An enhanced-sensitivity pure absorption gradient 4D 15N, 13C-edited NOESY experiment

Summary An enhanced-sensitivity gradient 4D ¹⁵N, ¹³C-edited NOESY experiment is presented. Gradients are employed to suppress artifacts, eliminate the intense H₂O signal and select for the coherence transfer pathway involving ¹⁵N magnetization. The latter use of the gradients results in a decrease in the number of phase cycle steps by a factor of two relative to the previously published 4D sequence (Kay et al., 1990) allowing for the recording of spectra with increased resolution per unit measuring time. Theoretical sensitivity enhancements of as much as a factor of can be expected over the previously published sequence, neglecting the effects of relaxation and pulse imperfections.     

Covariance NMR in higher dimensions: application to 4D NOESY spectroscopy of proteins

Elucidation of high-resolution protein structures by NMR spectroscopy requires a large number of distance constraints that are derived from nuclear Overhauser effects between protons (NOEs). Due to the high level of spectral overlap encountered in 2D NMR spectra of proteins, the measurement of high quality distance constraints requires higher dimensional NMR experiments. Although four-dimensional Fourier transform (FT) NMR experiments can provide the necessary kind of spectral information, the associated measurement times are often prohibitively long. Covariance NMR spectroscopy yields 2D spectra that exhibit along the indirect frequency dimension the same high resolution as along the direct dimension using minimal measurement time. The generalization of covariance NMR to 4D NMR spectroscopy presented here exploits the inherent symmetry of certain 4D NMR experiments and utilizes the trace metric between donor planes for the construction of a high-resolution spectral covariance matrix. The approach is demonstrated for a 4D (13)C-edited NOESY experiment of ubiquitin. The 4D covariance spectrum narrows the line-widths of peaks strongly broadened in the FT spectrum due to the necessarily short number of increments collected, and it resolves otherwise overlapped cross peaks allowing for an increase in the number of NOE assignments to be made from a given dataset. At the same time there is no significant decrease in the positive predictive value of observing a peak as compared to the corresponding 4D Fourier transform spectrum. These properties make the 4D covariance method a potentially valuable tool for the structure determination of larger proteins and for high-throughput applications in structural biology.     

Optimizing resolution in multidimensional NMR by three-way decomposition

Resolution depends on the number of points sampled in a FID; in indirectly detected dimensions it is an important determinant of the total experiment time. Based on the high redundancy present in NMR data, we propose the following timesaving scheme for three-dimensional spectra. An extensive grid of discrete t1- and t2-values is used, which increases resolution while preserving the spectral width. Total experiment time is reduced by avoiding the recording of t3-FIDs for selected pairs of t1 and t2; typically the recording is omitted for about 75% of the (t1,t2) combinations. These data sets are referred to as sparse, and post-experimental processing making optimal use of spectral redundancy provides the missing, non-recorded data. We have previously shown that three-way decomposition (TWD) within the MUNIN approach provides a practical way to process dense NMR data sets. Here, a novel TWD algorithm [Ibraghimov, (2002) Numer. Linear Algebra Appl. 9, 551–565] is used to complement a sparselyrecorded time-domain data set by providing the missing FIDs for all (t1,t2) combinations omitted in the experiment. A necessary condition is that for each t1-value at least a few FIDs are recorded, and similar for each t2-value. The method is demonstrated on non-uniformly sampled ¹⁵N-NOESY-HSQC data sets recorded for the 14 kD protein azurin. The spectra obtained by TWD, reconstruction and ordinary transform to frequency-domain are, in spite of the large number of signals and the high dynamic range typical for NOESYs, highly similar to a corresponding reference spectrum, for which all (t1,t2) combinations were recorded.     

New applications of 2D filtered/edited NOESY for assignment and structure elucidation of RNA and RNA-protein complexes

NMR spectra of large RNAs are difficult to assign because of extensive spectral overlap and unfavorable relaxation properties. Here we present a new approach to facilitate assignment of RNA spectra using a suite of four 2D-filtered/edited NOESY experiments in combination with base-type-specific isotopically labeled RNA. The filtering method was developed for use in 3D filtered NOESY experiments (Zwahlen et al., 1997), but the 2D versions are both more sensitive and easier to interpret for larger RNAs than their 3D counterparts. These experiments are also useful for identifying intermolecular NOEs in RNA-protein complexes. Applications to NOE assignment of larger RNAs and an RNA-protein complex are presented.     

