The 13C Chemical-Shift Index: A simple method for the identification of protein secondary structure using 13C chemical-shift data

Summary A simple technique for identifying protein secondary structures through the analysis of backbone ¹³C chemical shifts is described. It is based on the Chemical-Shift Index [Wishart et al. (1992) Biochemistry, 31, 1647–1651] which was originally developed for the analysis of ¹H chemical shifts. By extending the Chemical-Shift Index to include ¹³C, ¹³C and carbonyl ¹³C chemical shifts, it is now possible to use four independent chemical-shift measurements to identify and locate protein secondary structures. It is shown that by combining both ¹H and ¹³C chemical-shift indices to produce a consensus estimate of secondary structure, it is possible to achieve a predictive accuracy in excess of 92%. This suggests that the secondary structure of peptides and proteins can be accurately obtained from ¹H and ¹³C chemical shifts, without recourse to NOE measurements.Supplementary material is available in the form of a 10-page table (Table S1) describing the exact location of secondary structures in all 20 proteins as determined using the methods described in this paper. Requests for Table S1 should be directed to the authors.     

Determination of the three-dimensional structure of oligosaccharides in the solid state from experimental 13C NMR data and ab initio chemical shift surfaces

A novel method for the determination of the three-dimensional (3D) structure of oligosaccharides in the solid state using experimental 13C NMR data is presented. The approach employs this information, combined with 13C chemical shift surfaces (CSSs) for the glycosidic bond carbons in the generation of NMR pseudopotential energy functions suitable for use as constraints in molecular modeling simulations. Application of the method to trehalose, cellobiose, and cellotetraose produces 3D models that agree remarkably well with the reported X-ray structures, with phi and psi dihedral angles that are within 10 degrees from the ones observed in the crystals. The usefulness of the approach is further demonstrated in the determination of the 3D structure of the cellohexaose, an hexasaccharide for which no X-ray data has been reported, as well as in the generation of accurate structural models for cellulose II and amylose V6.     

Characterization of threonine side chain dynamics in an antifreeze protein using natural abundance 13C NMR spectroscopy

The dynamics of threonine side chains of the Tenebrio molitor antifreeze protein (TmAFP) were investigated using natural abundance (13)C NMR. In TmAFP, the array of threonine residues on one face of the protein is responsible for conferring its ability to bind crystalline ice and inhibit its growth. Heteronuclear longitudinal and transverse relaxation rates and the [(1)H]-(13)C NOE were determined in this study. The C alpha H relaxation measurements were compared to the previously measured (15)N backbone parameters and these are found to be in agreement. For the analysis of the threonine side chain motions, the model of restricted rotational diffusion about the chi(1) dihedral angle was employed [London and Avitabile (1978) J. Am. Chem. Soc., 100, 7159-7165]. We demonstrate that the motion experienced by the ice binding threonine side chains is highly restricted, with an approximate upper limit of less than +/-25 degrees.     

Assigning stereodiversity of the 27-Me group of furostane-type steroidal saponins via NMR chemical shifts

Applicability of (13)C and (1)H NMR chemical shifts for the assignment of the 25R/25S configuration of the 27-methyl group in the case of furostane-type steroidal saponins has been investigated. A comparative study of (13)C NMR data suggest that chemical shift values for C-20, C-21, C-22, C-23, C-24, C-25, C-26 and C-27 resonances were not much influenced by R/S configuration of the 27-Me group, thus reflecting limited application of (13)C NMR chemical shifts for such stereochemical determinations. In contrast, (1)H NMR chemical shifts (delta(a), delta(b)) for geminal protons of glycosyloxy methylene (H(2)-26) exhibit pronounced dependence and the difference (Delta(ab)=delta(a)-delta(b)) among their chemical shifts [Delta(ab)= or <0.48 for 25R; Delta(ab)= or >0.57 for 25S] seems to be of general applicability for ascertaining 25R/25S orientation of the 27-methyl group of furostane-type steroidal saponins.     

23Na NMR study of Fibrobacter succinogenes S85: comparison of three chemical shift reagents and calculation of sodium concentration using ionophores

In order to measure intracellular sodium concentrations in resting cells of Fibrobacter succinogenes S85 by (23)Na NMR spectrometry, two methodological aspects were studied. First, three different shift reagents (Dy(PPP(i))(7-)(2), Tm(DOTP)(5-), and Dy(TTHA)(3-)) were tested for their ability to separate internal and external (23)Na NMR resonances. Their toxicity toward F. succinogenes cells was evaluated by in vivo(13)C NMR experiments. Tm(DOTP)(5-) was found to be the most efficient shift reagent while being nontoxic. Second, a new methodology was developed to calculate intracellular sodium concentration in F. succinogenes by using ionophores. This approach avoided the problem of intracellular volume measurement and that of sodium visibility determination.     

