1H(C) and 1H(N) total NOE correlations in a single 3D NMR experiment. 15N and 13C time-sharing in t1 and t2 dimensions for simultaneous data acquisition

Simultaneous data acquisition in time-sharing (TS) multi-dimensional NMR experiments has been shown an effective means to reduce experimental time, and thus to accelerate structure determination of proteins. This has been accomplished by spin evolution time-sharing of the X and Y heteronuclei, such as ¹⁵N and ¹³C, in one of the time dimensions. In this work, we report a new 3D TS experiment, which allows simultaneous ¹³C and ¹⁵N spin labeling coherence in both t ₁ and t ₂ dimensions to give four NOESY spectra in a single 3D experiment. These spectra represent total NOE correlations between ¹H_N and ¹H_C resonances. This strategy of double time-sharing (2TS) results in an overall four-fold reduction in experimental time compared with its conventional counterpart. This 3D 2TS CN-CN-H HSQC-NOESY-HSQC pulse sequence also demonstrates improvements in water suppression, ¹⁵N spectral resolution and sensitivity, which were developed based on 2D TS CN-H HSQC and 3D TS H-CN-H NOESY-HSQC experiments. Combining the 3D TS and the 3D 2TS NOESY experiments, NOE assignment ambiguities and errors are considerably reduced. These results will be useful for rapid protein structure determination to complement the effort of discerning the functions of diverse genomic proteins.     

Joint non-uniform sampling of all incremented time delays for quicker acquisition in protein relaxation studies

NMR relaxometry plays crucial role in studies of protein dynamics. The measurement of longitudinal and transverse relaxation rates of \(^{15}\)N is the main source of information on backbone motions. However, even the most basic approach exploiting a series of \(^{15}\)N HSQC spectra can require several hours of measurement time. Standard non-uniform sampling (NUS), i.e. random under-sampling of indirect time domain, typically cannot reduce this by more than 2–4\(\times\) due to relatively low “compressibility” of these spectra. In this paper we propose an extension of NUS to relaxation delays. The two-dimensional space of \(t_1\)/\(t_{relax}\) is sampled in a way similar to NUS of \(t_1\)/\(t_2\) domain in 3D spectra. The signal is also processed in a way similar to that known from 3D NUS spectra i.e. using one of the most popular compressed sensing algorithms, iterative soft thresholding. The 2D Fourier transform matrix is replaced with mixed inverse Laplace-Fourier transform matrix. The peak positions in resulting 3D spectrum are characterized by two frequency coordinates and relaxation rate and thus no additional fitting of exponential curves is required. The method is tested on three globular proteins, providing satisfactory results in a time corresponding to acquisition of two conventional \(^{15}\)N HSQC spectra.     

Simultaneous acquisition of [13C,15N]- and [15N,15N]-separated 4D gradient-enhanced NOESY spectra in proteins

Summary The simultaneous acquisition of a 4D gradient-enhanced and sensitivity-enhanced [¹³C,¹⁵N]/[¹⁵N,¹⁵N]-separated NOESY is presented for the 74-residue [¹³C,¹⁵N]-labeled N-terminal SH3 domain of mGrb2 complexed with a peptide gragment from mSOS-2 in 90% H₂O. The method readily accommodates different ¹³C and ¹⁵N spectral widths, but requires that the same number of increments be collected for both ¹³C and ¹⁵N in the simultaneous dimension (F₂). For purposes of display and analysis, the two 4D spectra can be deconvolved during the processing stage by the appropriate linear combination of separately stored FIDs. Compared to collecting each of these two 4D data sets separately, the presented method is a factor (2)^1/2 more efficient in sensitivity per unit acquisition time. The interleaved nature of this method may also lead to improved peak registration between the two 4D spectra.     

