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     

Effect of deuteration on some structural parameters of methyl groups in proteins as evaluated by residual dipolar couplings

One bond methyl ¹H-¹³C and ¹³C_methyl–¹³C scalar and residual dipolar couplings have been measured at sites in an ¹⁵N, ¹³C, 50% ²H labeled sample of the B1 immunoglobulin binding domain of peptostreptococcal protein L to investigate changes in the structure of methyl groups in response to deuterium substitution. Both one bond methyl ¹H-¹³C and ¹³C_methyl–¹³C scalar coupling constants have been found to decrease slightly with increasing deuterium content. Previous studies have shown that ¹H-¹³C couplings in methyl groups are exquisitely sensitive to electronic structure, with decreases in coupling values as a function of deuteration consistent with a slight lengthening of the remaining H-C bonds. Changes in the H_methylC_methylC angle are found to be small, with average differences on the order of 0.3 ± 0.1° and 0.4 ± 0.2° between CH₃, CH₂D and CH₃, CHD₂ isotopomers, respectively. Knowledge of methyl geometry is a prerequisite for the extraction of accurate dynamics parameters from spin relaxation studies involving these groups.     

Deuterium isotope effects and fractionation factors of hydrogen-bonded A:T base pairs of DNA

Deuterium isotope effects and fractionation factors of N1...H3–N3 hydrogen bonded Watson–Crick A:T base pairs of two DNA dodecamers are presented here. Specifically, two-bond deuterium isotope effects on the chemical shifts of ¹³C2 and ¹³C4, ²¹³C2 and ²¹³C4, and equilibrium deuterium/protium fractionation factors of H3, , were measured and seen to correlate with the chemical shift of the corresponding imino proton, _H3. Downfield-shifted imino protons associated with larger values of ²¹³C2 and ²¹³C4 and smaller values, which together suggested that the effective H3–N3 vibrational potentials were more anharmonic in the stronger hydrogen bonds of these DNA molecules. We anticipate that ²¹³C2, ²¹³C4 and values can be useful gauges of hydrogen bond strength of A:T base pairs.     

Determination of 13Cα relaxation times in uniformly 13C/15N-enriched proteins

Summary Relaxation times of ¹³C carbons of uniformly ¹³C/¹⁵N-enriched probes have been investigated. The relaxation behaviour was analyzed in terms of a multispin system. Pulse sequences for the determination of T₁, T₂ and the heteronuclear NOE of ¹³C in uniformly ¹³C/¹⁵N-enriched ribonuclease T1 are presented. The experiments performed in order to obtain T₁ and the heteronuclear NOE were similar to those of the corresponding ¹⁵N experiments published previously. The determination of T₂ for the C-carbon in a completely labeled protein is more complicated, since the magnetization transfer during the T₂ evolution period owing to the scalar coupling of C–C must be suppressed. Various different pulse sequences for the T₂ evolution period were simulated in order to optimize the bandwidth for which reliable T₂ relaxation times can be obtained. A proof for the quality of these pulse sequences is given by fitting the intensity decay of individual ¹H–¹³C cross peaks, in a series of (¹H, ¹³C)-ct-HSQC spectra with a modified CPMG sequence as well as a T_1p sequence for the transverse relaxation time, to a single exponential using a simplex algorithm.     

Detection of amplified DNA sequences by reverse chromosome painting using genomic tumor DNA as probe

A modification of reverse chromosome painting was carried out using genomic DNA from tumor cells as a complex probe for chromosomal in situ suppression hybridization to normal metaphase chromsome spreads. Amplified DNA sequences contained in such probes showed specific signals, revealing the normal chromosome positions from which these sequences were derived. As a model system, genomic DNAs were analyzed from three tumor cell lines with amplification units including the proto-oncogene c-myc. The smallest amplification unit was about 90 kb and was present in 16–24 copies; the largest unit was bigger than 600 kb and was present in 16–32 copies. Specific signals that co-localized with a differently labeled c-myc probe on chromosome band 8q24 were obtained with genomic DNA from each cell line. In further experiments, genomic DNA derived from primary tumor material was used in the case of a male patient with glioblastoma multiforme (GBM). Southern blot analysis using an epidermal growth factor receptor gene (EGFR) probe that maps to 7p13 indicated the amplification of sequences from this gene. Using reverse chromosome painting, signals were found both on band 7p13 and bands 12q13–q15. Notably, the signal on 12q13–q15 was consistently stronger. The weaker 7p13 signal showed co-localization with the major signal of the differently labeled EGFR probe. A minor signal of this probe was seen on 12q13, suggesting cross-hybridization to ERB3 sequences homologous to EGFR. The results indicate co-amplification of sequences from bands 12q13–q15, in addition to sequences from band 7p13. Several oncogenes map to 12q13–q15 providing candidate genes for a tumor-associated proto-oncogene amplification. Although the nature of the amplified sequences needs to be clarified, this experiment demonstrates the potential of reverse chromosome painting with genomic tumor DNA for rapidly mapping the normal chromosomal localization of the DNA from which the amplified sequences were derived. In addition, a weaker staining of chromosomes 10 and X was consistently observed indicating that these chromosomes were present in only one copy in the GBM genome. This rapid approach can be used to analyze cases where no metaphase spreads from the tumor material are available. It does not require any preknowledge of amplified sequences and can be applied to screen large numbers of tumors.     

