Salt induced deprotonation of initially salt-free transfer RNA.

¹H NMR double resonance studies of valinomycin in (CD₃)₂SO were conducted at 90 MHz (FT-mode) and 250 MHz (correlation mode) to determine the mechanism of intramolecular nuclear Overhauser effects (NOE). These studies set specific constraints on any model for the conformation of valinomycin in (CD₃)₂SO and illustrate that NOE experiments of this type hold considerable promise for conformational studies of peptides, proteins and other biomolecules. The NOE's are positive at the lower frequency and negative at the higher frequency. Consideration of the theoretical dependence of the NOE on the proton-proton internuclear correlation time and on the resonance frequency indicates that these results are explained by a predominantly dipolar relaxation mechanism.     

Complete 1H, 13C and 15N NMR assignments and secondary structure of the 269-residue serine protease PB92 from Bacillus alcalophilus

Summary The ¹H, ¹³C and ¹⁵N NMR resonances of serine protease PB92 have been assigned using 3D tripleresonance NMR techniques. With a molecular weight of 27 kDa (269 residues) this protein is one of the largest monomeric proteins assigned so far. The side-chain assignments were based mainly on 3D H(C)CH and 3D (H)CCH COSY and TOCSY experiments. The set of assignments encompasses all backbone carbonyl and CH_n carbons, all amide (NH and NH₂) nitrogens and 99.2% of the amide and CH_n protons. The secondary structure and general topology appear to be identical to those found in the crystal structure of serine protease PB92 [Van der Laan et al. (1992) Protein Eng., 5, 405–411], as judged by chemical shift deviations from random coil values, NH exchange data and analysis of NOEs between backbone NH groups.Abbreviations 2D/3D/4D two-/three-/four-dimensional - HSQC heteronuclear single-quantum coherence - HMQC heteronuclear multiple-quantum coherence - COSY correlation spectroscopy - TOCSY total correlation spectroscopy - NOE nuclear Overhauser enhancement (connectivity) - NOESY 2D NOE spectroscopy Experiment nomenclature (H(C)CH, etc.) follows the conventions used elsewhere [e.g. Ikura et al. (1990) Biochemistry, 29, 4659–4667].     

Resonance assignment and structural analysis of acid denatured E. coli [U-15N]-glutaredoxin 3

Virtually complete sequence specific ¹H and ¹⁵N resonance assignments are presented for acid denatured reduced E. coli glutaredoxin 3. The sequential resonance assignments of the backbone rely on the combined use of 3D F₁-decoupled ROESY-¹⁵N-HSQC and 3D ¹⁵N-HSQC-(TOCSY-NOESY)-¹⁵N-HSQC using a single uniformly ¹⁵N labelled protein sample. The sidechain resonances were assigned from a 3D TOCSY-¹⁵N-HSQC and a homonouclear TOCSY spectrum. The presented assignment strategy works in the absence of chemical exchange peaks with signals from the native conformation and without ¹³C/¹⁵N double labelling. Chemical shifts, ³J(H, NH) coupling constants and NOEs indicate extensive conformational averaging of both backbone and side chains in agreement with a random coil conformation. The only secondary structure element persisting at pH 3.5 appears to be a short helical segment comprising residues 37 to 40.Abbreviations HSQC heteronuclear single quantum coherence - NMR nuclear magnetic resonance - NOE nuclear Overhauser effect - NOESY two-dimensional NOE spectroscopy - ROE nuclear Overhauser effect in the rotating frame - ROESY two-dimensional ROE spectroscopy - TOCSY total correlation spectroscopy - TPPI time proportional phase incrementation Correspondence to: G. Otting     

Solution conformation study of substance P methyl ester and [Nle10]-neurokinin A (4–10) by nmr spectroscopy

