Transverse relaxation optimized triple-resonance NMR experiments for nucleic acids

Triple resonance HCN and HCNCH experiments are reliable methods of establishing sugar-to-base connectivity in the NMR spectra of isotopicaly labeled oligonucleotides. However, with larger molecules the sensitivity of the experiments is drastically reduced due to relaxation processes. Since the polarization transfer between ¹³C and ¹⁵N nuclei relies on rather small heteronuclear coupling constants (11–12 Hz), the long evolution periods (up to 30–40 ms) in the pulse sequences cannot be avoided. Therefore any effort to enhance sensitivity has to concentrate on manipulating the spin system in such a way that the spin–spin relaxation rates would be minimized. In the present paper we analyze the efficiency of the two known approaches of relaxation rate control, namely the use of multiple-quantum coherence (MQ) and of the relaxation interference between chemical shift anisotropy and dipolar relaxation – TROSY. Both theoretical calculations and experimental results suggest that for the sugar moiety (H1-C1-N1/9) the MQ approach is clearly preferable. For the base moiety (H6/8-C6/8-N1/9), however, the TROSY shows results superior to the MQ suppression of the dipole–dipole relaxation at moderate magnetic fields (500 MHz) and the sensitivity improvement becomes dramatically more pronounced at very high fields (800 MHz). The pulse schemes of the triple-resonance HCN experiments with sensitivity optimized performance for unambiguous assignments of intra-residual sugar-to-base connectivities combining both approaches are presented.     

Histidine side-chain dynamics and protonation monitored by 13C CPMG NMR relaxation dispersion

The use of ¹³C NMR relaxation dispersion experiments to monitor micro-millisecond fluctuations in the protonation states of histidine residues in proteins is investigated. To illustrate the approach, measurements on three specifically ¹³C labeled histidine residues in plastocyanin (PCu) from Anabaena variabilis (A.v.) are presented. Significant Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion is observed for ¹³C^ε1 nuclei in the histidine imidazole rings of A.v. PCu. The chemical shift changes obtained from the CPMG dispersion data are in good agreement with those obtained from the chemical shift titration experiments, and the CPMG derived exchange rates agree with those obtained previously from ¹⁵N backbone relaxation measurements. Compared to measurements of backbone nuclei, ¹³C^ε1 dispersion provides a more direct method to monitor interchanging protonation states or other kinds of conformational changes of histidine side chains or their environment. Advantages and shortcomings of using the ¹³C^ε1 dispersion experiments in combination with chemical shift titration experiments to obtain information on exchange dynamics of the histidine side chains are discussed.     

13C spin relaxation measurements in RNA: Sensitivity and resolution improvement using spin-state selective correlation experiments

A set of new NMR pulse sequences has been designed for the measurement of ¹³C relaxation rate constants in RNA and DNA bases: the spin-lattice relaxation rate constant R(C_z), the spin-spin relaxation rate constant R(C₊), and the CSA-dipolar cross-correlated relaxation rate constant . The use of spin-state selective correlation techniques provides increased sensitivity and spectral resolution. Sensitivity optimised C-C filters are included in the pulse schemes for the suppression of signals originating from undesired carbon isotopomers. The experiments are applied to a 15% ¹³C-labelled 33-mer RNA–theophylline complex. The measured ratios indicate that ¹³C CSA tensors do not vary significantly for the same type of carbon (C₂, C₆, C₈), but that they differ from one type to another. In addition, conformational exchange effects in the RNA bases are detected as a change in the relaxation decay of the narrow ¹³C doublet component when varying the spacing of a CPMG pulse train. This new approach allows the detection of small exchange effects with a higher precision compared to conventional techniques.     