Feeding level and individual metabolic rate affect δ13C and δ15N values in carp: implications for food web studies

Stable isotope analyses are often used to calculate relative contributions of multiple food sources in an animals diet. One prerequisite for a precise calculation is the determination of the diet-tissue fractionation factor. Isotopic ratios in animals are not only affected by the composition of the diet, but also by the amount of food consumed. Previous findings regarding the latter point are controversial. As stable isotope analyses have often been used to investigate aquatic food webs, an experiment with carp (Cyprinus carpio L.) was carried out to test the influence of the feeding level and individual metabolic rate on ¹³C and ¹⁵N values of the whole body. After an initial phase, 49 carp were assigned randomly to four groups and fed the same diet at different levels for 8 weeks. For 15 fish, the energy budget was determined by indirect calorimetry. Feed and individual fish were analysed for their proximate composition, gross energy content and ¹³C and ¹⁵N values. ¹³C and ¹⁵N values differed significantly at different feeding levels. While ¹³C values of the lipids and ¹⁵N values decreased with increasing feeding rate, ¹³C values of the lipid-free matter showed a non-linear pattern. Data obtained from fish held in the respirometric system revealed a relationship between ¹³C values and the percentage retention of metabolizable energy. Our results show that reconstructing the diets of fish from the isotopic ratios when the feeding level and individual metabolic rates are unknown would introduce an error into the data used for back-calculation of up to 1 for both ¹³C and ¹⁵N values and may have substantial effects on the results of calculated diets. As other workers have pointed out, the development and application of stable isotopes to nutritional ecology studies is a field in its infancy and gives rise to erroneous, misleading results without nutritional, physiological and ecological knowledge.     

Improved 3D triple resonance experiments, HNN and HN(C)N, for HN and 15N sequential correlations in (13C, 15N) labeled proteins: application to unfolded proteins

Two triple resonance experiments, HNN and HN(C)N, are presented which correlate H^N and ¹⁵N resonances sequentially along the polypeptide chain of a doubly (¹³C, ¹⁵N) labeled protein. These incorporate several improvements over the previously published sequences for a similar purpose and have several novel features. The spectral characteristics enable direct identification of certain triplets of residues, which provide many starting points for the sequential assignment procedure. The experiments are sensitive and their utility has been demonstrated with a 22 kDa protein under unfolding conditions where most of the standard triple resonance experiments such as HNCA, CBCANH etc. have limited success because of poor amide, C and C chemical shift dispersions.     

Optimized labeling of 13CHD2 methyl isotopomers in perdeuterated proteins: Potential advantages for 13C relaxation studies of methyl dynamics of larger proteins

¹³CHD₂ methyl isotopomers are particularly useful to study methyl dynamics in proteins because, as compared with other methyl isotopomers, the ¹³C relaxation mechanism for this isotopomer is straightforward. However, in the case of proteins, where ()² 1, the refocused INEPT pulse sequence does not completely suppress unwanted ¹³CH₃ signals. The presence of weak ¹³CH₃ peaks is usually not a serious problem for smaller proteins because there are relatively few methyl signals and they are sharp; however, signal overlap becomes more common as the size of the protein increases. We overcome this problem by preparing a protein using a 98% D₂O cell culture medium containing 3-¹³C pyruvic acid, 50–60% deuterated at the 3-position, and 4-¹³C 2-ketobutyric acid, 98% and 62% deuterated at the 3- and 4-positions, respectively. This approach significantly reduces the population of the CH₃ isotopomer while optimizing the production of ¹³CHD₂, the isotopomer desired for ¹³C relaxation measurements. In larger proteins where the deuterium T₂ may be too short to measure accurately, we also suggest the alternative measurement of the proton T₂ of the ¹³CH₂D methyl isotopomer, because these protons are well-isolated from other protons in these highly deuterated samples.     