Projected [<Superscript>1</Superscript>H,<Superscript>15</Superscript>N]-HMQC-[<Superscript>1</Superscript>H,<Superscript>1</Superscript>H]-NOESY for large molecular systems: application to a 121 kDa protein-DNA complex

We present a projected [¹H,¹⁵N]-HMQC-[¹H,¹H]-NOESY experiment for observation of NOE interactions between amide protons with degenerate ¹⁵N chemical shifts in large molecular systems. The projection is achieved by simultaneous evolution of the multiple quantum coherence of the nitrogen spin and the attached proton spin. In this way NOE signals can be separated from direct-correlation peaks also in spectra with low resolution by fully exploiting both ¹H and ¹⁵N frequency differences, such that sensitivity can be increased by using short maximum evolution times. The sensitivity of the experiment is not dependent on the projection angle for projections up to 45° and no additional pulses or delays are required as compared to the conventional 2D [¹H,¹⁵N]-HMQC-NOESY. The experiment provides two distinct 2D spectra corresponding to the positive and negative angle projections, respectively. With a linear combination of 1D cross-sections from the two projections the unavoidable sensitivity loss in projection spectra can be compensated for each particular NOE interaction. We demonstrate the application of the novel projection experiment for the observation of an NOE interaction between two sequential glycines with degenerate ¹⁵N chemical shifts in a 121.3 kDa complex of the linker H1 histone protein with a 152 bp linear DNA.     

Automated assignment and 3D structure calculations using combinations of 2D homonuclear and 3D heteronuclear NOESY spectra

The NOAH/DIAMOD suite uses feedback filtering and self-correcting distance geometry to generate 3D structures from unassigned NOESY spectra. In this study we determined the minimum set of experiments needed to generate a high quality structure bundle. Different combinations of 3D ¹⁵N-edited, ¹³C-edited HSQC-NOESY and 2D homonuclear ¹H-¹H NOESY spectra of the 77 amino acid protein, myeloid progenitor inhibitory factor-1 (MPIF-1) were used as input for NOAH/DIAMOD calculations. The quality of the assignments of NOESY cross peaks and the accuracy of the automatically generated 3D structures were compared to those obtained with a conventional manual procedure. Combining data from two types of experiments synergistically increased the number of peaks assigned unambiguously in both individual spectra. As a general trend for the accuracy of the structures we observed structural variations in the backbone fold of the final structures of about 2 Å for single spectral data, of 1 Å to 1.5 Å for double spectral data, and of 0.6 Å for triple spectral data sets. The quality of the assignments and 3D structures from the optimal data using all three spectra were similar to those obtained from traditional assignment methods with structural variations within the bundle of 0.6 Å and 1.3 Å for backbone and heavy atoms, respectively. Almost all constraints (97%) of the automatic NOESY cross peak assignments were cross compatible with the structures from the conventional manual assignment procedure, and an even larger proportion (99%) of the manually derived constraints were compatible with the automatically determined 3D structures. The two mean structures determined by both methods differed only by 1.3 Å rmsd for the backbone atoms in the well-defined regions of the protein. Thus NOAD/DIAMOD analysis of spectra from labeled proteins provides a reliable method for high throughput analysis of genomic targets.     

Addressing the overlap problem in the quantitative analysis of two dimensional NMR spectra: Application to <Superscript>15</Superscript>N relaxation measurements

A quantitative analysis of 2D ¹H-¹⁵N spectra is often complicated by resonance overlap. Here a simple method is presented for resolving overlapped correlations by recording 2D projection planes from HNCO data sets. Applications are presented involving the measurement of ¹⁵N T₁ relaxation rates in a high molecular weight protein, malate synthase G, and in a system that exchanges between folded and unfolded states, the drkN SH3 domain. By supplementing relaxation data recorded in the conventional way as a series of 2D ¹H-¹⁵N data sets with a series of a pair of projection planes the number of dynamics probes is increased significantly for both systems studied.     