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.     

Dynamics and metal ion binding in the U6 RNA intramolecular stem-loop as analyzed by NMR

The U6 RNA intramolecular stem-loop (ISL) is a conserved component of the spliceosome, and contains an essential metal ion binding site centered between a protonated adenine, A79, and U80. Correlated with protonation of A79, U80 undergoes a base-flipping conformational change accompanied by significant helical movement. We have investigated the dynamics of the U6 ISL by analyzing the power dependence of 13C NMR relaxation rates in the rotating frame. The data provide evidence that the conformational transition is centered around an exchange lifetime of 84 micros. The U80 nucleotide displays low internal mobility on the picosecond time-scale at pH 7.0 but high internal mobility at pH 6.0, in agreement with the global transition resulting in the base of U80 adopting a looped-out conformation with increased dynamic disorder. A kinetic analysis suggests that the conformational change, rather than adenine protonation, is the rate-limiting step in the pathway of the conformational transition. Two nucleotides, U70 and U80, were found from chemical shift perturbation mapping to interact with the magnesium ion, with apparent K(d) values in the micromolar to millimolar range. These nucleotides also displayed metal ion-induced elevation of R1 rates, which can be explained by a model that assumes dynamic metal ion coordination concomitant with an induced higher shielding anisotropy for the base 13C nuclei. Addition of Mg2+ shifts the conformational equilibrium toward the high-pH (base-stacked) structure, accompanied by a significant drop in the apparent pK(a) of A79.     

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     

(13)C-labeled indolequinone-DTPA-Gd conjugate for NMR probing cytochrome:P450 reductase-mediated one-electron reduction

We designed and synthesized a new class of ¹³C-labeled NMR probe, ¹³C-IQ-Gd, to monitor one-electron reductions by cytochrome:P450 (CYP450) reductase under hypoxic conditions. ¹³C-IQ-Gd consisted of a Gd³⁺-diethylene triamine pentaacetic acid (DTPA) complex unit and an indolequinone (¹³C-IQ) unit bearing a ¹³C-labeled methoxy group. The ¹³C NMR signal of ¹³C-IQ-Gd was suppressed because of the intramolecular paramagnetic effect of Gd³⁺, whereas enzymatic reduction mediated by CYP450 reductase under hypoxic conditions yielded an intensed ¹³C NMR signal due to enzymatic activation of the IQ unit followed by release of the DTPA-Gd unit from ¹³C-IQ-Gd. This ¹³C NMR spectral change allowed the monitoring of CYP450 reducatase-mediated one-electron reduction.     

Editing and diagonal peak suppression in three-dimensional HCCH protein NMR correlation experiments

A novel three-dimensional (3D) HCCH NMR experiment is introduced. It involves ¹³C-¹³C COSY or TOCSY coherence transfer plus two independent editing steps according to the number of protons attached to the individual carbons before and after the ¹³C-¹³C homonuclear mixing. This double editing leads to simplification of HCCH protein side chain spectra that otherwise are prone to spectral overlap. Another interesting feature is amino acid selectivity, i.e. that the presence of certain correlations in a doubly edited HCCH subspectrum gives a clue as to assignment to a particular subgroup of amino acids or segments thereof. Finally, the selection of two different multiplicities in the two editing steps leads to diagonal peak suppression in the ¹H-¹H (3D spectrum recorded with two ¹H and one ¹³C dimension) or the ¹³C-¹³C (3D spectrum recorded with one ¹H and two ¹³C dimensions) two-dimensional projection. The new experiment is demonstrated using a ¹³C,¹⁵N-labeled protein sample, chymotrypsin inhibitor 2, at 500 MHz.     

A suite of Mathematica notebooks for the analysis of protein main chain 15N NMR relaxation data

A suite of Mathematica notebooks has been designed to ease the analysis of protein main chain ¹⁵N NMR relaxation data collected at a single magnetic field strength. Individual notebooks were developed to perform the following tasks: nonlinear fitting of ¹⁵N-T ₁ and -T ₂ relaxation decays to a two parameter exponential decay, calculation of the principal components of the inertia tensor from protein structural coordinates, nonlinear optimization of the principal components and orientation of the axially symmetric rotational diffusion tensor, model-free analysis of ¹⁵N-T ₁, -T ₂, and {¹H}–¹⁵N NOE data, and reduced spectral density analysis of the relaxation data. The principle features of the notebooks include use of a minimal number of input files, integrated notebook data management, ease of use, cross-platform compatibility, automatic visualization of results and generation of high-quality graphics, and output of analyses in text format.L. Spyracopoulos is an AHFMR Medical Research Senior Scholar     