Internal motions of apo-neocarzinostatin as studied by 13C NMR methine relaxation at natural abundance

Summary Dynamics of the backbone and some side chains of apo-neocarzinostatin, a 10.7 kDa carrier protein, have been studied from ¹³C relaxation rates R1, R2 and steady-state ¹³C-{¹H} NOEs, measured at natural abundance. Relaxation data were obtained for 79 nonoverlapping C resonances and for 11 threonine C single resonances. Except for three C relaxation rates, all data were analysed from a simple two-parameter spectral density function using the model-free approach of Lipari and Szabo. The corresponding C–H fragments exhibit fast (_e < 40 ps) restricted libration motions (S²=0.73 to 0.95). Global examination of the microdynamical parameters S² and _e along the amino acid sequence gives no immediate correlation with structural elements. However, different trends for the three loops involved in the binding site are revealed. The -ribbon comprising residues 37 to 47 is spatially restricted, with relatively large _e values in its hairpin region. The other -ribbon (residues 72 to 87) and the large disordered loop ranging between residues 97–107 experience small-amplitude motions on a much faster (picosecond) time scale. The two N-terminal residues, Ala¹ and Ala², and the C-terminal residue Asn¹¹³, exhibit an additional slow motion on a subnanosecond time scale (400–500 ps). Similarly, the relaxation data for eight threonine side-chain C must be interpreted in terms of a three-parameter spectral density function. They exhibit slower motions, on the nanosecond time scale (500–3000 ps). Three threonine (Thr⁶⁵, Thr⁶⁸, Thr⁸¹) side chains do not display a slow component, but an exchange contribution to the observed transverse relaxation rate R2 could not be excluded at these sites. The microdynamical parameters (S², _e and R2_ex) or (S _infslow ^sup2 , S _inffast ^sup2 and _slow) were obtained from a straightforward solution of the equations describing the relaxation data. They were calculated assuming an overall isotropic rotational correlation time _e for the protein of 5.7 ns, determined using standard procedures from R2/R1 ratios. However, it is shown that the product (1–S²)× _e is nearly independent of _e for residues not exhibiting slow motions on the nanosecond time scale. In addition, this parameter very closely follows the heteronuclear NOEs, which therefore could be good indices for local fast motions on the picosecond time scale.     

Residue Specific Ribose and Nucleobase Dynamics of the cUUCGg RNA Tetraloop Motif by MNMR 13C Relaxation

The dynamics of the nucleobase and the ribose moieties in a 14-nt RNA cUUCGg hairpin-loop uniformly labeled with ¹³C and ¹⁵N were studied by ¹³C spin relaxation experiments. R₁, R_1ρ and the ¹³C-{¹H} steady-state NOE of C₆ and C_1′ in pyrimidine and C₈ and C_1′ in purine residues were obtained at 298 K. The relaxation data were analyzed by the model-free formalism to yield dynamic information on timescales of pico-, nano- and milli-seconds. An axially symmetric diffusion tensor with an overall rotational correlation time τ_c of 2.31±0.13 ns and an axial ratio of 1.35±0.02 were determined. Both findings are in agreement with hydrodynamic calculations. For the nucleobase carbons, the validity of different reported ¹³C chemical shift anisotropy values (Stueber, D. and Grant, D. M., 2002 J. Am. Chem. Soc. 124, 10539–10551; Fiala et al., 2000 J. Biomol. NMR 16, 291–302; Sitkoff, D. and Case, D. A., 1998 Prog. NMR Spectroscopy 32, 165–190) is discussed. The resulting dynamics are in agreement with the structural features of the cUUCGg motif in that all residues are mostly rigid (0.82 < S² < 0.96) in both the nucleobase and the ribose moiety except for the nucleobase of U7, which is protruding into solution (S² = 0.76). In general, ribose mobility follows nucleobase dynamics, but is less pronounced. Nucleobase dynamics resulting from the analysis of ¹³C relaxation rates were found to be in agreement with ¹⁵N relaxation data derived dynamic information (Akke et al., 1997 RNA 3, 702–709). Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Internal mobility of cyclic RGD hexapeptides studied by 13C NMR relaxation and the model-free approach

