Nuclear Overhauser Effect Cross-peaks Research Articles

Kinetic, Binding, and NMR Studies of Perdeuterated Yeast Phosphoglycerate Kinase and Its Interactions with Substrates

Perdeuterated yeast phosphoglycerate kinase (2HPGK) was prepared from yeast cells grown in 99.9% 2H2O and an acid hydrolysate from deuterated algal cells. Kinetic and binding studies suggested that perdeuterated enzyme was similar to the isotopically normal PGK. The use of 2HPGK not only eliminated the spectral overlap between the enzyme and substrate nuclear Overhauser effect (NOE) cross-peaks, but also permitted observation of weak transfer NOE cross-peaks between the substrate protons that are greater than 4 Å apart. Intensity of NOE cross-peaks was used to determine the interproton distances of enzyme bound Mg(II)dATP. These distances were then used in model building studies to determine the conformation of Mg(II)dATP. The average conformation of enzyme bound dATP is anti with O4′/C2′ endo ribose pucker and trans, gauche about the C4′-C5′ bond. Although many spin diffusion pathways were eliminated by protein deuteration, spin diffusion was still observed between the protons of the substrate at mixing times longer than 25 ms.

Calculation of Protein Structures with Ambiguous Distance Restraints. Automated Assignment of Ambiguous NOE Crosspeaks and Disulphide Connectivities

The distances derived from nuclear Overhauser effect (NOE) spectra are usually converted into three-dimensional structures by computer algorithms loosely termed distance geometry. To a varying degree, these methods require that the distance data is unambiguously assigned to pairs of atoms. Typically, however, there are many NOE crosspeaks that cannot be assigned without some knowledge of the structure. These crosspeaks have to be assigned in an iterative manner, using preliminary structures calculated from the unambiguous crosspeaks. In this paper, I present an alternative to this iterative approach. The ambiguity of an NOE crosspeak is correctly described in terms of the distances between all pairs of protons that may be involved. A simple restraining term is defined in terms of “ambiguous” distance restraints that can allow all possible assignments. A new minimization procedure based on simulated annealing is described that is capable of using highly ambiguous data for ab initiostructure calculations. In particular, it is feasible to specify the restraint list directly in terms of the proton chemical shift assignment and the NOE peak table, without having assigned NOE crosspeaks to proton pairs. While the primary aim of this paper is determining the global fold of proteins from NMR data, similar strategies can be used for other types of ambiguous distance data. The application to one example, disulphide bridges with unknown connectivity, is described. Model NOE data were generated from the X-ray crystal structure of a small protein with known chemical shift assignments. Varying degrees of ambiguity in the data were assumed. The method obtained the correct polypeptide fold even when all distance restraints were ambiguous. Thus, the new approach may facilitate structure calculations with data derived from very overlapped spectra. It is also a step towards automating the calculation of structures from NMR data. This could prove especially valuable for data derived from three- and four-dimensional experiments. The approach may also prove useful for model building studies and tertiary structure prediction.

Buffers for cell culture

The structure of a synthesized peptide with the sequence of NHILPNESAYDQKNIRRRVYDALNVLMAMNIISK that corresponds to residues 151–184 of transcription factor DP-1 (Girling et al., Nature 362 (1993) 83–87) was determined by 1H-nuclear magnetic resonance in water and 40% d3-trifluoroethanol/water, respectively. Nuclear Overhauser effect cross peaks, αH chemical shifts and J-coupling constants of αH–NH show that the peptide consists a helix from Ser-8 to Ser-33 in solution. Fifty structures were constructed with 288 upper distance limits and 21 angle constraints by DIANA (Guntert et al., J. Mol. Biol. 217 (1991) 517–530). Although the N-terminal of the peptide exhibits a random conformation, the 20 best structures show a root mean square deviation of 0.89±0.36 Å for backbone atoms and 1.80±0.34 Å for heavy atoms from residue Ser-8 to Ser-33. This result supports the proposal that DP-1 and E2F-1 may dimerize with a coiled-coil type interaction.

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

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

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

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

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

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

Three-dimensional structure of the wild-type lac pribnow promoter DNA in solution : Two-dimensional nuclear magnetic resonance studies and distance geometry calculations

The solution structure of a 12 base-pair DNA duplex containing the wt- lac promoter Pribnow sequence TATGTT has been studied by two-dimensional nuclear magnetic resonance spectroscopy. Proton assignments for the 24 sugar and base residues were obtained from two-dimensional correlated nuclear magnetic resonance and two-dimensional nuclear Overhauser effect spectra in both 2H 2O and H 2O, and by two-dimensional relayed coherence transfer nuclear magnetic resonance spectroscopy experiments. Time-dependent, two-dimensional nuclear Overhauser effect spectra were used to determine the initial crossrelaxation rates between 212 pairs of assigned protons, leading to 212 interproton distances in the double helix (8 to 9 per nucleotide). These distance constraints, and known bond lengths and angles, were entered into a distance matrix. After smoothing the bounds of the distance matrix, 12 trial matrices within the bounds constraints were independently generated and embedded in three-dimensional space using a distance geometry algorithm, to generate 12 trial structures. These trial structures were then refined until they no longer violated the distance matrix. The resulting structures are very similar at the local base-pair and nearest-neighbor base-pair level, but exhibit increasing variation at more distant and global levels. At the nearest-neighbor level, the A to T step and the G to T step within the Pribnow hexamer, as well as the G to T step preceding the hexamer, all exhibit very low screw pitch, i.e. 5(±6) °. Conversely, the T to G step in the center of the promoter has a large screw pitch (47(±2) °) and the T to G step at the 3′ end of the promoter has a very large screw pitch (60(±3) °). The limitations of nuclear magnetic resonance spectroscopy distance determination of structure are discussed in terms of resolution and spectral overlap of two-dimensional nuclear Overhauser effect crosspeaks. In the present duplex, the inability to measure several 1′-2′ and 1′-2″ distances resulted in underdetermination of the precise local sugar conformation for seven of the 24 residues, although the spatial position of all sugars was well defined.

One- and two-dimensional NMR relaxation studies of dynamics and structure in bile salt-phosphatidylcholine mixed micelles

A series of one- and two-dimensional 1H-NMR relaxation measurements has been conducted on simple and mixed micellar aggregates of taurocholate, diphenylvaleroylphosphatidylcholine (diPVPC) and egg yolk phosphatidylcholine (egg PC). The results are interpreted to provide structural and dynamic comparisons between micelles and vesicles, between phospholipids of varying chain length, and between different lipid components within the same micellar aggregate. Both chemical shift changes and two-dimensional nuclear Overhauser effect cross peaks suggest direct interaction of taurocholate and PC chemical sites, although the latter observations may also be accounted for by PC-PC interactions. These experiments demonstrate the promise of NMR relaxation techniques for investigations of molecular organization in model substrate for lipolytic enzymes.

Observation of 2D nuclear overhauser effect crosspeaks involving amide protons in H 2O solutions of proteins

Observation of 2D nuclear overhauser effect crosspeaks involving amide protons in H 2O solutions of proteins