1H,13C-1H,1H Dipolar Cross-correlated Spin Relaxation in Methyl Groups

Relaxation in methyl groups is strongly influenced by cross-correlated interactions involving the methyl dipoles. One of the major interference effects results from intra-methyl (1)H-(13)C, (1)H-(1)H dipolar interactions, leading to significant differences in the relaxation of certain multiplet components that contribute to double- and zero-quantum (1)H-(13)C spectra. NMR experiments are presented for the measurement of this differential relaxation effect. It is shown that this difference in relaxation between double- and zero-quantum multiplet components can be used as a sensitive reporter of side chain dynamics and that accurate methyl axis order parameters can be measured in proteins that tumble with correlation times greater than approximately 5 ns.     

Measurement of Scaled Residual Dipolar Couplings in Proteins Using Variable-angle Sample Spinning

NMR spectra of ubiquitin in the presence of bicelles at a concentration of 25% w/v have been recorded under sample spinning conditions for different angles of rotation. For an axis of rotation equal to the magic angle, the (1)H/(15)N HSQC recorded without any (1)H decoupling in the indirect dimension corresponds to the classical spectrum obtained on a protein in an isotropic solution and allows the measurement of scalar J-couplings (1) J (NH). For an angle of rotation smaller than the magic angle, the bicelles orient with their normal perpendicular to the spinning axis, whereas for an angle of rotation greater than the magic angle the bicelles orient with their normal along the spinning axis. This bicelle alignment creates anisotropic conditions that give rise to the observation of residual dipolar couplings in ubiquitin. The magnitude of these dipolar couplings depends directly on the angle that the rotor makes with the main magnetic field. By changing this angle in a controlled manner, residual dipolar couplings can be either scaled up or down thus offering the possibility to study simultaneously a wide range of dipolar couplings in the same sample.     

A Thorough Dynamic Interpretation of Residual Dipolar Couplings in Ubiquitin

The presence of slow motions with large amplitudes, as detected by measurements based on residual dipolar couplings [Peti, W., Meiler, J., Brueschweiler, R. and Griesinger, C. (2002) J. Am. Chem. Soc., 124, 5822–5833], has stirred up much discussion in recent years. Based on ubiquitin NH residual dipolar couplings (rdcs) measured in 31 different alignment conditions, a model-free analysis of structure and dynamics [Meiler, J., Peti, W., Prompers, J., Griesinger, C. and Brueschweiler, R. (2001) J. Am. Chem. Soc., 123, 6098–6107] is presented. Starting from this broad experimental basis, rdc-based order parameters with so far unattained accuracy were determined. These rdc-based order parameters underpin the presence of new modes of motion slower than the inverse overall tumbling correlation time. Amplitudes and anisotropies of the motion were derived. The effect of structural noise on the results was proven to be negligible. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Estimating the Accuracy of Protein Structures using Residual Dipolar Couplings

It has been commonly recognized that residual dipolar coupling data provide a measure of quality for protein structures. To quantify this observation, a database of 100 single-domain proteins has been compiled where each protein was represented by two independently solved structures. Backbone ¹H–¹⁵N dipolar couplings were simulated for the target structures and then fitted to the model structures. The fits were characterized by an R-factor which was corrected for the effects of non-uniform distribution of dipolar vectors on a unit sphere. The analyses show that favorable values virtually guarantee high accuracy of the model structure (where accuracy is defined as the backbone coordinate rms deviation). On the other hand, unfavorable values do not necessarily suggest low accuracy. Based on the simulated data, a simple empirical formula is proposed to estimate the accuracy of protein structures. The method is illustrated with a number of examples, including PDZ2 domain of human phosphatase hPTP1E. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

A set of HA-detected experiments for measuring scalar and residual dipolar couplings

A new set of HCACO based three-dimensional NMR experiments for measuring residual dipolar couplings in proteins is presented. Using spin-state selection and editing in three dimensions, the experiments allow accurate measurement of intraresidual , and scalar and residual dipolar couplings of ¹⁵N/¹³C labeled proteins in D₂O and dilute liquid crystals with minimal spectral crowding. The presented experiments are especially suitable for small or medium sized proline-rich proteins, or proteins that require high pH solvent conditions, making ¹H^N detected experiments unattractive. In addition, the tetrahedral coordination of C is superior to the planar peptide bond for determination of local alignments in partially structured polypeptides. For the efficient use of spectrometer time and to avoid complications arising from the varying magnitude of the alignment tensor during relatively long experiments, the and couplings can also be measured simultaneously in an E.COSY like manner with high accuracy. The pulse sequences are balanced for cross-correlation effects and minimized for relaxation losses. The pulse sequences are tested with a sample of ¹⁵N/¹³C human ubiquitin. We find internuclear vector directions determined from the dipolar couplings to have an excellent correlation with those of ubiquitins refined solution structure.     