Summary The internal mobility of three isomeric cyclic RGD hexapeptides designed to contain two -turns in defined positions, cyclo(Arg-Gly-Asp-Gly-d-Pro-Pro) (I), cyclo(Arg-Gly-Asp-d-Pro-Gly-Pro) (II) and cyclo(Arg-Gly-Asp-d-Pro-Pro-Gly) (III), have been studied by ¹³C NMR longitudinal and transverse relaxation experiments and measurements of steady-state heteronuclear {¹H}-¹³C NOE enhancement with ¹³C at natural abundance. The data were interpreted according to the model-free formalism of Lipari and Szabo, which is usually applied to data from macromolecules or larger sized peptides with overall rotational correlation times exceeding 1 ns, to yield information about internal motions on the 10–100 ps time scale. The applicability of the model-free analysis with acceptable uncertainties to these small peptides, with overall rotational correlation times slightly below 0.3 ns, was demonstrated for this specific instance. Chemical exchange contributions to T₂ from slower motions were also identified in the process. According to the order parameters obtained for its backbone -carbon atoms, II has the most rigid backbone conformation on the 10–100 ps time scale, and I the most flexible. This result coincides with the results of earlier NMR-constrained conformational searches, which indicated greatest uncertainty in the structure of I and least in II.     

Motional properties of a pentasaccharide containing a 2,6-branched mannose residue as studied by 13C nuclear spin relaxation

Summary ¹³C relaxation data obtained at three different magnetic fields, 9.4, 11.8 and 14.1 T, and at two temperatures, 303 and 318 K, are reported for the pentasaccharide p-trifluoroacetamidophenyl 2,6-di-O-[-d-galactopyranosyl-(14)-O-2-acetamido-2-deoxy--d-glucopyranosyl]-d-mannopyranoside. The pentasaccharide consists of two disaccharide units, attached at position 2 and 6 to the central mannopyranoside residue. The relaxation data were interpreted with the Lipari-Szabo model-free approach. For the central mannose residue in the molecule a high order parameter (S²=0.91) was found and the relaxation data could be interpreted with the truncated form of the Lipari-Szabo model. The motional behavior of the two 2-acetamido-2-deoxy-glucopyranoside residues was found to differ. The one attached at the primary hydroxylic position displayed more extensive local motion (S²=0.75–0.77) than the one attached at the secondary hydroxylic position (S²=0.83–0.85). More extensive local motion for the two outer galactopyranoside residues was found (S²=0.56–0.59), but no significant difference in motional behavior between the two residues could be observed. Analysis of the relaxation data for the exocyclic carbons confirmed the results for the rings. For the mannose C6, the same motional parameters as obtained for the substituting 2-acetamido-2-deoxy-glucopyranoside residue were found. The two exocyclic carbons on the 2-acetamido-2-deoxy-glucopyranoside residues showed more extensive local motion, with lower order parameters (S²=0.59–0.66).To whom correspondence should be addressed.     

13C NMR relaxation studies of RNA base and ribose nuclei reveal a complex pattern of motions in the RNA binding site for human U1A protein

The widespread importance of induced fit and order-disorder transition in RNA recognition by proteins and small molecules makes it imperative that RNA motional properties are characterized quantitatively. Until now, however, very few studies have been dedicated to the systematic characterization of RNA motion and to their changes upon protein or small-molecule binding. The U1A protein-RNA complexes provide some of the best-studied examples of the role of RNA motional changes upon protein binding. Here, we report (13)C NMR relaxation studies of base and ribose dynamics for the RNA internal loop target of human U1A protein located within the 3'-untranslated region (3'-UTR) of the mRNA coding for U1A itself. We also report the semi-quantitative analysis of both fast (nano- to picosecond) and intermediate (micro- to millisecond) motions for this paradigmatic RNA system. We measure (13)C T(1), T(1rho) and heteronuclear nuclear Overhauser effects (NOEs) for sugar and base nuclei, as well as the power dependence of T(1rho) at 500 MHz and 750 MHz, and analyze these results using the model-free formalism. The results provide a much clearer picture of the type of motions experienced by this RNA in the absence of the protein than was provided by the analysis of the structure based solely on NOEs and scalar couplings. They define a model where the RNA internal loop region "breathes" on a micro- to millisecond timescale with respect to the double-helical regions. Superimposed on this slower motion, the residues at the very tip of the loop undergo faster (nano- to picosecond) motions. We hypothesize that these motions allow the RNA to sample multiple conformations so that the protein can select a structure within the ensemble that optimizes intermolecular contacts.     