High-resolution proton spectra at 500 MHz of two tachykinin peptides, substance P methyl ester (SPOMe) and [Nle¹⁰]-neurokinin A (4–10), have been obtained in dimethylsulfoxide (DMSO), and for SPOMe, also in 2, 2, 2-trifluoroethanol (TFE)/water mixtures. Complete chemical shift assignments for these peptides were made based on two-dimensional (2D) nmr techniques, correlated spectroscopy and total COSY. J coupling measurement and nuclear Overhauser effect spectroscopy (NOESY) were then used to determine the conformation of these peptides in the various solvents. Based on the J coupling, NOE correlations, and temperature coefficients of the NH resonances, it is concluded that these two peptides exist in DMSO at room temperature as a mixture of conformers that are primarily extended. For SPOMe in TFE/water with high TFE content, however, helical structures are found to be present, and they become quite clear at temperatures between 270 and 280 K. The variation of the ¹³C chemical shifts of the C_α (the secondary shift) with TFE contents corroborates this conclusion. The NOE and C_α shifts show that the main helical region for SPOMe lies between ⁴P and ⁹G. The C-terminus segment L? M? NH₂ is found to be quite flexible, which appears to be quite common for neurokinin-1 selective peptides. © 1993 John Wiley & Sons, Inc.     

A type II β-turn in a flexible peptide: Proton assignment and conformational analysis of the α-factor from saccharomyces cerevisiae in solution

Two-dimensional ¹H-nmr spectra of the α-mating factor [in dimethyl sulfoxide-d₆ (DMSO) and in water] and several dodecapeptide analogues (in DMSO) were obtained. Homonuclear correlated spectroscopy resulted in the complete and unequivocal assignment of all backbone and side-chain resonances of the peptides. The solution conformation of the pheromones was probed using two-dimensional (2D) nuclear Overhauser effect spectroscopy (NOESY) and rotating frame nuclear Overhauer effect spectroscopy (ROESY). The 2D NOE results, and results of complementary one-dimensional experiments, suggest that a type II β-turn is assumed by the central portion of active pheromones in both DMSO and water. Inactive analogues of the α-factor do not exhibit this structural feature. Except for this one β-turn, the nmr parameters for α-factor are indicative of a conformationally flexible molecule in both solvents. This conclusion is in contrast to that of other researchers who have proposed a highly structured conformation of α-factor in aqueous solution.     

Graph-theoretical assignment of secondary structure in multidimensional protein NMR spectra: Application to the lac repressor headpiece

Summary A novel procedure is presented for the automatic identification of secondary structures in proteins from their corresponding NOE data. The method uses a branch of mathematics known as graph theory to identify prescribed NOE connectivity patterns characteristic of the regular secondary structures. Resonance assignment is achieved by connecting these patterns of secondary structure together, thereby matching the connected spin systems to specific segments of the protein sequence. The method known as SERENDIPITY refers to a set of routines developed in a modular fashion, where each program has one or several well-defined tasks. NOE templates for several secondary structure motifs have been developed and the method has been successfully applied to data obtained from NOESY-type spectra. The present report describes the application of the SERENDIPITY protocol to a 3D NOESY-HMQC spectrum of the ¹⁵N-labelled lac repressor headpiece protein. The application demonstrates that, under favourable conditions, fully automated identification of secondary structures and semi-automated assignment are feasible.Abbreviations 2D, 3D two-, three-dimensional - NOESY nuclear Overhauser enhancement spectroscopy - HMQC heteronuclear multiple quantum coherence - SSE secondary structure element - SERENDIPITY SEcondary structuRE ideNtification in multiDImensional ProteIn specTra analYsis Supplementary Material available from the authors: Two tables containing the total number of mappings resulting from the graph search procedure for simulated and experimental NOE data.     

Unique backbone-water interaction detected in sphingomyelin bilayers with 1H/31P and 1H/13C HETCOR MAS NMR spectroscopy

Two-dimensional ¹H/³¹P dipolar heteronuclear correlation (HETCOR) magic-angle spinning nuclear magnetic resonance (NMR) is used to investigate the correlation of the lipid headgroup with various intra- and intermolecular proton environments. Cross-polarization NMR techniques involving ³¹P have not been previously pursued to a great extent in lipid bilayers due to the long ¹H-³¹P distances and high degree of headgroup mobility that averages the dipolar coupling in the liquid crystalline phase. The results presented herein show that this approach is very promising and yields information not readily available with other experimental methods. Of particular interest is the detection of a unique lipid backbone-water intermolecular interaction in egg sphingomyelin (SM) that is not observed in lipids with glycerol backbones like phosphatidylcholines. This backbone-water interaction in SM is probed when a mixing period allowing magnetization exchange between different ¹H environments via the nuclear Overhauser effect (NOE) is included in the NMR pulse sequence. The molecular information provided by these ¹H/³¹P dipolar HETCOR experiments with NOE mixing differ from those previously obtained by conventional NOE spectroscopy and heteronuclear NOE spectroscopy NMR experiments. In addition, two-dimensional ¹H/¹³C INEPT HETCOR experiments with NOE mixing support the ¹H/³¹P dipolar HETCOR results and confirm the presence of a H₂O environment that has nonvanishing dipolar interactions with the SM backbone.     