Applications of Variable-angle Sample Spinning Experiments to the Measurement of Scaled Residual Dipolar Couplings and <Superscript>15</Superscript>N CSA in Soluble Proteins

NMR spectra of ubiquitin in the presence of bicelles at a concentration of 32% w/v have been recorded at 700 MHz under sample spinning conditions at the magic angle (54.7°) and at an angle of 45.5°. At the magic angle, the ¹H–¹⁵N HSQC spectrum of ubiquitin in bicelles is virtually indistinguishable from the one recorded on the protein in solution. Spinning the sample at the magic angle creates an isotropic environment with no preferred bicelle orientations, thus allowing the determination of scalar coupling constants. For an angle of rotation of 45.5°, the bicelles orient with their normal perpendicular to the spinning axis leading to the observation of strong residual dipolar couplings and chemical shift variations of the ¹⁵N resonances. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

TROSY experiment for refinement of backbone psi and phi by simultaneous measurements of cross-correlated relaxation rates and 3,4J(H alpha HN) coupling constants

The TROSY principle has been introduced into a HNCA experiment, which is designed for measurements of the intraresidual and sequential H-C/H^N-N dipole/dipole and H-C/N dipole/CSA cross-correlated relaxation rates. In addition, the new experiment provides values of the ^3,4 J _{H HN} coupling constants measured in an E.COSY manner. The conformational restraints for the and angles are obtained through the use of the cross-correlated relaxation rates together with the Karplus-type dependencies of the coupling constants. Improved signal-to-noise is achieved through preservation of all coherence transfer pathways and application of the TROSY principle. The application of the [¹⁵N,¹³C]-DQ/ZQ-[¹⁵N,¹H]-TROSY-E.COSY experiment to the 16 kDa apo-form of the E. coli Heme Chaperon protein CcmE is described. Overall good agreement is achieved between and angles measured with the new experiment and the average values determined from an ensemble of 20 NMR conformers.     

Application of Correlated Residual Dipolar Couplings to the Determination of the Molecular Alignment Tensor Magnitude of Oriented Proteins and Nucleic Acids

Residual dipolar couplings (RDC) between nuclear spins in partially aligned samples offer unique insights into biomacromolecular structure and dynamics. To fully benefit from the RDC data, accurate knowledge of the magnitude ( D (a)) and rhombicity ( R ) of the molecular alignment tensor, A, is important. An extended histogram method (EHM) is presented which extracts these parameters more effectively from dipolar coupling data. The method exploits the correlated nature of RDCs for structural elements of planar geometry, such as the one-bond (13)C'(i)-(13)C(i)(alpha), (13)C'(i)-(15)N(i+1), and (15)N(i+1)-(1)H(N)(i+1) couplings in peptide bonds of proteins, or suitably chosen combinations of (1) D (C1'H1'), (1) D (C2'H2'), (1) D (C1'C2'), (2) D (C2'H1'), (2) D (C1'H2'), and (3) D (H1'H2') couplings in nucleic acids, to generate an arbitrarily large number of synthetic RDCs. These synthetic couplings result in substantially improved histograms and resulting values of D (a) and R, compared with histograms generated solely from the original sets of correlated RDCs, particularly when the number of planar fragments for which couplings are available is small. An alternative method, complementary to the EHM, is also described, which uses a systematic grid search procedure, based on least-squares fitting of sets of correlated RDCs to structural elements of known geometry, and provides an unambiguous lower limit for the degree of molecular alignment.     

Sensitivity-optimized experiment for the measurement of residual dipolar couplings between amide protons

High signal to noise is a necessity for the quantification of NMR spectral parameters to be translated into accurate and precise restraints on protein structure and dynamics. An important source of long-range structural information is obtained from ¹H–¹H residual dipolar couplings (RDCs) measured for weakly aligned molecules. For sensitivity reasons, such measurements are generally performed on highly deuterated protein samples. Here we show that high sensitivity is also obtained for protonated protein samples if the pulse schemes are optimized in terms of longitudinal relaxation efficiency and J-mismatch compensated coherence transfer. The new sensitivity-optimized quantitative J-correlation experiment yields important signal gains reaching factors of 1.5 to 8 for individual correlation peaks when compared to previously proposed pulse schemes. Paul Schanda and Ewen Lescop contributed equally to this work.     