Lysine side-chain dynamics derived from 13C-multiplet NMR relaxation studies on di- and tripeptides

Summary ¹³C NMR relaxation data have been used to determine dipolar auto- and cross-correlation times for the di- and tripeptides GK, KG and GKG, primarily to analyze lysine side-chain motional dynamics. In general, correlation times are largest for backbone positions and decrease on going through the lysine side chain, consistent with the idea of increased mobility at C and C methylenes. Correlation times, however, vary with the peptide ionization state. In the zwitterionic state of GK, for example, both auto-and cross-correlation times are at their maximum values, indicating reduced internal motions probably resulting from intramolecular electrostatic interactions. Modifying the charge state increases motional fluctuations. Activation energies determined from the temperature dependence of CH rotational autocorrelation times at neutral pH are approximately equal for glycine and lysine C and lysine C and C positions (4.1±0.2 to 4.5±0.2 kcal/mol) and tend to decrease slightly for lysine C and C (3.8±0.2 to 4.3±0.2 kcal/mol). The sign of lysine side-chain cross-correlations could not be explained by using any available rotational model, including one parameterized for multiple internally restricted rotations and anisotropic overall tumbling. Molecular and stochastic dynamics calculations were performed to obtain insight into correlated internal rotations and coupled overall tumbling and internal motions. Relatively strong correlations were found for i,i+1 backbone and lysine side-chain internal bond rotations. Stochastic dynamics calculations were more successful at explaining experimentally observed correlation times. In the fully charged state, a preferred conformation was detected with an all-trans lysine side chain.Abbreviations rf radio frequency - GK dipeptide glycine-lysine - KG dipeptide lysine-glycine - GKG tripeptide glycine-lysine-glycine     

Refinement of protein structure against non-redundant carbonyl <Superscript>13</Superscript>C NMR relaxation

Carbonyl ¹³C′ relaxation is dominated by the contribution from the ¹³C′ chemical shift anisotropy (CSA). The relaxation rates provide useful and non-redundant structural information in addition to dynamic parameters. It is straightforward to acquire, and offers complimentary structural information to the ¹⁵N relaxation data. Furthermore, the non-axial nature of the ¹³C′ CSA tensor results in a T₁/T₂ value that depends on an additional angular variable even when the diffusion tensor of the protein molecule is axially symmetric. This dependence on an extra degree of freedom provides new geometrical information that is not available from the NH dipolar relaxation. A protocol that incorporates such structural restraints into NMR structure calculation was developed within the program Xplor-NIH. Its application was illustrated with the yeast Fis1 NMR structure. Refinement against the ¹³C′ T₁/T₂ improved the overall quality of the structure, as evaluated by cross-validation against the residual dipolar coupling as well as the ¹⁵N relaxation data. In addition, possible variations of the CSA tensor were addressed. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Optimization of 13C direct detection NMR methods

(13)C-detected experiments are still limited by their inherently lower sensitivity, as compared to the equivalent (1)H-detected experiments. Improving the sensitivity of (13)C detection methods remains a significant area of NMR research that may provide better means for studying large macromolecular systems by NMR. In this communication, we show that (13)C-detected experiments are less sensitive to the salt concentration of the sample solution than (1)H-detected experiments. In addition, acquisition can be started with anti-phase coherence, resulting in higher sensitivity due to the elimination of the final INEPT transfer step.     

