Measuring <Superscript>13</Superscript>C<Superscript>β</Superscript> chemical shifts of invisible excited states in proteins by relaxation dispersion NMR spectroscopy

A labeling scheme is introduced that facilitates the measurement of accurate ¹³C^β chemical shifts of invisible, excited states of proteins by relaxation dispersion NMR spectroscopy. The approach makes use of protein over-expression in a strain of E. coli in which the TCA cycle enzyme succinate dehydrogenase is knocked out, leading to the production of samples with high levels of ¹³C enrichment (30–40%) at C^β side-chain carbon positions for 15 of the amino acids with little ¹³C label at positions one bond removed (≈5%). A pair of samples are produced using [1-¹³C]-glucose/NaH¹²CO₃ or [2-¹³C]-glucose as carbon sources with isolated and enriched (>30%) ¹³C^β positions for 11 and 4 residues, respectively. The efficacy of the labeling procedure is established by NMR spectroscopy. The utility of such samples for measurement of ¹³C^β chemical shifts of invisible, excited states in exchange with visible, ground conformations is confirmed by relaxation dispersion studies of a protein–ligand binding exchange reaction in which the extracted chemical shift differences from dispersion profiles compare favorably with those obtained directly from measurements on ligand free and fully bound protein samples.     

<Superscript>1</Superscript>H–<Superscript>15</Superscript>N correlation spectroscopy of nanocrystalline proteins

The limits of resolution that can be obtained in ¹H–¹⁵N 2D NMR spectroscopy of isotopically enriched nanocrystalline proteins are explored. Combinations of frequency switched Lee–Goldburg (FSLG) decoupling, fast magic angle sample spinning (MAS), and isotopic dilution via deuteration are investigated as methods for narrowing the amide ¹H resonances. Heteronuclear decoupling of ¹⁵N from the ¹H resonances is also studied. Using human ubiquitin as a model system, the best resolution is most easily obtained with uniformly ²H and ¹⁵N enriched protein where the amides have been exchanged in normal water, MAS at 20 kHz, and WALTZ-16 decoupling of the ¹⁵N nuclei. The combination of these techniques results in average ¹H lines of only 0.26 ppm full width at half maximum. Techniques for optimizing instrument stability and ¹⁵N decoupling are described for achieving the best possible performance in these experiments.     

Impact of two-bond <Superscript>15</Superscript>N–<Superscript>15</Superscript>N scalar couplings on <Superscript>15</Superscript>N transverse relaxation measurements for arginine side chains of proteins

NMR relaxation of arginine (Arg) ¹⁵N_ε nuclei is useful for studying side-chain dynamics of proteins. In this work, we studied the impact of two geminal ¹⁵N–¹⁵N scalar couplings on measurements of transverse relaxation rates (R ₂ ) for Arg side-chain ¹⁵N_ε nuclei. For 12 Arg side chains of the DNA-binding domain of the Antp protein, we measured the geminal ¹⁵N–¹⁵N couplings ( ² J _NN ) of the ¹⁵N_ε nuclei and found that the magnitudes of the ² J _NN coupling constants were virtually uniform with an average of 1.2 Hz. Our simulations, assuming ideal 180° rotations for all ¹⁵N nuclei, suggested that the two ² J _NN couplings of this magnitude could in principle cause significant modulation in signal intensities during the Carr–Purcell-Meiboom–Gill (CPMG) scheme for Arg ¹⁵N_ε R ₂ measurements. However, our experimental data show that the expected modulation via two ² J _NN couplings vanishes during the ¹⁵N CPMG scheme. This quenching of J modulation can be explained by the mechanism described in Dittmer and Bodenhausen (Chemphyschem 7:831–836, 2006). This effect allows for accurate measurements of R ₂ relaxation rates for Arg side-chain ¹⁵N_ε nuclei despite the presence of two ² J _NN couplings. Although the so-called recoupling conditions may cause overestimate of R ₂ rates for very mobile Arg side chains, such conditions can readily be avoided through appropriate experimental settings.     