Measurement of small scalar and dipolar couplings in purine and pyrimidine bases*

A suite of spin-state-selective excitation (S³E) NMR experiments for the measurements of small one-bond (¹³C-¹³C, ¹⁵N-¹³C) and two-bond (¹H-¹³C, ¹H-¹⁵N) coupling constants in ¹³C,¹⁵N labeled purine and pyrimidine bases is presented. The incorporation of band-selective shaped pulses, elimination of the cross talk between and sub-spectra, and accuracy and precision of the proposed approach are discussed. Merits of using S³E rather than /-half-filter are demonstrated using results obtained on isotopically labeled DNA oligonucleotides.     

Quantitative NMR studies of high molecular weight proteins: application to domain orientation and ligand binding in the 723 residue enzyme malate synthase G

A high-resolution multidimensional NMR study of ligand-binding to Escherichia coli malate synthase G (MSG), a 723-residue monomeric enzyme (81.4 kDa), is presented. MSG catalyzes the condensation of glyoxylate with an acetyl group of acetyl-CoA, producing malate, an intermediate in the citric-acid cycle. We show that despite the size of the protein, important structural and dynamic information about the molecule can be obtained on a per-residue basis. 15N-1HN residual dipolar couplings and carbonyl chemical shift changes upon alignment in Pf1 phage establish that there are no significant domain reorientations in the molecule upon ligand binding, in contrast to what was anticipated on the basis of both the X-ray structure of the glyoxylate-bound form of the enzyme and structural studies of a related set of proteins. The chemical shift changes of 1HN, 15N and 13CO nuclei upon binding of pyruvate, a glyoxylate-mimicking inhibitor, and acetyl-CoA have been mapped onto the three-dimensional structure of the molecule. Binding constants of pyruvate, glyoxylate, and acetyl-CoA (in the presence of pyruvate) have been measured, along with the kinetic parameters for glyoxylate and pyruvate binding. The on-rates of pyruvate and glyoxalate binding, approximately 1.2 x 10(6)M(-1)s(-1) and approximately 2.7 x 10(6)M(-1)s(-1), respectively, are significantly lower than what is anticipated from a simple diffusion-controlled process. Some structural implications of the chemical shift perturbations upon binding and the estimated ligand on-rates are discussed.     

NMR experiments for the measurement of proton-proton and carbon-carbon residual dipolar couplings in uniformly labelled oligosaccharides

A 2D-HSQC-carbon selective/proton selective-constant time COSY, 2D-HSQC-(sel C, sel H)-CT COSY experiment, which is applicable to uniformly ¹³C isotopically enriched samples (U-¹³C) of oligosaccharides or oligonucleotides is proposed for the measurement of proton–proton RDC in crowded regions of 2D-spectra. In addition, a heteronuclear constant time-COSY experiment, ¹³C-¹³C CT-COSY, is proposed for the measurement of one bond carbon–carbon RDC in these molecules. These two methods provide an extension, to U-¹³C molecules, of the original homonuclear constant time-COSY experiment proposed by Tian et al. (1999) for saccharides. The combination of a number of these RDC with NOE data may provide the method of choice to study oligosaccharide conformation in the free and receptor-bound state.     

Structure and orientation of a G protein fragment in the receptor bound state from residual dipolar couplings

Residual dipolar couplings for a ligand that is in fast exchange between a free state and a state where it is bound to a macroscopically ordered membrane protein carry precise information on the structure and orientation of the bound ligand. The couplings originate in the bound state but can be detected on the free ligand using standard high resolution NMR. This approach is used to study an analog of the C-terminal undecapeptide of the alpha-subunit of the heterotrimeric G protein transducin when bound to photo-activated rhodopsin. Rhodopsin is the major constituent of disk-shaped membrane vesicles from rod outer segments of bovine retinas, which align spontaneously in the NMR magnet. Photo-activation of rhodopsin triggers transient binding of the peptide, resulting in measurable dipolar contributions to 1J(NH) and 1J(CH) splittings. These dipolar couplings report on the time-averaged orientation of bond vectors in the bound peptide relative to the magnetic field, i.e. relative to the membrane normal. Approximate distance restraints of the bound conformation were derived from transferred NOEs, as measured from the difference of NOESY spectra recorded prior to and after photo-activation. The N-terminal eight residues of the bound undecapeptide adopt a near-ideal alpha-helical conformation. The helix is terminated by an alpha(L) type C-cap, with Gly9 at the C' position in the center of the reverse turn. The angle between the helix axis and the membrane normal is 40 degrees (+/-4) degrees. Peptide protons that make close contact with the receptor are identified by analysis of the NOESY cross-relaxation pattern and include the hydrophobic C terminus of the peptide.     