Anisotropic rotational diffusion of perdeuterated HIV protease from 15N NMR relaxation measurements at two magnetic fields

Summary ¹⁵N NMR relaxation times in perdeuterated HIV-1 protease, complexed with the sub-nanomolar inhibitor DMP323, have been measured at 600 and 360 MHz ¹H frequency. The relative magnitudes of the principal components of the inertia tensor, calculated from the X-ray coordinates of the protein-drug complex, are 1.0:0.85:0.44. The relation between the T₁/T₂ ratios observed for the individual backbone amides and their N-H orientation within the 3D structure of the protease dimer yields a rotational diffusion tensor oriented nearly collinear to the inertia tensor. The relative magnitudes of its principal components (1.00:1.11:1.42) are also in good agreement with hydrodynamic modeling results. The orientation and magnitude of the diffusion tensors derived from relaxation data obtained at 360 and 600 MHz are nearly identical. The anisotropic nature of the rotational diffusion has little influence on the order parameters derived from the ¹⁵N T₁ and T₂ relaxation times; however, if anisotropy is ignored, this can result in erroneous identification of either exchange broadening or internal motions on a nanosecond time scale. The average ratio of the T₁ values measured at 360 and 600 MHz is 0.50±0.015, which is slightly larger than the value of 0.466 expected for an isotropic rigid rotor with _c = 10.7 ns. The average ratio of the T₂ values measured at 360 and 600 MHz is 1.14±0.04, which is also slightly larger than the expected ratio of 1.11. This magnetic field dependence of the T₁ and T₂ relaxation times suggests that the spectral density contribution from fast internal motions is not negligible, and that the chemical shift anisotropy of peptide backbone amides, on average, is larger than the 160 ppm value commonly used in ¹⁵N relaxation studies of proteins.     

Analysis of peptide rotational diffusion by homonuclear NMR

The analysis of the rotational diffusion of a molecule using homonuclear NMR is investigated. The homonuclear longitudinal and transverse cross-relaxation rates, which can be quantitatively measured using off-Resonance Rotating frame nuclear Overhauser Effect Spectroscopy (ROESY), are used to build a distribution, which exhibits a solid-state-like pattern characteristic of the diffusion tensor. The distributions of the antimicrobial peptide ranalexin in water and in 30% of trifluoracetic acid (TFE) are compared, and the peptide rotational diffusion is shown to be more isotropic in water than in 30% TFE. This difference is further supported by the analysis of NMR ranalexin conformers in 30% TFE, and by the analysis of a molecular dynamics simulation of peptide in water.     

Improved labeling strategy for 13C relaxation measurements of methyl groups in proteins

Selective incorporation of ¹³C into the methyl groupsof protein side chains is described as a means for simplifying themeasurement and interpretation of ¹³C relaxation parameters.High incorporation (>90%) is accomplished by using pyruvate(3-¹³C, 99%) as the sole carbon source in the growthmedia for protein overexpression in E. coli. This improved labeling schemeincreases the sensitivity of the relaxation experiments by approximatelyfivefold when compared to randomly fractionally ¹³C-labeledprotein, allowing high-quality measurements on relatively dilute (<1 mM)protein samples at a relatively low cost.     

Kavalactones from Piper methysticum, and their 13C NMR spectroscopic analyses

The kavalactone, 11-methoxy-5,6-dihydroyangonin, and eight previously reported analogs along with four other aromatic compounds were isolated from the root extracts of Piper methysticum (Kava Kava). Their structural elucidations were made by 1H and 13C NMR spectroscopic assignments using COSY, HMBC and HMQC experiments.     