ABACUS, a direct method for protein NMR structure computation via assembly of fragments

The ABACUS algorithm obtains the protein NMR structure from unassigned NOESY distance restraints. ABACUS works as an integrated approach that uses the complete set of available NMR experimental information in parallel and yields spin system typing, NOE spin pair identities, sequence specific resonance assignments, and protein structure, all at once. The protocol starts from unassigned molecular fragments (including single amino acid spin systems) derived from triple-resonance (1)H/(13)C/(15)N NMR experiments. Identifications of connected spin systems and NOEs precede the full sequence specific resonance assignments. The latter are obtained iteratively via Monte Carlo-Metropolis and/or probabilistic sequence selections, molecular dynamics structure computation and BACUS filtering (A. Grishaev and M. Llinás, J Biomol NMR 2004;28:1-10). ABACUS starts from scratch, without the requirement of an initial approximate structure, and improves iteratively the NOE identities in a self-consistent fashion. The procedure was run as a blind test on data recorded on mth1743, a 70-amino acid genomic protein from M. thermoautotrophicum. It converges to a structure in ca. 15 cycles of computation on a 3-GHz processor PC. The calculated structures are very similar to the ones obtained via conventional methods (1.22 A backbone RMSD). The success of ABACUS on mth1743 further validates BACUS as a NOESY identification protocol.     

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.     

Constant-time NOESY: An aid in the analysis of protein NMR spectra

Summary A constant-time version of the homonuclear NOESY experiment (CT-NOESY) is described. The experiment yields simplified protein spectra, in which cross peaks arising from protons with zero or small couplings are differentiated from other cross peaks, thus partially overcoming the problem of signal overlap. In addition, the CT-NOESY spectrum provides information on the magnitude of³J_NH- and³J coupling' constants, and is thus useful to determine torsion angle constraints and to perform stereospecific assignments of CHH protons in the case of³J constants.     

2D and 3D TROSY-enhanced NOESY of 15N labeled proteins

Recently, several TROSY-based experiments have been designed for backbone chemical shift assignment and measurement of the NOEs of ²H, ¹³C and ¹⁵N labeled proteins. Here, we present TROSY-enhanced NOESY experiments, namely the 2D S3E-NOESY-S3E, 3D TROSY-NOESY-S3E and S3E-NOESY-TROSY experiments. These experiments use the spin-state selective excitation method (S3E), and have the TROSY effect in all the indirectly and directly detected dimensions, and so provide optimal resolution for amide protons. The first two experiments provide an additional useful feature in that the diagonal peaks of the amide proton region are cancelled or greatly reduced, allowing clear identification of NOE cross peaks that are close to diagonal peaks.     

Towards miniaturization of a structural genomics pipeline using micro-expression and microcoil NMR

In structural genomics centers, nuclear magnetic resonance (NMR) screening is in increasing use as a tool to identify folded proteins that are promising targets for three-dimensional structure determination by X-ray crystallography or NMR spectroscopy. The use of 1D ¹H NMR spectra or 2D [¹H,¹⁵N]-correlation spectroscopy (COSY) typically requires milligram quantities of unlabeled or isotope-labeled protein, respectively. Here, we outline ways towards miniaturization of a structural genomics pipeline with NMR screening for folded globular proteins, using a high-density micro-fermentation device and a microcoil NMR probe. The proteins are micro-expressed in unlabeled or isotope-labeled media, purified, and then subjected to 1D ¹H NMR and/or 2D [¹H,¹⁵N]-COSY screening. To demonstrate that the miniaturization is functioning effectively, we processed nine mouse homologue protein targets and compared the results with those from the “macro-scale” Joint Center of Structural Genomics (JCSG) high-throughput pipeline. The results from the two pipelines were comparable, illustrating that the data were not compromised in the miniaturized approach.     