Development of analytical methods for NMR spectra and application to a 13C toxicology study

Metabolomics offers the potential to assess the effects of toxicants on metabolite levels. To fully realize this potential, a robust analytical workflow for identifying and quantifying treatment-elicited changes in metabolite levels by nuclear magnetic resonance (NMR) spectrometry has been developed that isolates and aligns spectral regions across treatment and vehicle groups to facilitate analytical comparisons. The method excludes noise regions from the resulting reduced spectra, significantly reducing data size. Principal components analysis (PCA) identifies data clusters associated with experimental parameters. Cluster-centroid scores, derived from the principal components that separate treatment from vehicle samples, are used to reconstruct the mean spectral estimates for each treatment and vehicle group. Peak amplitudes are determined by scanning the reconstructed mean spectral estimates. Confidence levels from Mann–Whitney order statistics and amplitude change ratios are used to identify treatment-related changes in peak amplitudes. As a demonstration of the method, analysis of ¹³C NMR data from hepatic lipid extracts of immature, ovariectomized C57BL/6 mice treated with 30 μg/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or sesame oil vehicle, sacrificed at 72, 120, or 168 h, identified 152 salient peaks. PCA clustering showed a prominent treatment effect at all three time points studied, and very little difference between time points of treated animals. Phenotypic differences between two animal cohorts were also observed. Based on spectral peak identification, hepatic lipid extracts from treated animals exhibited redistribution of unsaturated fatty acids, cholesterols, and triacylglycerols. This method identified significant changes in peaks without the loss of information associated with spectral binning, increasing the likelihood of identifying treatment-elicited metabolite changes.     

ORB, a homology-based program for the prediction of protein NMR chemical shifts

A computer program (ORB) has been developed to predict ¹H,¹³C and ¹⁵N NMR chemical shifts of previouslyunassigned proteins. The program makes use of the information contained in achemical shift database of previously assigned proteins supplemented by astatistically derived averaged chemical shift database in which the shifts arecategorized according to their residue, atom and secondary structure type[Wishart et al. (1991) J. Mol. Biol., 222, 311–333]. The predictionprocess starts with a multiple alignment of all previously assigned proteinswith the unassigned query protein. ORB uses the sequence and secondarystructure alignment program XALIGN for this task [Wishart et al. (1994)CABIOS, 10, 121–132; 687–688]. The prediction algorithm in ORB isbased on a scoring of the known shifts for each sequence. The scores dependon global sequence similarity, local sequence similarity, structuralsimilarity and residue similarity and determine how much weight one particularshift is given in the prediction process. In situations where no applicablepreviously assigned chemical shifts are available, the shifts derived from theaveraged database are used. In addition to supplying the user with predictedchemical shifts, ORB calculates a confidence value for every prediction. Theseconfidence values enable the user to judge which predictions are the mostaccurate and they are particularly useful when ORB is incorporated into acomplete autoassignment package. The usefulness of ORB was tested on threemedium-sized proteins: an interleukin-8 analog, a troponin C synthetic peptideheterodimer and cardiac troponin C. Excellent results are obtained if ORB isable to use the chemical shifts of at least one highly homologous sequence.ORB performs well as long as the sequence identity between proteins with knownchemical shifts and the new sequence is not less than 30%.     

Dynamic pictures of membrane proteins in two-dimensional crystal, lipid bilayer and detergent as revealed by site-directed solid-state 13C NMR

We have compared site-directed 13C solid-state NMR spectra of [3-13C]Ala- and/or [1-13C]Val-labeled membrane proteins, including bacteriorhodopsin (bR), pharaonis phoborhodopin (ppR), its cognate transducer (pHtrII) and Escherichia coli diacylglycerol kinase (DGK), in two-dimensional (2D) crystal, lipid bilayers, and detergent. Restricted fluctuation motions of these membrane proteins due to oligomerization of bR by specific protein-protein interactions in the 2D crystalline lattice or protein complex between ppR and pHtrII provide the most favorable environment to yield well-resolved, fully visible 13C NMR signals for [3-13C]Ala-labeled proteins. In contrast, several signals from such membrane proteins were broadened or lost owing to interference of inherent fluctuation frequencies (10(4)-10(5)Hz) with frequency of either proton decoupling or magic angle spinning, if their 13C NMR spectra were recorded as a monomer in lipid bilayers at ambient temperature. The presence of such protein dynamics is essential for the respective proteins to achieve their own biological functions. Finally, spectral broadening found for bR and DGK in detergents were discussed.     