13C NMR chemical shifts can predict disulfide bond formation

The presence of disulfide bonds can be detected unambiguously only by X-ray crystallography, and otherwise must be inferred by chemical methods. In this study we demonstrate that ¹³C NMR chemical shifts are diagnostic of disulfide bond formation, and can discriminate between cysteine in the reduced (free) and oxidized (disulfide bonded) state. A database of cysteine ¹³C C and C chemical shifts was constructed from the BMRB and Sheffield databases, and published journals. Statistical analysis indicated that the C shift is extremely sensitive to the redox state, and can predict the disulfide-bonded state. Further, chemical shifts in both states occupy distinct clusters as a function of secondary structure in the C/C chemical shift map. On the basis of these results, we provide simple ground rules for predicting the redox state of cysteines; these rules could be used effectively in NMR structure determination, predicting new folds, and in protein folding studies.     

Synthesis of methyl 4'-O-methyl-beta-D-cellobioside-13C12 from D-glucose-13C6. Part 2: solid-state NMR studies

Double Quantum (DQ) NMR, which utilizes the magnetic dipole interaction between the (13)C atoms, was used for the complete assignment of the (13)C NMR resonances to the corresponding carbon ring positions for the monoclinic and triclinic allomorphs of methyl 4'-O-methyl-beta-D-cellobioside-(13)C(12)(1-(13)C(12)), a cellodextrin model compound of cellulose (13)C-perlabeled at the cellobiose core. The through-space interactions were used to identify the direct chemical bonds between adjacent carbon atoms in the rings. More importantly, the (13)C NMR signals of the carbon sites C1' and C4 involved in the glycosidic bond were identified. This allowed for the complete (13)C chemical shift assignment, that when combined with the X-ray crystallography data provides a complete characterization.     

A13C NMR study of the hinge region of a mouse monoclonal antibody

Summary A¹³C NMR study is reported of the hinge region of an intact mouse monoclonal antibody with a molecular weight of 150 K. Cys, Ile, and Pro analogs of the antibody labeled with¹³C at the carbonyl carbon were prepared by growing hybridoma cells in the serum-free media. Resonance assignments have been performed as described previously [Kato, K., Matsunaga, C., Igarashi, T., Kim, H., Odaka, A., Shimada, I. and Arata, Y. (1991)Biochemistry,30, 270–278]. The spectral data obtained show that¹³C NMR can give detailed information about the structure of the hinge region of the intact antibody molecule. Prospects for the future role of¹³C NMR in the structural analyses of larger proteins are briefly discussed.Dedicated to the memory of Professor V.F. Bystrov     

1H, 13C and 15N random coil NMR chemical shifts of the common amino acids. I. Investigations of nearest-neighbor effects

Summary In this study we report on the ¹H, ¹³C and ¹⁵N NMR chemical shifts for the random coil state and nearest-neighbor sequence effects measured from the protected linear hexapeptide Gly-Gly-X-Y-Gly-Gly (where X and Y are any of the 20 common amino acids). We present data for a set of 40 peptides (of the possible 400) including Gly-Gly-X-Ala-Gly-Gly and Gly-Gly-X-Pro-Gly-Gly, measured under identical aqueous conditions. Because all spectra were collected under identical experimental conditions, the data from the Gly-Gly-X-Ala-Gly-Gly series provide a complete and internally consistent set of ¹H, ¹³C and ¹⁵N random coil chemical shifts for all 20 common amino acids. In addition, studies were also conducted into nearest-neighbor effects on the random coil shift arising from a variety of X and Y positional substitutions. Comparisons between the chemical shift measurements obtained from Gly-Gly-X-Ala-Gly-Gly and Gly-Gly-X-Pro-Gly-Gly reveal significant systematic shift differences arising from the presence of proline in the peptide sequence. Similarly, measurements of the chemical shift changes occurring for both alanine and proline (i.e., the residues in the Y position) are found to depend strougly on the type of amino acid substituted into the X position. These data lend support to the hypothesis that sequence effects play a significant role in determining peptide and protein chemical shifts.