Computation of relaxation matrix elements from incomplete NOESY data sets

Summary The structural determination of biological molecules in solution by NMR relies on the determination of a set of interatomic distances obtained by measurement of intramolecular nuclear Overhauser effects (NOE). It is shown in this paper that it is possible to obtain the accurate relaxation rate (and hence the interatomic distance) from the direct measurement of a single NOE signal. The precise analysis of a NOESY peak evolution with respect to the mixing time allows the evaluation of the relaxation parameters for the pair of spins under consideration. This is done without any assumption on the relaxation of unmeasured spins, or on the movement of the molecule. The theoretical basis of this method is presented. In order to evaluate the proposed method, a simulated case on the protein BPTI is studied, which shows that the method performs very well even in the case of noisy data sets.     

High resolution 4-D spectroscopy with sparse concentric shell sampling and FFT-CLEAN

Recent efforts to reduce the measurement time for multidimensional NMR experiments have fostered the development of a variety of new procedures for sampling and data processing. We recently described concentric ring sampling for 3-D NMR experiments, which is superior to radial sampling as input for processing by a multidimensional discrete Fourier transform. Here, we report the extension of this approach to 4-D spectroscopy as Randomized Concentric Shell Sampling (RCSS), where sampling points for the indirect dimensions are positioned on concentric shells, and where random rotations in the angular space are used to avoid coherent artifacts. With simulations, we show that RCSS produces a very low level of artifacts, even with a very limited number of sampling points. The RCSS sampling patterns can be adapted to fine rectangular grids to permit use of the Fast Fourier Transform in data processing, without an apparent increase in the artifact level. These artifacts can be further reduced to the noise level using the iterative CLEAN algorithm developed in radioastronomy. We demonstrate these methods on the high resolution 4-D HCCH-TOCSY spectrum of protein G's B1 domain, using only 1.2% of the sampling that would be needed conventionally for this resolution. The use of a multidimensional FFT instead of the slow DFT for initial data processing and for subsequent CLEAN significantly reduces the calculation time, yielding an artifact level that is on par with the level of the true spectral noise.     

Novel projected 4D triple resonance experiments for polypeptide backbone chemical shift assignment

Here we present a novel suite of projected 4D triple-resonance NMR experiments for efficient sequential assignment of polypeptide backbone chemical shifts in ¹³C/¹⁵N doubly labeled proteins. In the 3D HNN[CAHA] and 3D HNN(CO)[CAHA] experiments, the ¹³C and ¹H chemical shifts evolve in a common dimension and are simultaneously detected in quadrature. These experiments are particularly useful for the assignment of glycine-rich polypeptide segments. Appropriate setting of the ¹H radiofrequency carrier allows one to place cross peaks correlating either backbone ¹⁵N/¹H^N/¹³C or ¹⁵N/¹H^N/¹H chemical shifts in separate spectral regions. Hence, peak overlap is not increased when compared with the conventional 3D HNNCA and HNN(CA)HA. 3D HNN[CAHA] and 3D HNN(CO)[CAHA] are complemented by 3D reduced-dimensionality (RD) HNN COCA and HNN CACO, where ¹³C and ¹³C chemical shifts evolve in a common dimension. The ¹³C shift is detected in quadrature, which yields peak pairs encoding the ¹³C chemical shift in an in-phase splitting. This suite of four experiments promises to be of value for automated high-throughput NMR structure determination in structural genomics, where the requirement to independently sample many indirect dimensions in a large number of NMR experiments may prevent one from accurately adjusting NMR measurement times to spectrometer sensitivity.     

Mixed-time parallel evolution in multiple quantum NMR experiments: sensitivity and resolution enhancement in heteronuclear NMR

A new strategy is demonstrated that simultaneously enhances sensitivity and resolution in three- or higher-dimensional heteronuclear multiple quantum NMR experiments. The approach, referred to as mixed-time parallel evolution (MT-PARE), utilizes evolution of chemical shifts of the spins participating in the multiple quantum coherence in parallel, thereby reducing signal losses relative to sequential evolution. The signal in a given PARE dimension, t ₁, is of a non-decaying constant-time nature for a duration that depends on the length of t ₂, and vice versa, prior to the onset of conventional exponential decay. Line shape simulations for the ¹H–¹⁵N PARE indicate that this strategy significantly enhances both sensitivity and resolution in the indirect ¹H dimension, and that the unusual signal decay profile results in acceptable line shapes. Incorporation of the MT-PARE approach into a 3D HMQC-NOESY experiment for measurement of H^N–H^N NOEs in KcsA in SDS micelles at 50°C was found to increase the experimental sensitivity by a factor of 1.7±0.3 with a concomitant resolution increase in the indirectly detected ¹H dimension. The method is also demonstrated for a situation in which homonuclear ¹³C–¹³C decoupling is required while measuring weak H3′–2′OH NOEs in an RNA oligomer. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification and distribution of protein families in 120 completed genomes using Gene3D