Use of 13C chemical shift surfaces in the study of carbohydrate conformation. Application to cyclomaltooligosaccharides (cyclodextrins) in the solid state and in solution

The anomeric carbon chemical shifts of free cyclomaltohexaose, -heptaose, -octaose, -decaose, and -tetradecaose (alpha-, beta-, gamma-, epsilon-, and eta-cyclodextrin, respectively), and of alpha-cyclodextrin inclusion complexes, both in the solid state and in solution, were computed using ab initio 13C chemical shift surfaces for the D-Glcp-alpha-(1-->4)-D-Glcp linkage as a function of the glycosidic bond dihedral angles. Chemical shift calculations in the solid state used angle pairs measured from cyclodextrin X-ray structures as input. For estimations in the liquid state two different approaches were employed to account for dynamic averaging. In one, the computed solid-state anomeric carbon chemical shifts for each cyclodextrin D-Glcp monomer were simply averaged to obtain an estimate of the 13C shifts in solution. In the other, chemical shifts for the anomeric carbons were determined by averaging back-calculated 13C shift trajectories derived from a series of 5 ns molecular dynamic simulations for the oligosaccharides with explicit representation of water. Good agreement between calculated and experimental 13C shifts was found in all cases. Furthermore, our results show that the ab initio 13C chemical shift surfaces are sufficiently sensitive to reproduce the small variations observed for the anomeric 13C shifts of the different cyclodextrin D-Glcp units in the solid state with excellent accuracy. The use of chemical shift surfaces as tools in conformational studies of oligosaccharides is discussed.     

A C-detection NMR approach for large glycoproteins

NMR spectroscopy has great potential to provide us with information on structure and dynamics at atomic resolution of glycoproteins in solution. In larger glycoproteins, however, the detrimental fast ¹H transverse relaxation renders the conventional ¹H-detected NMR experiments difficult. ¹³C direct detection potentially offers a valuable alternative to ¹H detection to overcome the fast T₂ relaxation. Here, we applied ¹³C-detected NMR methods to observe the NMR signals of ¹³C-labeled glycans attached to the Fc fragment of immunoglobulin G with a molecular mass of 56 kDa. Spectral analysis revealed that a ¹³C-detected ¹³C-¹³C NOESY experiment is highly useful for spectral assignments of the glycans of large glycoproteins. This approach would be, in part, complementary to ¹³C-¹³C TOCSY and ¹H-detection experiments.     

Crystal structure and solid-state 13C NMR analysis of N-o-, N-m- and N-p-nitrophenyl-2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosylamines, and their N-acetyl derivatives

The X-ray diffraction analysis of N-o-nitrophenyl-2,3,4,6-tetra-O-acetyl-β-d-glucopyranosylamine (1), N-m-nitrophenyl-2,3,4,6-tetra-O-acetyl-β-d-glucopyranosylamines, N-p-nitrophenyl-2,3,4,6-tetra-O-acetyl-β-d-glucopyranosylamines, and their N-acetyl derivatives was performed. The sugar moieties always adopt ⁴C₁ conformations, however, due to crystal packing forces they are always slightly distorted. It was found that except N-acetyl, N-m-nitrophenyl-2,3,4,6-tetra-O-acetyl-β-d-glucopyranosylamine (5), none of the glucopyranosylamines studied in this paper form strong hydrogen bonds in the crystal lattice. Additionally, (5) crystallizes with a molecule of water, which occupies a special crystallographic position (on the twofold axis) and links two sugar molecules by hydrogen bonds. The CP MAS NMR spectra confirmed the presence of the intermolecular hydrogen bond involving the molecule of water in (5). Moreover, it was proved that in (1) an intramolecular hydrogen bond is formed between the glycosidic linkage and the nitro group.     

Time-saving methods for heteronuclear multidimensional NMR of (13C, 15N) doubly labeled proteins

Summary Heteronuclear 2D (¹³C, ¹H) and (¹⁵N, ¹H) correlation spectra of (¹³C, ¹⁵N) fully enriched proteins can be acquired simultaneously with virtually no sensitivity loss or increase in artefact levels. Three pulse sequences are described, for 2D time-shared or TS-HSQC, 2D TS-HMQC and 2D TS-HSMQC spectra, respectively. Independent spectral widths can be sampled for both heteronuclei. The sequences can be greatly improved by combining them with field-gradient methods. By applying the sequences to 3D and 4D NMR spectroscopy, considerable time savings can be obtained. The method is demonstrated for the 18 kDa HU protein.Abbreviations HMQC heteronuclear multiple-quantum coherence spectroscopy - HSQC heteronuclear single-quantum coherence spectroscopy - HSMQC heteronuclear single- and multiple-quantum coherence spectroscopy - NOESY nuclear Overhauser enhancement spectroscopy