A simple urea leaf-feeding method for the production of 13C and 15N labelled plant material

The use of ¹³C isotope tracer techniques in terrestrial ecology has been restricted by the technical requirements and high costs associated with the production of ¹³C enriched plant material by ¹³CO₂ release in labelling chambers. We describe a novel, simple and relatively inexpensive method for the small-scale production of ¹³C and ¹⁵N labelled plant material. The method is based on foliar feeding of plants with a urea solution (97 atom% ¹³C, 2 atom% ¹⁵N) by daily misting. Maize was grown in a greenhouse in a compost–soil mixture and enclosed in clear polythene bags between urea applications. Final enrichment in 27 d old maize shoots was 211 ¹³C (1.34 atom% ¹³C) and 434 ¹⁵N (0.52 atom% ¹⁵N). Enrichments of hot-water extractable fractions (289 ¹³C, 469 ¹⁵N) were only slightly higher than those observed in plant bulk material, which suggests that daily urea applications ensured fairly uniform labelling of different biochemical fractions and plant tissues. Recovery of applied excess ¹³C and ¹⁵N in plant shoots was 22% and 42%, respectively. Roots were less enriched (21 ¹³C and 277 ¹⁵N), but no attempts were made to recover roots quantitatively.     

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.     

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.     

[13c]-Constant-Time [15n,1h]-Trosy-Hnca for Sequential Assignments of Large Proteins

The greatly improved sensitivity resulting from the use of TROSY during 15N evolution and amide proton acquisition enables the recording of HNCA spectra of large proteins with constant-time 13C evolution. In [13C]-ct-[15N,1H]-TROSY-HNCA experiments with a 2H/13C/15N-labeled 110 kDa protein, 7,8-dihydroneopterin aldolase from Staphylococcus aureus, nearly all correlation peaks seen in the [15N,1H]-TROSY-HNCA spectrum were also detected. The improved resolution in the 13C dimension then enabled a significant number of sequential assignments that could not be obtained with [15N,1H]-TROSY-HNCA without [13C]-constant-time period.     

TROSY-based HNCO pulse sequences for the measurement of 1HN-15N, 15N-13CO, 1HN-13CO, 13CO-13Cα and 1HN-13Cα dipolar couplings in 15N, 13C, 2H-labeled proteins

HNCO-based 3D pulse schemes are presented for measuring ¹HN-¹⁵N,¹⁵N-¹³CO, ¹HN-¹³CO,¹³CO-¹³C and ¹HN-¹³C dipolar couplings in ¹⁵N,¹³C,²-labeled proteins. The experiments are based on recently developed TROSY methodology for improving spectral resolution and sensitivity. Data sets recorded on a complex of Val, Leu, Ile (1 only) methyl protonated ¹⁵N,¹³C,²H-labeled maltose binding protein and -cyclodextrin as well as ¹⁵N,¹³C,²H-labeled human carbonic anhydrase II demonstrate that precise dipolar couplings can be obtained on proteins in the 30–40 kDa molecular weight range. These couplings will serve as powerful restraints for obtaining global folds of highly deuterated proteins.     

Improved pulse sequences for measuring coupling constants in 13C, 15N-labeled proteins

Summary NMR pulse sequences for measuring coupling constants in ¹³C, ¹⁵N-labeled proteins are presented. These pulse sequences represent improvements over earlier experiments with respect to resolution and number of radiofrequency pulses. The experiments are useful for measuring J_NH , J_NCO, J_NC , J_H ^N _CO and J_H ^N _H . Applications to chymotrypsin inhibitor 2 (CI-2) are shown.     

Differential deuterium isotope shifts and one-bond 1H−13C scalar couplings in the conformational analysis of protein glycine residues

Summary The one-bond deuterium isotope shift effect for glycine C resonances exhibits a conformational dependence comparable to that of the corresponding ¹J_HC scalar coupling in both magnitude (11 Hz at 14.1 T) and dihedral angle dependence. The similarity in the conformational dependence of the ¹J_HC and deuterium isotope shift values suggests a common physical basis. Given the known distribution of (,) main-chain dihedral angles for glycine residues, the deuterium isotope shifts and the ¹J_HC scalar couplings can determine conformations in the left-and right-handed helical-to-bridge regions of the (,) plane to an accuracy of approximately 13°. In the absence of stereochemical assignments, the differential deuterium isotope shifts and the ¹J_HC scalar couplings can be combined with limited independent structural information (e.g., the sign of ) to determine the chirality of the deuterium substitution.     