Using a new protocol, PFscape, we undertake a systematic identification of protein families and domain architectures in 120 complete genomes. PFscape clusters sequences into protein families using a Markov clustering algorithm (Enright et al., Nucleic Acids Res 2002;30:1575-1584) followed by complete linkage clustering according to sequence identity. Within each protein family, domains are recognized using a library of hidden Markov models comprising CATH structural and Pfam functional domains. Domain architectures are then determined using DomainFinder (Pearl et al., Protein Sci 2002;11:233-244) and the protein family and domain architecture data are amalgamated in the Gene3D database (Buchan et al., Genome Res 2002;12:503-514). Using Gene3D, we have investigated protein sequence space, the extent of structural annotation, and the distribution of different domain architectures in completed genomes from all kingdoms of life. As with earlier studies by other researchers, the distribution of domain families shows power-law behavior such that the largest 2,000 domain families can be mapped to approximately 70% of nonsingleton genome sequences; the remaining sequences are assigned to much smaller families. While approximately 50% of domain annotations within a genome are assigned to 219 universal domain families, a much smaller proportion (< 10%) of protein sequences are assigned to universal protein families. This supports the mosaic theory of evolution whereby domain duplication followed by domain shuffling gives rise to novel domain architectures that can expand the protein functional repertoire of an organism. Functional data (e.g. COG/KEGG/GO) integrated within Gene3D result in a comprehensive resource that is currently being used in structure genomics initiatives and can be accessed via http://www.biochem.ucl.ac.uk/bsm/cath/Gene3D/.     

Transverse relaxation optimized 3D and 4D 15N/15N separated NOESY experiments of 15N labeled proteins

Distance constraints are an important complement to orientational constraints. While a high-resolution monomer structure of the ion channel forming polypeptide, gramicidin A, has been solved with 120 orientational constraints, the precise geometry of the dimer interface has not been characterized. Here, using both ¹³C and ¹⁵N labeled gramicidin A samples in hydrated phospholipid bilayers, both inter- and intramolecular distances have been measured with a recently developed simultaneous frequency and amplitude modulation (SFAM) solid-state NMR scheme. Using this approach ¹⁵N-¹³C₁ residual dipolar couplings across a hydrogen bond as small as 20 ± 2 Hz have been characterized. While such distances are on the order of 4.2 ± 0.2 Å, the spectroscopy is complicated by rapid global motion of the molecular structure about the bilayer normal and channel axis. Consequently, the nominal 40 Hz dipolar coupling is averaged depending on the orientation of the internuclear vector with respect to the motional axis. The intermolecular distance confirmed the previously described monomeric structure, while the intramolecular distance across the monomer–monomer interface defined this junction and confirmed the previous model of this interface.     

Automated combined assignment of NOESY spectra and three-dimensional protein structure determination

A procedure for automated protein structure determination is presented that is based on an iterative procedure during which the NOESY peak list assignment and the structure calculation are performed simultaneously. The input consists of a list of NOESY peak positions and a list of chemical shifts as obtained from sequence-specific resonance assignment. For the present applications of this approach the previously introduced NOAH routine was implemented in the distance geometry program DIANA. As an illustration, experimental 2D and 3D NOESY cross-peak lists of six proteins have been analyzed, for which complete sequence-specific ¹H assignments are available for the polypeptide backbone and the amino acid side chains. The automated method assigned 70–90% of all NOESY cross peaks, which is on average 10% less than with the interactive approach, and only between 0.8% and 2.4% of the automatically assigned peaks had a different assignment than in the corresponding manually assigned peak lists. The structures obtained with NOAH/DIANA are in close agreement with those from manually assigned peak lists, and with both approaches the residual constraint violations correspond to high-quality NMR structure determinations. Systematic comparisons of the bundles of conformers that represent corresponding automatically and interactively determined structures document the absence of significant bias in either approach, indicating that an important step has been made towards automation of structure determination from NMR spectra.     