An NMR Study of the Spatial Structure and Intramolecular Dynamics of Modified Analogues of Steroid Hormones

Special features of the use of homo- and heteronuclear correlation methods of NMR in one and two dimensions for studying the spatial structure and intramolecular dynamics of modified analogues of steroid hormones (MASH) are considered. The application of these methods to the assignment of resonances in the high-field ¹H NMR region and to the determination of the most stereospecifically important parameters, such as the vicinal constants of spin–spin coupling (³ J _H–H) and nuclear Overhauser effects (NOE), are discussed using the example of NMR studies of some estrogens and androgens at 300 MHz and on the basis of literature data. The most efficient combination of the methods and the necessary modification of each of them may be chosen considering the spectral and relaxation parameters of MASH in liquid medium, including the anisotropy of the overall diffusive motion. The characteristics of MASH are the wide use of correlations through long-range couplings (COSY-45 and DQF-COSY), the application of the ^4,5 J _H–H constants for the determination of spatial structure, and the advantage of heteronuclear HSQC methods with and without ¹³C decoupling over the corresponding HMQC methods in both resolution and sensitivity. In the conformationally rigid MASH molecules, the anisotropy of the MASH diffusive motion in liquid adversely affects the determination of interproton distances by the calibrating processing method for the NOE difference and NOESY spectra: it results in both overestimated and underestimated distance values depending on the polar angle ratios of the reference and the determined distances. Under certain conditions, conformationally mobile MASH demonstrate the additional contribution of the scalar relaxation mechanism between the indirectly (scalarly) bound protons. This mechanism is responsible for the underestimated values of NOE and the corresponding errors in the distance determination.     

3d haro-Noesy-ch3nh and caro-Noesy-ch3nh Experiments for Double Labeled Proteins

Precision in the determination of the 3D structures of proteins by NMR depends on obtaining an adequate number of NOE restraints. Ambiguity in the assignment of NOE cross peaks between aromatic and other protons is an impediment to high quality structure determination. Two pulse sequences, 3D H_aro-NOESY-CH₃NH and 3D C_aro-NOESY-CH₃NH, based on a modification of a technique for simultaneous detection of ¹³C-¹H (of CH₃) and ¹⁵N-¹H correlations in one measurement, are proposed in the present work. These 3D experiments, which are optimized for resolution in the ¹³C and ¹⁵N dimensions, provide NOE information between aromatic protons and methyl or amide protons. CH₂ moieties are filtered out and the CH groups in aromatic rings are selected, allowing their NOE cross peaks to be unambiguously assigned. Unambiguous NOEs connecting aromatic and methyl or amide protons will provide important restraints for protein structure calculations.     

A complete algorithm to resolve ambiguity for intersubunit NOE assignment in structure determination of symmetric homo-oligomers

Assignment of nuclear Overhauser effect (NOE) data is a key bottleneck in structure determination by NMR. NOE assignment resolves the ambiguity as to which pair of protons generated the observed NOE peaks, and thus should be restrained in structure determination. In the case of intersubunit NOEs in symmetric homo-oligomers, the ambiguity includes both the identities of the protons within a subunit, and the identities of the subunits to which they belong. This paper develops an algorithm for simultaneous intersubunit NOE assignment and C(n) symmetric homo-oligomeric structure determinations, given the subunit structure. By using a configuration space framework, our algorithm guarantees completeness, in that it identifies structures representing, to within a user-defined similarity level, every structure consistent with the available data (ambiguous or not). However, while our approach is complete in considering all conformations and assignments, it avoids explicit enumeration of the exponential number of combinations of possible assignments. Our algorithm can draw two types of conclusions not possible under previous methods: (1) that different assignments for an NOE would lead to different structural classes, or (2) that it is not necessary to uniquely assign an NOE, since it would have little impact on structural precision. We demonstrate on two test proteins that our method reduces the average number of possible assignments per NOE by a factor of 2.6 for MinE and 4.2 for CCMP. It results in high structural precision, reducing the average variance in atomic positions by factors of 1.5 and 3.6, respectively.     