Improved accuracy of <Superscript>15</Superscript>N–<Superscript>1</Superscript>H scalar and residual dipolar couplings from gradient-enhanced IPAP-HSQC experiments on protonated proteins

The presence of dipole-dipole cross-correlated relaxation as well as unresolved E.COSY effects adversely impacts the accuracy of ¹ J _NH splittings measured from gradient-enhanced IPAP-HSQC spectra. For isotropic samples, the size of the systematic errors caused by these effects depends on the values of ² J _NHα , ³ J _NHβ and ³ J _HNHα . Insertion of band-selective ¹H decoupling pulses in the IPAP-HSQC experiment eliminates these systematic errors and for the protein GB3 yields ¹ J _NH splittings that agree to within a root-mean-square difference of 0.04 Hz with values measured for perdeuterated GB3. Accuracy of the method is also highlighted by a good fit to the GB3 structure of the ¹H-¹⁵N RDCs extracted from the minute differences in ¹J_NH splitting measured at 500 and 750 MHz ¹H frequencies, resulting from magnetic susceptibility anisotropy. A nearly complete set of ² J _NHα couplings was measured in GB3 in order to evaluate whether the impact of cross-correlated relaxation is dominated by the ¹⁵N–¹H ^α or ¹⁵N–¹H ^β dipolar interaction. As expected, we find that ² J _NHα  ≤ 2 Hz, with values in the α-helix (0.86 ± 0.52 Hz) slightly larger than in β-sheet (0.66 ± 0.26 Hz). Results indicate that under isotropic conditions, N–H^N/N–H ^β cross-correlated relaxation often dominates. Unresolved E.COSY effects under isotropic conditions involve ³ J _HNHα and J _NHα , but when weakly aligned any aliphatic proton proximate to both N and H^N can contribute. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C,and <Superscript>15</Superscript>N resonance assignments for the pro-inflammatory cytokine interleukin-36α

Interleukin-36α (IL-36α) is a recently characterised member of the interleukin-1 superfamily. It is involved in the pathogenesis of inflammatory arthritis in one third of psoriasis patients. By binding of IL-36α to its receptor IL-36R via the NF-κB pathway other cytokines involved in inflammatory and apoptotic cascade are activated. The efficacy of complex formation is controlled by N-terminal processing. To obtain a more detailed view on the structure function relationship we performed a heteronuclear multidimensional NMR investigation and here report the ¹H, ¹³C, and ¹⁵N resonance assignments for the backbone and side chain nuclei of the pro-inflammatory cytokine interleukin-36α.     

Carbonyl carbon label selective (CCLS) <Superscript>1</Superscript>H–<Superscript>15</Superscript>N HSQC experiment for improved detection of backbone <Superscript>13</Superscript>C–<Superscript>15</Superscript>N cross peaks in larger proteins

We present a highly sensitive pulse sequence, carbonyl carbon label selective ¹H–¹⁵N HSQC (CCLS-HSQC) for the detection of signals from ¹H–¹⁵N units involved in ¹³C′–¹⁵N linkages. The CCLS-HSQC pulse sequence utilizes a modified ¹⁵N CT evolution period equal to 1/( ) (∼33 ms) to select for ¹³C′–¹⁵N pairs. By collecting CCLS-HSQC and HNCO data for two proteins (8 kDa ubiquitin and 20 kDa HscB) at various temperatures (5–40°C) in order to vary correlation times, we demonstrate the superiority of the CCLS-HSQC pulse sequence for proteins with long correlation times (i.e. higher molecular weight). We then show that the CCLS-HSQC experiment yields assignments in the case of a 41 kDa protein incorporating pairs of ¹⁵N- and ¹³C′-labeled amino acids, where a TROSY 2D-HN(CO) had failed. Although the approach requires that the ¹H–¹⁵N HSQC cross peaks be observable, it does not require deuteration of the protein. The method is suitable for larger proteins and is less affected by conformational exchange than HNCO experiments, which require a longer period of transverse ¹⁵N magnetization. The method also is tolerant to the partial loss of signal from isotopic dilution (scrambling). This approach will be applicable to families of proteins that have been resistant to NMR structural and dynamic analysis, such as large enzymes, and partially folded or unfolded proteins.     

Sequential nearest-neighbor effects on computed <Superscript>13</Superscript>C<Superscript>α</Superscript> chemical shifts

To evaluate sequential nearest-neighbor effects on quantum-chemical calculations of ¹³C^α chemical shifts, we selected the structure of the nucleic acid binding (NAB) protein from the SARS coronavirus determined by NMR in solution (PDB id 2K87). NAB is a 116-residue α/β protein, which contains 9 prolines and has 50% of its residues located in loops and turns. Overall, the results presented here show that sizeable nearest-neighbor effects are seen only for residues preceding proline, where Pro introduces an overestimation, on average, of 1.73 ppm in the computed ¹³C^α chemical shifts. A new ensemble of 20 conformers representing the NMR structure of the NAB, which was calculated with an input containing backbone torsion angle constraints derived from the theoretical ¹³C^α chemical shifts as supplementary data to the NOE distance constraints, exhibits very similar topology and comparable agreement with the NOE constraints as the published NMR structure. However, the two structures differ in the patterns of differences between observed and computed ¹³C^α chemical shifts, Δ _ca,i , for the individual residues along the sequence. This indicates that the Δ _ca,i -values for the NAB protein are primarily a consequence of the limited sampling by the bundles of 20 conformers used, as in common practice, to represent the two NMR structures, rather than of local flaws in the structures.     

