COSY experiments, the temperature profiles and the analysis of the coupling data it is concluded that: (a) the compound adopts a B-DNA-type helix in solution; (b) the sugar rings in the intact duplex display limited conformational freedom; (c) methylation of the cytosine bases at the C5 position has no measurable effect on the conformational behaviour of the octamer, nor on the conformation of the sugar rings; however, methylation does increase the stability of the duplex in aqueous solution under conditions of low salt concentration.