Quantitative two-dimensional nmr study of dermenkephalin,a highly potent and selective δ-opioid peptide

Dermenkephalin, H-Tyr-(D ) Met-Phe-His-Leu-Met-Asp-NH₂, a highly potent and selective δ-opioid peptide isolated from frog skin, was studied in DMSO-d₆ solution by two-dimensional nmr spectroscopy, including the determination of NH temperature coefficients, the evaluation of ³J coupling constants from phase-sensitive correlated spectroscopy (COSY) and the volumes of nuclear Overhauser effect (NOE) correlations. The two-dimensional NOE spectroscopy (NOESY) spectrum of dermenkephalin revealed sequential, medium-, and long-range effects. To put this information on a quantitative basis, special attention was devoted to J cross-peak suppression, quantification of the NOE volumes and analysis of the overlaps, normalization of the NOEs against diagonal peaks and H_ββ′ geminal interactions. Although most of the dihedral angles deduced from the ³J_Nα coupling constants together with several N_iα_i and α_iN_{i + 1} NOEs pointed to a partially extended peptide backbone, several N_i N_{i + 1} NOEs and β_i N_{i + 1} interactions argued in favor of a folded structure. Moreover, several long-range correlations of strong intensities were found that supported a close spatial proximity between the side chains of D -Met² and Met⁶, Tyr¹ and His⁴, Tyr¹ and Asp⁷, and His⁴ and the C-terminal amide group. In Phe, the g^? rotamer in the side chain is deduced from the ³J_αβ coupling constants and αβ and Nβ NOE correlations. Whereas the amide proton dependency was not indicative of stable hydrogen bonds, the nonuniform values of the temperature coefficient may reflect an equilibrium mixture of folded and extended conformers. The overall data should provide realistic starting models for energy minimization and modelization studies. © 1993 John Wiley & Sons, Inc.     

Metropolis Monte Carlo calculations of DNA structure using internal coordinates and NMR distance restraints: An alternative method for generating a high-resolution solution structure

Summary A new method, a restrained Monte Carlo (rMC) calculation, is demonstrated for generating high-resolution structures of DNA oligonucleotides in solution from interproton distance restraints and bounds derived from complete relaxation matrix analysis of two-dimensional nuclear Overhauser effect (NOE) spectral peak intensities. As in the case of restrained molecular dynamics (rMD) refinement of structures, the experimental distance restraints and bounds are incorporated as a pseudo-energy term (or penalty function) into the mathematical expression for the molecular energy. However, the use of generalized helical parameters, rather than Cartesian coordinates, to define DNA conformation increases efficiency by decreasing by an order of magnitude the number of parameters needed to describe a conformation and by simplifying the potential energy profile. The Metropolis Monte Carlo method is employed to simulate an annealing process. The rMC method was applied to experimental 2D NOE data from the octamer duplex d(GTA-TAATG)·d(CATTATAC). Using starting structures from different locations in conformational space (e.g. A-DNA and B-DNA), the rMC calculations readily converged, with a root-mean-square deviation (RMSD) of <0.3 Å between structures generated using different protocols and starting structures. Theoretical 2D NOE peak intensities were calculated for the rMC-generated structures using the complete relaxation matrix program CORMA, enabling a comparison with experimental intensities via residual indices. Simulation of the vicinal proton coupling constants was carried out for the structures generated, enabling a comparison with the experimental deoxyribose ring coupling constants, which were not utilized in the structure determination in the case of the rMC simulations. Agreement with experimental 2D NOE and scalar coupling data was good in all cases. The rMC structures are quite similar to that refined by a traditional restrained MD approach (RMSD<0.5 Å) despite the different force fields used and despite the fact that MD refinement was conducted with additional restraints imposed on the endocyclic torsion angles of deoxyriboses. The computational time required for the rMC and rMD calculations is about the same. A comparison of structural parameters is made and some limitations of both methods are discussed with regard to the average nature of the experimental restraints used in the refinement.Abbreviations MC Monte Carlo - rMC restrained Monte Carlo - MD molecular dynamics - rMD restrained molecular dynamics - DG distance geometry - EM energy minimization - 2D NOE two-dimensional nuclear Overhauser effect - DQF-COSY double-quantum-filtered correlation spectroscopy - RMSD root-mean-square deviation To whom correspondence should be addressed.     