Iterative algorithm of discrete Fourier transform for processing randomly sampled NMR data sets

Spectra obtained by application of multidimensional Fourier Transformation (MFT) to sparsely sampled nD NMR signals are usually corrupted due to missing data. In the present paper this phenomenon is investigated on simulations and experiments. An effective iterative algorithm for artifact suppression for sparse on-grid NMR data sets is discussed in detail. It includes automated peak recognition based on statistical methods. The results enable one to study NMR spectra of high dynamic range of peak intensities preserving benefits of random sampling, namely the superior resolution in indirectly measured dimensions. Experimental examples include 3D ¹⁵N- and ¹³C-edited NOESY-HSQC spectra of human ubiquitin.     

1H and 15N resonance assignments and secondary structure of the carbon monoxide complex of sperm whale myoglobin

Summary Sequence-specific backbone ¹H and ¹⁵N resonance assignments have been made for 95% of the amino acids in sperm whale myoglobin, complexed with carbon monoxide (MbCO). Many assignments for side-chain resonances have also been obtained. Assignments were made by analysis of an extensive series of homonuclear 2D spectra, measured with unlabeled protein, and both 2D and 3D ¹H-¹⁵N-correlated spectra obtained from uniformly ¹⁵N-labeled myoglobin. Patterns of medium-range NOE connectivities indicate the presence of eight helices in positions that are very similar to those found in the crystal structures of sperm whale myoglobin. The resonance assignments of MbCO form the basis for determination of the solution structure and for hydrogen-exchange measurements to probe the stability and folding pathways of myoglobin. They will also form a basis for assignment of the spectra of single-site mutants with altered ligand-binding properties.     

Optimization of resolution and sensitivity of 4D NOESY using Multi-dimensional Decomposition

Highly resolved multi-dimensional NOE data are essential for rapid and accurate determination of spatial protein structures such as in structural genomics projects. Four-dimensional spectra contain almost no spectral overlap inherently present in lower dimensionality spectra and are highly amenable to application of automated routines for spectral resonance location and assignment. However, a high resolution 4D data set using conventional uniform sampling usually requires unacceptably long measurement time. Recently we have reported that the use of non-uniform sampling and multi-dimensional decomposition (MDD) can remedy this problem. Here we validate accuracy and robustness of the method, and demonstrate its usefulness for fully protonated protein samples. The method was applied to 11 kDa protein PA1123 from structural genomics pipeline. A systematic evaluation of spectral reconstructions obtained using 15–100% subsets of the complete reference 4D ¹H–¹³C–¹³C–¹H NOESY spectrum has been performed. With the experimental time saving of up to six times, the resolution and the sensitivity per unit time is shown to be similar to that of the fully recorded spectrum. For the 30% data subset we demonstrate that the intensities in the reconstructed and reference 4D spectra correspond with a correlation coefficient of 0.997 in the full range of spectral amplitudes. Intensities of the strong, middle and weak cross-peaks correlate with coefficients 0.9997, 0.9965, and 0.83. The method does not produce false peaks. 2% of weak peaks lost in the 30% reconstruction is in line with theoretically expected noise increase for the shorter measurement time. Together with good accuracy in the relative line-widths these translate to reliable distance constrains derived from sparsely sampled, high resolution 4D NOESY data.     

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     

High-Resolution 1H NMR Spectroscopy of Fish Muscle,Eggs and Small Whole Fish via Hadamard-Encoded Intermolecular Multiple-Quantum Coherence

Background and Purpose

Nuclear magnetic resonance (NMR) spectroscopy has become an important technique for tissue studies. Since tissues are in semisolid-state, their high-resolution (HR) spectra cannot be obtained by conventional NMR spectroscopy. Because of this restriction, extraction and high-resolution magic angle spinning (HR MAS) are widely applied for HR NMR spectra of tissues. However, both of the methods are subject to limitations. In this study, the feasibility of HR ¹H NMR spectroscopy based on intermolecular multiple-quantum coherence (iMQC) technique is explored using fish muscle, fish eggs, and a whole fish as examples.