Exact Solutions for Internuclear Vectors and Backbone Dihedral Angles from NH Residual Dipolar Couplings in Two Media, and their Application in a Systematic Search Algorithm for Determining Protein Backbone Structure

We have derived a quartic equation for computing the direction of an internuclear vector from residual dipolar couplings (RDCs) measured in two aligning media, and two simple trigonometric equations for computing the backbone (phi,psi) angles from two backbone vectors in consecutive peptide planes. These equations make it possible to compute, exactly and in constant time, the backbone (phi,psi) angles for a residue from RDCs in two media on any single backbone vector type. Building upon these exact solutions we have designed a novel algorithm for determining a protein backbone substructure consisting of alpha-helices and beta-sheets. Our algorithm employs a systematic search technique to refine the conformation of both alpha-helices and beta-sheets and to determine their orientations using exclusively the angular restraints from RDCs. The algorithm computes the backbone substructure employing very sparse distance restraints between pairs of alpha-helices and beta-sheets refined by the systematic search. The algorithm has been demonstrated on the protein human ubiquitin using only backbone NH RDCs, plus twelve hydrogen bonds and four NOE distance restraints. Further, our results show that both the global orientations and the conformations of alpha-helices and beta-strands can be determined with high accuracy using only two RDCs per residue. The algorithm requires, as its input, backbone resonance assignments, the identification of alpha-helices and beta-sheets as well as sparse NOE distance and hydrogen bond restraints.     

Structure of calmodulin complexed with an olfactory CNG channelfragment and role of the central linker: Residual dipolar couplingsto evaluate calmodulin binding modes outside the kinase family

The NMR high-resolution structure of calmodulin complexed with a fragment of the olfactory cyclic-nucleotide gated channel is described. This structure shows features that are unique for this complex, including an active role of the linker connecting the N- and C-lobes of calmodulin upon binding of the peptide. Such linker is not only involved in the formation of an hydrophobic pocket to accommodate a bulky peptide residue, but it also provides a positively charged region complementary to a negative charge of the target. This complex of calmodulin with a target not belonging to the kinase family was used to test the residual dipolar coupling (RDC) approach for the determination of calmodulin binding modes to peptides. Although the complex here characterized belongs to the (1--14) family, high Q values were obtained with all the 1:1 complexes for which crystalline structures are available. Reduction of the RDC data set used for the correlation analysis to structured regions of the complex allowed a clear identification of the binding mode. Excluded regions comprise calcium binding loops and loops connecting the EF-hand motifs.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s10858-005-0165-1.     

Accurate determination of leucine and valine side-chain conformations using U-[15N/13C/2H]/[1H-(methine/methyl)-Leu/Val] isotope labeling, NOE pattern recognition, and methine Cgamma-Hgamma/Cbeta-Hbeta residual dipolar couplings: application to the 34-kDa enzyme IIA(chitobiose)

An isotope labeling scheme is described in which specific protonation of methine and methyl protons of leucine and valine is obtained on a ¹⁵N/¹³C labeled background with uniform deuteration of all other non-exchangeable protons. The presence of a protonated methine group has little effect on the favorable relaxation properties of the methyl protons of Leu and Val. This labeling scheme permits the rotameric state of leucine side-chains to be readily determined by simple inspection of the pattern of Hγ(i)–H_N(i) and Hγ(i)–H_N(i+1) NOEs in a 3D ¹⁵N-separated NOE spectrum free of complications arising from spectral overlap and spin-diffusion. In addition, one-bond residual dipolar couplings for the methine ¹³C–¹H bond vectors of Leu and Val can be accurately determined from an intensity J-modulated constant-time HCCH-COSY experiment and used to accurately orient the side-chains of Leu and Val. Incorporation of these data into structure refinement improves the accuracy with which the conformations of Leu and Val side-chains can be established. This is important to ensure optimal packing both within the protein core and at intermolecular interfaces. The impact of the method on protein structure determination is illustrated by application to enzyme IIA^Chitobiose, a 34 kDa homotrimeric phosphotransferase protein.     