Antiestrogen binding sites in rat liver nuclei

Isolated, intact rat liver nuclei have high-affiity (K_d=10⁻⁹ M) binding sites that are highly specific for nonsteroidal antiestrogens, especially for compounds of the triphenylethylene series. Nuclear [³H]tamoxifen binding capacity is thermolabile, being most stable at 4°C and rapidly lost at 37°C. More [³H]tamoxifen, however, is specifically bound at incubation temperatures of 25°C and 37°C than at 4°C although prewarming nuclei has no effect, suggesting exchange of [³H]tamoxifen for an unidentified endogenous ligand. Nuclear antiestrogen binding sites are destroyed by trypsin but not by deoxyribonuclease I or ribonuclease A. The nuclear antiestrogen binding protein is not solubilized by 0.6 M potassium chloride, 2 M sodium chloride, 0.6 M sodium thiocyanate, 3 M urea, 20 mM pyridoxal phosphate, 1% (w/v) digitonin or 2% (w/v) sodium cholate but is extractable by sonication, indicating that it is tightly bound within the nucleus. Rat liver nuclear matrix contains high-affinity (K_d=10⁻⁹ M) [³H]tamoxifen binding sites present in 5-fold higher concentrations (4.18 pmol/mg DNA) than in intact nuclei (0.78±0.10 (S.D.) pmol/mg DNA). Low-speed rat liver cytosol (20 000×g, 30 min) contains high-capacity (955±405 (S.D.) fmol/mg protein), low-affinity (K_d=10.9±4.5 (S.D.) nM) antiestrogen binding sites. In contrast, high-speed cytosol (100 000×g, 60 min) contains low-capacity (46±15 (S.D.) fmol/mg protein), high-affinity (K_d=0.61± 0.20 (S.D.) nM) binding sites. Low-affinity cytosolic sites constitute more than 90% of total liver binding sites, high-affinity cytosolic sites 0.3%–3.2%, and nuclear sites less than 0.5% of total sites.     

High-resolution solution structure of siamycin II: Novel amphipathic character of a 21-residue peptide that inhibits HIV fusion

Summary The 21-amino acid peptides siamycin II (BMY-29303) and siamycin I (BMY-29304), derived from Streptomyces strains AA3891 and AA6532, respectively, have been found to inhibit HIV-1 fusion and viral replication in cell culture. The primary sequence of siamycin II is CLGIGSCNDFAGCGYAIVCFW. Siamycin I differs by only one amino acid; it has a valine residue at position 4. In both peptides, disulfide bonds link Cys¹ with Cys¹³ and Cys⁷ with Cys¹⁹, and the side chain of Asp⁹ forms an amide bond with the N-terminus. Siamycin II, when dissolved in a 50:50 mixture of DMSO and H₂O, yields NOESY spectra with exceptional numbers of cross peaks for a peptide of this size. We have used 335 NOE distance constraints and 13 dihedral angle constraints to generate an ensemble of 30 siamycin II structures; these have average backbone atom and all heavy atom rmsd values to the mean coordinates of 0.24 and 0.52 Å, respectively. The peptide displays an unusual wedge-shaped structure, with one face being predominantly hydrophobic and the other being predominantly hydrophilic. Chemical shift and NOE data show that the siamycin I structure is essentially identical to siamycin II. These peptides may act by preventing oligomerization of the HIV transmembrane glycoprotein gp41, or by interfering with interactions between gp41 and the envelope glycoprotein gp120, the cell membrane or membrane-bound proteins [Frèchet, D. et al. (1994) Biochemistry, 33, 42–50]. The amphipathic nature of siamycin II and siamycin I suggests that a polar (or apolar) site on the target protein may be masked by the apolar (or polar) face of the peptide upon peptide/protein complexation.Abbreviations ABNR adopted basis Newton Raphson - AIDS acquired immunodeficiency syndrome - CW continuous wave - DMSO dimethylsulfoxide - DQF-COSY two-dimensional double-quantum-filtered correlation spectroscopy - HIV human immunodeficiency virus - HSQC heteronuclear single-quantum coherence - NOE nuclear Overhauser enhancement - NOESY two-dimensional nuclear Overhauser enhancement spectroscopy - ppm parts per million - P.E.-COSY two-dimensional primitive exclusive correlation spectroscopy - REDAC redundant dihedral angle constraint - rf radio frequency - rmsd root-mean-square difference - SIV simian immunodeficiency virus - sw spectral width - _m mixing time - TOCSY two-dimensional total correlation spectroscopy - TSP trimethylsilyl-2,2,3,3-²H₄-propionate - 2D two-dimensional     