Materials and Methods

Intact salmon muscle tissues, intact eggs from shishamo smelt and a whole fish (Siamese algae eater) are studied by using conventional 1D one-pulse sequence, Hadamard-encoded iMQC sequence, and HR MAS.

Results

When we use the conventional 1D one-pulse sequence, hardly any useful spectral information can be obtained due to the severe field inhomogeneity. By contrast, HR NMR spectra can be obtained in a short period of time by using the Hadamard-encoded iMQC method without shimming. Most signals from fatty acids and small metabolites can be observed. Compared to HR MAS, the iMQC method is non-invasive, but the resolution and the sensitivity of resulting spectra are not as high as those of HR MAS spectra.

Conclusion

Due to the immunity to field inhomogeneity, the iMQC technique can be a proper supplement to HR MAS, and it provides an alternative for the investigation in cases with field distortions and with samples unsuitable for spinning. The acquisition time of the proposed method is greatly reduced by introduction of the Hadamard-encoded technique, in comparison with that of conventional iMQC method.     

Solid state NMR of proteins at high MAS frequencies: symmetry-based mixing and simultaneous acquisition of chemical shift correlation spectra

We have carried out chemical shift correlation experiments with symmetry-based mixing sequences at high MAS frequencies and examined different strategies to simultaneously acquire 3D correlation spectra that are commonly required in the structural studies of proteins. The potential of numerically optimised symmetry-based mixing sequences and the simultaneous recording of chemical shift correlation spectra such as: 3D NCAC and 3D NHH with dual receivers, 3D NC??C and 3D C??NCA with sequential ¹³C acquisitions, 3D NHH and 3D NC??H with sequential ¹H acquisitions and 3D CANH and 3D C??NH with broadband ¹³C?C¹⁵N mixing are demonstrated using microcrystalline samples of the ??1 immunoglobulin binding domain of protein G (GB1) and the chicken ??-spectrin SH3 domain.     

A fast NMR method for resonance assignments: application to metabolomics

We present a new method for rapid NMR data acquisition and assignments applicable to unlabeled (¹²C) or ¹³C-labeled biomolecules/organic molecules in general and metabolomics in particular. The method involves the acquisition of three two dimensional (2D) NMR spectra simultaneously using a dual receiver system. The three spectra, namely: (1) G-matrix Fourier transform (GFT) (3,2)D [¹³C, ¹H] HSQC–TOCSY, (2) 2D ¹H–¹H TOCSY and (3) 2D ¹³C–¹H HETCOR are acquired in a single experiment and provide mutually complementary information to completely assign individual metabolites in a mixture. The GFT (3,2)D [¹³C, ¹H] HSQC–TOCSY provides 3D correlations in a reduced dimensionality manner facilitating high resolution and unambiguous assignments. The experiments were applied for complete ¹H and ¹³C assignments of a mixture of 21 unlabeled metabolites corresponding to a medium used in assisted reproductive technology. Taken together, the experiments provide time gain of order of magnitudes compared to the conventional data acquisition methods and can be combined with other fast NMR techniques such as non-uniform sampling and covariance spectroscopy. This provides new avenues for using multiple receivers and projection NMR techniques for high-throughput approaches in metabolomics.     

Combinatorial triple-selective labeling as a tool to assist membrane protein backbone resonance assignment