Rapid measurement of <Superscript>3</Superscript>J(H<Superscript>N</Superscript>–H<Superscript>α</Superscript>) and <Superscript>3</Superscript>J(N–H<Superscript>β</Superscript>) coupling constants in polypeptides

We present two NMR experiments, (3,2)D HNHA and (3,2)D HNHB, for rapid and accurate measurement of 3J(H N-H alpha) and 3J(N-H beta) coupling constants in polypeptides based on the principle of G-matrix Fourier transform NMR spectroscopy and quantitative J-correlation. These experiments, which facilitate fast acquisition of three-dimensional data with high spectral/digital resolution and chemical shift dispersion, will provide renewed opportunities to utilize them for sequence specific resonance assignments, estimation/characterization of secondary structure with/without prior knowledge of resonance assignments, stereospecific assignment of prochiral groups and 3D structure determination, refinement and validation. Taken together, these experiments have a wide range of applications from structural genomics projects to studying structure and folding in polypeptides.     

Predicting <Superscript>13</Superscript>C<Superscript>α</Superscript> chemical shifts for validation of protein structures

The ¹³C^α chemical shifts for 16,299 residues from 213 conformations of four proteins (experimentally determined by X-ray crystallography and Nuclear Magnetic Resonance methods) were computed by using a combination of approaches that includes, but is not limited to, the use of density functional theory. Initially, a validation test of this methodology was carried out by a detailed examination of the correlation between computed and observed ¹³C^α chemical shifts of 10,564 (of the 16,299) residues from 139 conformations of the human protein ubiquitin. The results of this validation test on ubiquitin show agreement with conclusions derived from computation of the chemical shifts at the ab initio Hartree–Fock level. Further, application of this methodology to 5,735 residues from 74 conformations of the three remaining proteins that differ in their number of amino acid residues, sequence and three-dimensional structure, together with a new scoring function, namely the conformationally averaged root-mean-square-deviation, enables us to: (a) offer a criterion for an accurate assessment of the quality of NMR-derived protein conformations; (b) examine whether X-ray or NMR-solved structures are better representations of the observed ¹³C^α chemical shifts in solution; (c) provide evidence indicating that the proposed methodology is more accurate than automated predictors for validation of protein structures; (d) shed light as to whether the agreement between computed and observed ¹³C^α chemical shifts is influenced by the identity of an amino acid residue or its location in the sequence; and (e) provide evidence confirming the presence of dynamics for proteins in solution, and hence showing that an ensemble of conformations is a better representation of the structure in solution than any single conformation. Electronic Supplementary Material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Facile measurement of <Superscript>1</Superscript>H–<Superscript>15</Superscript>N residual dipolar couplings in larger perdeuterated proteins

We present a simple method, ARTSY, for extracting ¹J_NH couplings and ¹H–¹⁵N RDCs from an interleaved set of two-dimensional ¹H–¹⁵N TROSY-HSQC spectra, based on the principle of quantitative J correlation. The primary advantage of the ARTSY method over other methods is the ability to measure couplings without scaling peak positions or altering the narrow line widths characteristic of TROSY spectra. Accuracy of the method is demonstrated for the model system GB3. Application to the catalytic core domain of HIV integrase, a 36 kDa homodimer with unfavorable spectral characteristics, demonstrates its practical utility. Precision of the RDC measurement is limited by the signal-to-noise ratio, S/N, achievable in the 2D TROSY-HSQC spectrum, and is approximately given by 30/(S/N) Hz.     

Comprehensive determination of <Superscript>3</Superscript>J<Subscript>HNHα</Subscript> for unfolded proteins using <Superscript>13</Superscript>C′-resolved spin-echo difference spectroscopy

An experiment is presented to determine ³J_HNHα coupling constants, with significant advantages for applications to unfolded proteins. The determination of coupling constants for the peptide chain using 1D ¹H, or 2D and 3D ¹H-¹⁵N correlation spectroscopy is often hampered by extensive resonance overlap when dealing with flexible, disordered proteins. In the experiment detailed here, the overlap problem is largely circumvented by recording ¹H-¹³C′ correlation spectra, which demonstrate superior resolution for unfolded proteins. J-coupling constants are extracted from the peak intensities in a pair of 2D spin-echo difference experiments, affording rapid acquisition of the coupling data. In an application to the cytoplasmic domain of human neuroligin-3 (hNlg3cyt) data were obtained for 78 residues, compared to 54 coupling constants obtained from a 3D HNHA experiment. The coupling constants suggest that hNlg3cyt is intrinsically disordered, with little propensity for structure.     