Proton nuclear magnetic resonance studies on huwentoxin-I from the venom of the spiderSelenocosmia huwena: 1. Sequence-specific1H-NMR assignments

The complete sequence-specific assignments of resonances in the¹H-NMR spectrum of huwentoxin-I from the Chinese bird spider,Selenocosmia huwena, is described. A combination of two-dimensional NMR experiments including 2D-COSY, 2D-NOESY, and 2D-TOCSY has been employed on samples of the toxin dissolved in D₂O and in H₂O for assignment purposes. Protons belonging to spin systems for each of the 33 amino acids were identified. The sequence-specific assignments were facilitated by the identification ofd _N connectivities on the fingerprint regions of the COSY and NOESY spectra and were supported by the identification ofd _NN andd _N connectivities in the TOCSY and NOESY spectra. These studies provide a basis for the determination of the solution-phase conformation of this toxin.Abbreviations HWTX-I huwentoxin-I - 2D two-dimensional - COSY 2D homonuclear correlation spectroscopy - NOE nuclear Overhauser enhancement - NOESY 2D nuclear Overhauser enhancement spectroscopy - TOCSY 2D total correlation spectroscopy - TPPI time-proportional phase incrementation - TSP sodium 3-(trimethyl-silyl)propionate-d4 - EDTA ethylenediaminetetraacetic acid     

Structural Studies of 5-Ethyl-2′-Deoxyuridine by Selective Pulse 1H DPFGSE NOE Spectroscopy and PM3 Calculations

Abstract

The solution conformation of 5-ethyl-2′-deoxyuridine (EDU) has been calculated from the vicinyl proton-proton NMR coupling constants and nuclear Overhauser (NOE) distances using excitation sculpting of selective pulses (Double Pulsed Field Gradient Spin Echo NOE) at 500 MHz and molecular modelling (PM3) studies.     

Projected [<Superscript>1</Superscript>H,<Superscript>15</Superscript>N]-HMQC-[<Superscript>1</Superscript>H,<Superscript>1</Superscript>H]-NOESY for large molecular systems: application to a 121 kDa protein-DNA complex

We present a projected [¹H,¹⁵N]-HMQC-[¹H,¹H]-NOESY experiment for observation of NOE interactions between amide protons with degenerate ¹⁵N chemical shifts in large molecular systems. The projection is achieved by simultaneous evolution of the multiple quantum coherence of the nitrogen spin and the attached proton spin. In this way NOE signals can be separated from direct-correlation peaks also in spectra with low resolution by fully exploiting both ¹H and ¹⁵N frequency differences, such that sensitivity can be increased by using short maximum evolution times. The sensitivity of the experiment is not dependent on the projection angle for projections up to 45° and no additional pulses or delays are required as compared to the conventional 2D [¹H,¹⁵N]-HMQC-NOESY. The experiment provides two distinct 2D spectra corresponding to the positive and negative angle projections, respectively. With a linear combination of 1D cross-sections from the two projections the unavoidable sensitivity loss in projection spectra can be compensated for each particular NOE interaction. We demonstrate the application of the novel projection experiment for the observation of an NOE interaction between two sequential glycines with degenerate ¹⁵N chemical shifts in a 121.3 kDa complex of the linker H1 histone protein with a 152 bp linear DNA.     