Obtaining NMR assignments for slowly tumbling molecules such as detergent-solubilized membrane proteins is often compromised by low sensitivity as well as spectral overlap. Both problems can be addressed by amino-acid specific isotope labeling in conjunction with ¹⁵N–¹H correlation experiments. In this work an extended combinatorial selective in vitro labeling scheme is proposed that seeks to reduce the number of samples required for assignment. Including three different species of amino acids in each sample, ¹⁵N, 1-¹³C, and fully ¹³C/¹⁵N labeled, permits identification of more amino acid types and sequential pairs than would be possible with previously published combinatorial methods. The new protocol involves recording of up to five 2D triple-resonance experiments to distinguish the various isotopomeric dipeptide species. The pattern of backbone NH cross peaks in this series of spectra adds a new dimension to the combinatorial grid, which otherwise mostly relies on comparison of [¹⁵N, ¹H]–HSQC and possibly 2D HN(CO) spectra of samples with different labeled amino acid compositions. Application to two α-helical membrane proteins shows that using no more than three samples information can be accumulated such that backbone assignments can be completed solely based on 3D HNCA/HN(CO)CA experiments. Alternatively, in the case of severe signal overlap in certain regions of the standard suite of triple-resonance spectra acquired on uniformly labeled protein, or missing signals due to a lack of efficiency of 3D experiments, the remaining gaps can be filled.     

Simultaneous NMR assignment of backbone and side chain amides in large proteins with IS-TROSY

A new strategy for the simultaneous NMR assignment of both backbone and side chain amides in large proteins with isotopomer-selective transverse-relaxation-optimized spectroscopy (IS-TROSY) is reported. The method considers aspects of both the NMR sample preparation and the experimental design. First, the protein is dissolved in a buffer with 50%H₂O/50%D₂O in order to promote the population of semideuterated NHD isotopomers in side chain amides of Asn/Gln residues. Second, a ¹³C′-coupled 2D ¹⁵N–¹H IS-TROSY spectrum provides a stereospecific distinction between the geminal protons in the E and Z configurations of the carboxyamide group. Third, a suite of IS-TROSY-based triple-resonance NMR experiments, e.g. 3D IS-TROSY-HNCA and 3D IS-TROSY-HNCACB, are designed to correlate aliphatic carbon atoms with backbone amides and, for Asn/Gln residues, at the same time with side chain amides. The NMR assignment procedure is similar to that for small proteins using conventional 3D HNCA/3D HNCACB spectra, in which, however, signals from NH₂ groups are often very weak or even missing due to the use of broad-band proton decoupling schemes and NOE data have to be used as a remedy. For large proteins, the use of conventional TROSY experiments makes resonances of side chain amides not observable at all. The application of IS-TROSY experiments to the 35-kDa yeast cytosine deaminase has established a complete resonance assignment for the backbone and stereospecific assignment for side chain amides, which otherwise could not be achieved with existing NMR experiments. Thus, the development of IS-TROSY-based method provides new opportunities for the NMR study of important structural and biological roles of carboxyamides and side chain moieties of arginine and lysine residues in large proteins as well as amino moieties in nucleic acids.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Partial site-specific assignment of a uniformly C, N enriched membrane protein, light-harvesting complex 1 (LH1), by solid state NMR

Partial site-specific assignments are reported for the solid state NMR spectra of light-harvesting complex 1, a 160 kDa integral membrane protein. The assignments were derived from 600 MHz ¹⁵N-¹³CO-¹³Cα and ¹⁵N-¹³Cα-¹³CX correlation spectra, using uniformly ¹³C, ¹⁵N enriched hydrated material, in an intact and precipitated form. Sequential assignments were verified using characteristic ¹⁵N-¹³Cα-¹³Cβ side chain chemical shifts observed in 3D experiments. Tertiary contacts found in 2D DARR spectra of the selectively ¹³C enriched sample provided further confirmatory evidence for the assignments. The assignments include the region of the Histidine ligands binding the Bacteriochlorophyll chromophore. The chemical shifts of Cα and Cβ resonances indicated the presence of typical α-helical secondary structure, consistent with previous studies.     