Resolution and sensitivity of high field nuclear magnetic resonance spectroscopy

NMR frequency assignments are usually considered a prerequisite for the analysis of NOESY spectra, in turn required for the calculation of biomolecular structures. In contrast, as we propose here, relatively high numbers of unambiguous NOE identities can be consistently achieved in an automated manner by relying only on grouping resonances into connected spin systems. To achieve this goal, we have developed for proteins two protocols, SPI and BACUS, based on Bayesian inference. SPI (Grishaev and Llinás, 2002c) produces a list of the (1)H resonance frequencies from homo- and hetero-nuclear multidimensional spectra, grouped into effective spin systems. BACUS automatically establishes probabilistic identities of NOESY cross-peaks in terms of the chemical shifts provided by SPI. BACUS requires neither assignment of resonances nor an initial structural model. It successfully copes with chemical shift overlap and does so without cycling through 3D structure calculations. The method exploits the self-consistency of the NOESY graph by taking advantage of a network of J- as well as NOE-connected "reporter" protons sorted via SPI. BACUS was validated by tests on experimental NOESY data recorded for the col 2 and kringle 2 domains.     

Improved simulation of NOESY spectra by RELAX-JT2 including effects of J-coupling, transverse relaxation and chemical shift anisotropy

RELAX-JT2 is an extension of RELAX, a program for the simulation of 1H 2D NOESY spectra and (15)N or (13)C edited 3D NOESY-HSQC spectra of biological macromolecules. In addition to the already existing NOE-simulation it allows the proper simulation of line shapes by the integrated calculation of T(2) times and multiplet structures caused by J-couplings. Additionally the effects of relaxation mediated by chemical shift anisotropy are taken into account. The new routines have been implemented in the program AUREMOL, which aims at the automated NMR structure determination of proteins in solution. For a manual or automatic assignment of experimental spectra that is based on the comparison with the corresponding simulated spectra, the additional line shape information now available is a valuable aid. The new features have been successfully tested with the histidine-containing phosphocarrier protein HPr from Staphylococcus carnosus.     

Stereochemical structure determination of p-cymene Ru(II) complexes containing the PPh2Py ligand with 2-D NOESY and HMQC NMR experiments

Complexes [Ru(p-cymene)Cl₂(PPh₂Py)] (1) and [Ru(p-cymene)Cl(PPh₂Py)]BF₄ (2) were studied by means of ¹H, ¹³C{¹H} 2-D NOESY and HMQC NMR spectral methods. NMR data agree with C₁ and C_s symmetries for complexes 1 and 2, respectively. NOESY cross-peaks allowed the assignment of signals to CH arene protons and in the case of complex 2 the determination of the molecular stereochemistry. These results are in agreement with the X-ray molecular structures of both complexes.     

Solution structure of thioredoxin h1 from Arabidopsis thaliana

Present in virtually every species, thioredoxins catalyze disulfide/dithiol exchange with various substrate proteins. While the human genome contains a single thioredoxin gene, plant thioredoxins are a complex protein family. A total of 19 different thioredoxin genes in six subfamilies has emerged from analysis of the Arabidopsis thaliana genome. Some function specifically in mitochondrial and chloroplast redox signaling processes, but target substrates for a group of eight thioredoxin proteins comprising the h subfamily are largely uncharacterized. In the course of a structural genomics effort directed at the recently completed A. thaliana genome, we determined the structure of thioredoxin h1 (At3g51030.1) in the oxidized state. The structure, defined by 1637 NMR-derived distance and torsion angle constraints, displays the conserved thioredoxin fold, consisting of a five-stranded beta-sheet flanked by four helices. Redox-dependent chemical shift perturbations mapped primarily to the conserved WCGPC active-site sequence and other nearby residues, but distant regions of the C-terminal helix were also affected by reduction of the active-site disulfide. Comparisons of the oxidized A. thaliana thioredoxin h1 structure with an h-type thioredoxin from poplar in the reduced state revealed structural differences in the C-terminal helix but no major changes in the active site conformation.