31P NMR relaxation measurements of the phosphate backbone of a double stranded hexadeoxynucleotide in solution: determination of the chemical shift anisotropy

The five phosphates of the deoxynucleotide d(CpGpTpApCpG)₂ have been assigned by two-dimensional heteronuclear NMR spectroscopy. The chemical shift anisotropy and correlation time of each phosphate group has been determined from measurements of the spin-lattice, spin-spin relaxation rate constants and the ³¹P-{¹H} nuclear Overhauser enhancement (NOE) at three magnetic field strengths (4.7 T, 9.4 T, and 11.75 T) and two temperatures (288 K and 298 K). As expected, the relaxation data require two mechanisms to account for the observed rate constants, i.e. dipole-dipole and chemical shift anisotropy. At 9.4 T and 11.75 T, the latter mechanism dominates the relaxation, leading to insignificant NOE intensities. The correlation time, chemical shift anisotropy and effective P-H distance were obtained from least-squares fitting to the data. Comparison of the fitted value for the correlation time with that obtained from ¹H measurements shows that the molecule behaves essentially as rigid rotor on the nanosecond timescale. Large amplitude motions observed in long segments of DNA are due to bending motions that do not contribute significantly to relaxation in short oligonucleotides.Abbreviations CSA chemical shift anisotropy - NOE nuclear Overhauser enhancement Offprint requests to: A. N. Lane     

Identification of HN-methyl NOEs in large proteins using simultaneous amide-methyl TROSY-based detection

A pair of HN-methyl NOESY experiments that are based on simultaneous TROSY-type detection of amide and methyl groups is described. The preservation of cross-peak symmetry in the simultaneous ¹H–¹⁵N/¹³CH₃ NOE spectra enables straightforward assignments of HN-methyl NOE cross-peaks in large and complex protein structures. The pulse schemes are designed to preserve the slowly decaying components of both ¹H–¹⁵N and methyl ¹³CH₃ spin-systems in the course of indirect evolution (t ₂) and acquisition period (t ₃) of 3D NOESY experiments. The methodology has been tested on {U-[¹⁵N,²H]; Ileδ1-[¹³CH₃]; Leu,Val-[¹³CH₃,¹²CD₃]}-labeled 82-kDa enzyme Malate Synthase G (MSG). A straightforward procedure that utilizes the symmetry of NOE cross-peaks in the time-shared 3D NOE data sets allows unambiguous assignments of more than 300 HN-methyl interactions in MSG from a single 3D data set providing important structural restraints for derivation of the backbone global fold.     

Complete relaxation matrix refinement of NMR structures of proteins using analytically calculated dihedral angle derivatives of NOE intensities

Summary A new method for refining three-dimensional (3D) NMR structures of proteins is described, which takes account of the complete relaxation pathways. Derivatives of the NOE intensities with respect to the dihedral angles are analytically calculated, and efficiently evaluated with the use of a filter technique for identifying the dominant terms of these derivatives. This new method was implemented in the distance geometry program DIANA. As an initial test, we refined 30 rigid distorted helical structures, using a simulated data set of NOE distance constraints for a rigid standard -helix. The final root-mean-square deviations of the refined structures relative to the standard helix were less than 0.1 Å, and the R-factors dropped from values between 7% and 32% to values of less than 0.5% in all cases, which compares favorably with the results from distance geometry calculations. In particular, because spin diffusion was not explicitly considered in the evaluation of exact¹H–¹H distances corresponding to the simulated NOE intensities, a group of nearly identical distance geometry structures was obtained which had about 0.5 Å root-mean-square deviation from the standard -helix. Further test calculations using an experimental NOE data set recorded for the protein trypsin inhibitor K showed that the complete relaxation matrix refinement procedure in the DIANA program is functional also with systems of practical interest.Abbreviations RMSD root-mean-square deviation - NOE nuclear Overhauser enhancement - NOESY 2-dimensional nuclear Overhauser enhancement spectroscopy - CPU central processing unit