Efficient χ-tensor determination and NH assignment of paramagnetic proteins

Anisotropic magnetic susceptibility tensors χ of paramagnetic metal ions are manifested in pseudocontact shifts, residual dipolar couplings, and other paramagnetic observables that present valuable long-range information for structure determinations of protein-ligand complexes. A program was developed for automatic determination of the χ-tensor anisotropy parameters and amide resonance assignments in proteins labeled with paramagnetic metal ions. The program requires knowledge of the three-dimensional structure of the protein, the backbone resonance assignments of the diamagnetic protein, and a pair of 2D ¹⁵N-HSQC or 3D HNCO spectra recorded with and without paramagnetic metal ion. It allows the determination of reliable χ-tensor anisotropy parameters from 2D spectra of uniformly ¹⁵N-labeled proteins of fairly high molecular weight. Examples are shown for the 185-residue N-terminal domain of the subunit ε from E. coli DNA polymerase III in complex with the subunit θ and La³⁺ in its diamagnetic and Dy³⁺, Tb³⁺, and Er³⁺ in its paramagnetic form.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.The first two authors contributed equally to the project.     

A spectral correlation function for efficient sequential NMR assignments of uniformly 15N-labeled proteins

Summary A new computer-based approach is described for efficient sequence-specific assignment of uniformly ¹⁵N-labeled proteins. For this purpose three-dimensional ¹⁵N-correlated [¹H, ¹H]-NOESY spectra are divided up into two-dimensional ¹H-¹H strips which extend over the entire spectral width along one dimension and have a width of ca. 100 Hz, centered about the amide proton chemical shifts along the other dimension. A spectral correlation function enables sorting of these strips according to proximity of the corresponding residues in the amino acid sequence. Thereby, starting from a given strip in the spectrum, the probability of its corresponding to the C-terminal neighboring residue is calculated for all other strips from the similarity of their peak patterns with a pattern predicted for the sequentially adjoining residue, as manifested in the scalar product of the vectors representing the predicted and measured peak patterns. Tests with five different proteins containing both -helices and -sheets, and ranging in size from 58 to 165 amino acid residues show that the discrimination achieved between the sequentially neighboring residue and all other residues compares well with that obtained with an unguided interactive search of pairs of sequentially neighboring strips, with important savings in the time needed for complete analysis of 3D ¹⁵N-correlated [¹H, ¹H]-NOESY spectra. The integration of this routine into the program package XEASY ensures that remaining ambiguities can be resolved by visual inspection of the strips, combined with reference to the amino acid sequence and information on spin-system types obtained from additional NMR spectra.Abbreviations 1D, 2D, 3D, 4D one-, two-, three-, four-dimensional - NOE nuclear Overhauser enhancement - NOESY nuclear Overhauser enhancement spectroscopy - COSY correlation spectroscopy - TOCSY total correlation spectroscopy     

Gradient-enhanced 3D NOESY-HMQC spectroscopy

Summary A gradient-enhanced 3D NOESY-HMQC experiment is presented and applied to¹⁵N-labeled Mnt repressor (1–76) in¹H₂O. Both coherence selection and¹H₂O suppression are achieved using gradients. A singlescan experiment can be recorded for each time increment, which greatly reduces recording time with respect to conventional methods using phase cycling. This can be of use for kinetic studies and for relatively unstable molecules.     

Time-shared HSQC-NOESY for accurate distance constraints measured at high-field in <Superscript>15</Superscript>N-<Superscript>13</Superscript>C-ILV methyl labeled proteins

We present a time-shared 3D HSQC-NOESY experiment that enables one to simultaneously record ¹³C- and ¹⁵N-dispersed spectra in Ile, Leu and Val (ILV) methyl-labeled samples. This experiment is designed to delineate the two spectra which would otherwise overlap with one another when acquired together. These spectra display nOe correlations in the detected proton dimension, i.e. with maximum resolution. This is in contrast to NOESY-HSQC types of experiments that provide cross-peaks in the indirect dimension with low resolution due to limits in experimental time. The technique is particularly advantageous at high field where even longer experimental times would be required for comparable resolution in NOESY-HSQC experiments. The method is demonstrated at 900 MHz and at 750 MHz on 37 and 31 kDa proteins, respectively. The resolution and time saving provided in this experiment was crucial for solving the structures of these two proteins.