Detection of C′,C<Superscript>α</Superscript> correlations in proteins using a new time- and sensitivity-optimal experiment

Sensitivity- and time-optimal experiment, called COCAINE (CO-CA In- and aNtiphase spectra with sensitivity Enhancement), is proposed to correlate chemical shifts of ¹³C and ¹³C spins in proteins. A comparison of the sensitivity and duration of the experiment with the corresponding theoretical unitary bounds shows that the COCAINE experiment achieves maximum possible transfer efficiency in the shortest possible time, and in this sense the sequence is optimal. Compared to the standard HSQC, the COCAINE experiment delivers a 2.7-fold gain in sensitivity. This newly proposed experiment can be used for assignment of backbone resonances in large deuterated proteins effectively bridging ¹³C and ¹³C resonances in adjacent amino acids. Due to the spin-state selection employed, the COCAINE experiment can also be used for efficient measurements of one-bond couplings (e.g. scalar and residual dipolar couplings) in any two-spin system (e.g. the N/H in the backbone of protein).     

Variability and directionality of temporal changes in δ<Superscript>13</Superscript>C and δ<Superscript>15</Superscript>N of aquatic invertebrate primary consumers

Seasonal oscillations in the carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope signatures of aquatic algae can cause seasonal enrichment–depletion cycles in the isotopic composition of planktonic invertebrates (e.g., copepods). Yet, there is growing evidence that seasonal enrichment–depletion cycles also occur in the isotope signatures of larger invertebrate consumers, taxa used to define reference points in isotope-based trophic models (e.g., trophic baselines). To evaluate the general assumption of temporal stability in non-zooplankton aquatic invertebrates, δ¹³C and δ¹⁵N time series data from the literature were analyzed for seasonality and the influence of biotic (feeding group) and abiotic (trophic state, climate regime) factors on isotope temporal patterns. The amplitude of δ¹³C and δ¹⁵N enrichment–depletion cycles was negatively related to body size, although all size-classes of invertebrates displayed a winter-to-summer enrichment in δ¹³C and depletion in δ¹⁵N. Among feeding groups, periphytic grazers were more variable and displayed larger temporal changes in δ¹³C than detritivores. For nitrogen, temporal variability and magnitude of directional change of δ¹⁵N was most strongly related to ecosystem trophic state (eutrophic > mesotrophic, oligotrophic). This study provides evidence of seasonality in the isotopic composition of aquatic invertebrates across very broad geographical and ecological gradients as well as identifying factors that are likely to modulate the strength and variability of seasonality. These results emphasize the need for researchers to recognize the likelihood of temporal changes in non-zooplankton aquatic invertebrate consumers at time scales relevant to seasonal studies and, if present, to account for temporal dynamics in isotope trophic models.     

H<Superscript>N</Superscript>, N,C<Superscript>α</Superscript> and C<Superscript>β</Superscript> assignments of the two periplasmic domains of <Emphasis Type="Italic">Neisseria meningitidis</Emphasis> DsbD

DsbD is a disulfide bond reductase present in the inner membrane of many Gamma-Proteobacteria. In the human pathogen Neisseria meningitidis, DsbD is required for viability and represents a potential target for the development of antibiotics. Here we report the chemical shift assignments (H^N, N, C^α and C^β) for the reduced and oxidized forms of the two periplasmic domains of N. meningitidis DsbD, n-NmDsbD and c-NmDsbD. The backbone amide resonances in all four forms were completely assigned, and the secondary structures for the core regions of the proteins were calculated using ¹³C^αβ shifts. The reduced and oxidized forms of each domain have similar secondary shifts suggesting they retain the same fold. We anticipate that these data will provide an important basis for studying the interaction between n-NmDsbD and c-NmDsbD, which is required for electron transfer across the bacterial cytoplasmic membrane.     