Direct NMR observation and DFT calculations of a hydrogen bond at the active site of a 44 kDa enzyme

A hydrogen bond between the amide backbone of Arg7 and the remote imidazole side chain of His106 has been directly observed by improved TROSY-NMR techniques in the 44 kDa trimeric enzyme chorismate mutase from Bacillus subtilis. The presence of this hydrogen bond in the free enzyme and its complexes with a transition state analog and the reaction product was demonstrated by measurement of ¹⁵N-¹⁵N and ¹H-¹⁵N trans-hydrogen bond scalar couplings, ^2h J _NN and ^1h J _HN, and by transfer of nuclear polarization across the hydrogen bond. The conformational dependences of these coupling constants were analyzed using sum-over-states density functional perturbation theory (SOS-DFPT). The observed hydrogen bond might stabilize the scaffold at the active site of BsCM. Because the Arg7-His106 hydrogen bond has not been observed in any of the high resolution crystal structures of BsCM, the measured coupling constants provide unique information about the enzyme and its complexes that should prove useful for structural refinement of atomic models.     

Quantitative estimation of magnitude and orientation of the CSA tensor from field dependence of longitudinal NMR relaxation rates

A method is presented that makes it possible to estimate both the orientation and the magnitude of the chemical shift anisotropy (CSA) tensor in molecules with a pair of spin 1/2 nuclei, typically ¹³C-¹H or ¹⁵ N-¹H. The method relies on the fact that the longitudinal cross-correlation rate as well as a linear combination of the autorelaxation rates of longitudinal heterospin magnetization, longitudinal two-spin order and longitudinal proton magnetization are proportional to the spectral density at the Larmor frequency of the heterospin. Therefore the ratio between the cross-correlation rate and the above linear combination is independent of the dynamics. From the field dependence of the ratio both the magnitude and the orientation of the CSA tensor can be estimated. The method is applicable to molecules in all motional regimes and is not limited to molecules in extreme narrowing or slow tumbling, nor is it sensitive to chemical exchange broadening. It is tested on the 22 amino acid residue peptide motilin, selectively ¹³ C labeled in the ortho positions in the ring of the single tyrosine residue. In the approximation of an axially symmetric ¹³C CSA tensor, the symmetry axis of the CSA tensor makes an angle of 23° ± 1° to the ¹³ C-¹H bond vector, and has a magnitude of 156 ± 5 ppm. This is in close agreement with solid-state NMR data on tyrosine powder [Frydman et al. (1992) Isr. J. Chem., 32, 161–164].     

Angular dependence of 1J(Ni,Calphai) and 2J(Ni,Calpha(i-1)) coupling constants measured in J-modulated HSQCs

A new method to measure ¹J(N_i,C _i) and ²J(N_i,C _{(i – 1)}) coupling constants in proteins based on a J-modulated sensitivity enhanced HSQC was introduced. Coupling constants were measured in the denatured and in the native state of ubiquitin and found to depend on the conformation of the protein backbone. Using a combined data set of experimental coupling constants from ubiquitin and staphylococcal nuclease (Delaglio et al., 1991), the angular dependence of the coupling constants on the backbone angles and was investigated. It was found that the size of ²J(N_i,C _{(i – 1)}) correlates strongly with the backbone conformation, while only a weak conformational dependence on the size of ¹J(N_i,C _i) coupling constants was observed. Coupling constants in the denatured state of ubiquitin were uniform along the sequence of the protein and not dependent on a given residue type. Furthermore it was shown that the observed coupling constants were in good agreement with predicted coupling constants using a simple model for the random coil.     

Molecular details of Itk activation by prolyl isomerization and phospholigand binding: the NMR structure of the Itk SH2 domain bound to a phosphopeptide

The Src homology 2 (SH2) domain of interleukin-2 tyrosine kinase (Itk) is a critical component of the regulatory apparatus controlling the activity of this immunologically important enzyme. To gain insight into the structural features associated with the activated form of Itk, we have solved the NMR structure of the SH2 domain bound to a phosphotyrosine-containing peptide (pY) and analyzed changes in trans-hydrogen bond scalar couplings ((3h)J(NC')) that result from pY binding. Isomerization of a single prolyl imide bond in this domain is responsible for simultaneous existence of two distinct SH2 conformers. Prolyl isomerization directs ligand recognition: the trans conformer preferentially binds pY. The structure of the SH2/pY complex provides insight into the ligand specificity; the BG loop in the ligand-free trans SH2 conformer is pre-arranged for optimal contacts with the pY+3 residue of the ligand. Analysis of (3h)J(NC') couplings arising from hydrogen bonds has revealed propagation of structural changes from the pY binding pocket to the CD loop containing conformationally heterogeneous proline as well as to the alphaB helix, on the opposite site of the domain. These findings offer a structural framework for understanding the roles of prolyl isomerization and pY binding in Itk regulation.