Alternative E.COSY techniques for the measurement of 3J(C i ′ −1,C i β ) and 3 J(H i N ,C i β ) coupling constants in proteins

Experiments are proposed for the measurement of the vicinal coupling constants between -carbons and either amide protons of the same or carbonyl carbons of the preceding amino acid residue in 13C/15N-labeled proteins. Both couplings depend on the backbone torsional angle . The three-dimensional pulse sequences give rise to E.COSY-like multiplet patterns in which heteronuclear one-bond couplings separate the doublet components corresponding to the two spin states of the respective passive nuclei. Thus, in contrast to previously published pulse schemes which employed the homonuclear 1J(C,C) interaction, difficulties due to overlap of spectral regions of active and passive spins are avoided. A major drawback of the novel sequences is their limited sensitivity. Nevertheless, application to Desulfovibrio vulgaris flavodoxin yielded coupling constants for more than 85% of all non-glycine and non-proline residues.     

Unambiguous NOE assignments in proteins by a combination of through-bond and through-space correlations

Heteronuclear editing has found widespread use in the detection ofproton–proton dipolar interactions in isotopically labelled proteins.However, in cases where both the resonances of protons and directly bound¹³C or ¹⁵N spins of two or more sites aredegenerate, unambiguous assignments are difficult to obtain by conventionalmethods. Here, we present simple extensions of well-known triple-resonancepulse sequences which improve the dispersion of NOESY spectra. In order torecord the chemical shifts of backbone nuclei which allow a resolution ofoverlapping cross peaks, the magnetization is relayed via the scalarcoupling network either before or after the NOE mixing period. The novelpulse sequences are applied to flavodoxin from the sulfate-reducing organismDesulfovibrio vulgaris. A number of previously unassigned NOE interactionsinvolving -, - and amide protons can be unequivocallyidentified, suggesting that the accuracy of protein structure determinationcan be improved.     

A new triple-resonance experiment for the sequential assignment of backbone resonances in proteins

Summary A new protocol is described for obtaining intraresidual and sequential correlations between carbonyl carbons and amide ¹H and ¹⁵N resonances of amino acids. Frequency labeling of ¹³CO spins occurs during a period required for the ¹³C-¹⁵N polarization transfer, leading to an optimized transfer efficiency. In a four-dimensional version of the experiment, ¹³C chemical shifts are used to improve the dispersion of signals. The resonance frequencies of all backbone nuclei can be detected in a 3D variant in which cross peaks are split along two frequency axes. This pulse scheme is the equivalent of a five-dimensional experiment. The novel pulse sequences are applied to flavodoxin from Desulfovibrio vulgaris.     

QUDeX-MS: hydrogen/deuterium exchange calculation for mass spectra with resolved isotopic fine structure

Background

Hydrogen/deuterium exchange (HDX) coupled to mass spectrometry permits analysis of structure, dynamics, and molecular interactions of proteins. HDX mass spectrometry is confounded by deuterium exchange-associated peaks overlapping with peaks of heavy, natural abundance isotopes, such as carbon-13. Recent studies demonstrated that high-performance mass spectrometers could resolve isotopic fine structure and eliminate this peak overlap, allowing direct detection and quantification of deuterium incorporation.

Results

Here, we present a graphical tool that allows for a rapid and automated estimation of deuterium incorporation from a spectrum with isotopic fine structure. Given a peptide sequence (or elemental formula) and charge state, the mass-to-charge ratios of deuterium-associated peaks of the specified ion is determined. Intensities of peaks in an experimental mass spectrum within bins corresponding to these values are used to determine the distribution of deuterium incorporated. A theoretical spectrum can then be calculated based on the estimated distribution of deuterium exchange to confirm interpretation of the spectrum. Deuterium incorporation can also be detected for ion signals without a priori specification of an elemental formula, permitting detection of exchange in complex samples of unidentified material such as natural organic matter. A tool is also incorporated into QUDeX-MS to help in assigning ion signals from peptides arising from enzymatic digestion of proteins. MATLAB-deployable and standalone versions are available for academic use at qudex-ms.sourceforge.net and agarlabs.com.

Conclusion

Isotopic fine structure HDX-MS offers the potential to increase sequence coverage of proteins being analyzed through mass accuracy and deconvolution of overlapping ion signals. As previously demonstrated, however, the data analysis workflow for HDX-MS data with resolved isotopic fine structure is distinct. QUDeX-MS we hope will aid in the adoption of isotopic fine structure HDX-MS by providing an intuitive workflow and interface for data analysis.