Accurate measurement of <Superscript>15</Superscript>N–<Superscript>13</Superscript>C residual dipolar couplings in nucleic acids

New 3D HCN quantitative J (QJ) pulse schemes are presented for the precise and accurate measurement of one-bond ¹⁵N_1/9–¹³C₁, ¹⁵N_1/9–¹³C_6/8, and ¹⁵N_1/9–¹³C_2/4 residual dipolar couplings (RDCs) in weakly aligned nucleic acids. The methods employ ¹H–¹³C multiple quantum (MQ) coherence or TROSY-type pulse sequences for optimal resolution and sensitivity. RDCs are obtained from the intensity ratio of H₁–C₁–N_1/9 (MQ-HCN-QJ) or H_6/8–C_6/8–N_1/9 (TROSY-HCN-QJ) correlations in two interleaved 3D NMR spectra, with dephasing intervals of zero (reference spectrum) and 1/(2J_NC) (attenuated spectrum). The different types of ¹⁵N–¹³C couplings can be obtained by using either the 3D MQ-HCN-QJ or TROSY-HCN-QJ pulse scheme, with the appropriate setting of the duration of the constant-time ¹⁵N evolution period and the offset of two frequency-selective ¹³C pulses. The methods are demonstrated for a uniformly ¹³C, ¹⁵N-enriched 24-nucleotide stem-loop RNA sequence, helix-35, aligned in the magnetic field using phage Pf1. For measurements of RDCs systematic errors are found to be negligible, and experiments performed on a 1.5 mM helix-35 sample result in an estimated precision of ca. 0.07 Hz for ¹D_NC, indicating the utility of the measured RDCs in structure validation and refinement. Indeed, for a complete set of ¹⁵N_1/9–¹³C₁, ¹⁵N_1/9–¹³C_6/8, and ¹⁵N_1/9–¹³C_2/4 dipolar couplings obtained for the stem nucleotides, the measured RDCs are in excellent agreement with those predicted for an NMR structure of helix-35, refined using independently measured observables, including ¹³C–¹H, ¹³C–¹³C and ¹H–¹H dipolar couplings.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s10858-005-0646-2.     

Simultaneous α/β spin-state selection for <Superscript>13</Superscript>C and <Superscript>15</Superscript>N from a time-shared HSQC-IPAP experiment

Two novel HSQC-IPAP approaches are proposed to achieve α/β spin-state editing simultaneously for ¹³C and ¹⁵N in a single NMR experiment. The pulse schemes are based on a time-shared (TS) 2D ¹H,¹³C/¹H,¹⁵N-HSQC correlation experiment that combines concatenated echo elements for simultaneous J(CH) and J(NH) coupling constants evolution, TS evolution of ¹³C and ¹⁵N chemical shifts in the indirect dimension and heteronuclear α/β-spin-state selection by means of the IPAP principle. Heteronuclear α/β-editing for all CH _n (n = 1–3) and NH _n (1–2) multiplicities can be achieved in the detected F2 dimension of a single TS-HSQC-F2-IPAP experiment. On the other hand, an alternative TS-HSQC-F1-IPAP experiment is also proposed to achieve α/β-editing in the indirect F1 dimension. Experimental and simulated data is provided to evaluate these principles in terms of sensitivity and performance simultaneously on backbone and side-chain CH, CH₂, CH₃, NH, and NH₂ spin systems in uniformly ¹³C/¹⁵N-labeled proteins and in small natural-abundance peptides.     

A <Superscript>13</Superscript>C-detected <Superscript>15</Superscript>N double-quantum NMR experiment to probe arginine side-chain guanidinium <Superscript>15</Superscript>N<Superscript>η</Superscript> chemical shifts

Arginine side-chains are often key for enzyme catalysis, protein–ligand and protein–protein interactions. The importance of arginine stems from the ability of the terminal guanidinium group to form many key interactions, such as hydrogen bonds and salt bridges, as well as its perpetual positive charge. We present here an arginine ¹³C^ζ-detected NMR experiment in which a double-quantum coherence involving the two ¹⁵N^η nuclei is evolved during the indirect chemical shift evolution period. As the precession frequency of the double-quantum coherence is insensitive to exchange of the two ¹⁵N^η; this new approach is shown to eliminate the previously deleterious line broadenings of ¹⁵N^η resonances caused by the partially restricted rotation about the C^ζ–N^ε bond. Consequently, sharp and well-resolved ¹⁵N^η resonances can be observed. The utility of the presented method is demonstrated on the L99A mutant of the 19 kDa protein T4 lysozyme, where the measurement of small chemical shift perturbations, such as one-bond deuterium isotope shifts, of the arginine amine ¹⁵N^η nuclei becomes possible using the double-quantum experiment.