Showing papers in "Journal of Biomolecular NMR in 1994"
TL;DR: NMR View provides structure analysis features and data transfer to and from structure generation programs, allowing for a tight coupling between spectral analysis and structure generation.
Abstract: NMR View is a computer program designed for the visualization and analysis of NMR data. It allows the user to interact with a practically unlimited number of 2D, 3D and 4D NMR data files. Any number of spectral windows can be displayed on the screen in any size and location. Automatic peak picking and facilitated peak analysis features are included to aid in the assignment of complex NMR spectra. NMR View provides structure analysis features and data transfer to and from structure generation programs, allowing for a tight coupling between spectral analysis and structure generation. Visual correlation between structures and spectra can be done with the Molecular Data Viewer, a molecular graphics program with bidirectional communication to NMR View. The user interface can be customized and a command language is provided to allow for the automation of various tasks.
2,949 citations
TL;DR: It is shown that by combining both 1H and 13C chemical-shift indices to produce a ‘consensus’ estimate of secondary structure, it is possible to achieve a predictive accuracy in excess of 92%.
Abstract: A simple technique for identifying protein secondary structures through the analysis of backbone 13C chemical shifts is described. It is based on the Chemical-Shift Index [Wishart et al. (1992) Biochemistry, 31, 1647–1651] which was originally developed for the analysis of 1Hα chemical shifts. By extending the Chemical-Shift Index to include 13Cα, 13Cβ and carbonyl 13C chemical shifts, it is now possible to use four independent chemical-shift measurements to identify and locate protein secondary structures. It is shown that by combining both 1H and 13C chemical-shift indices to produce a ‘consensus’ estimate of secondary structure, it is possible to achieve a predictive accuracy in excess of 92%. This suggests that the secondary structure of peptides and proteins can be accurately obtained from 1H and 13C chemical shifts, without recourse to NOE measurements.
2,017 citations
TL;DR: General pulse sequence elements that achieve sensitivity-enhanced coherence transfer from a heteronucleus to protons of arbitrary multiplicity are introduced and incorporated into heteronuclear correlation experiments, in conjunction with coherence selection by the formation of aheteronuclear gradient echo.
Abstract: General pulse sequence elements that achieve sensitivity-enhanced coherence transfer from a heteronucleus to protons of arbitrary multiplicity are introduced. The building blocks are derived from the sensitivity-enhancement scheme introduced by Cavanagh et al. ((1991) J. Magn. Reson., 91, 429-436), which was used in conjunction with gradient coherence selection by Kay et al. ((1992) J. Am. Chem. Soc., 114, 10663-10665), as well as from a multiple-pulse sequence effecting a heteronuclear planar coupling Hamiltonian. The building blocks are incorporated into heteronuclear correlation experiments, in conjunction with coherence selection by the formation of a heteronuclear gradient echo. This allows for efficient water suppression without the need for water presaturation. The methods are demonstrated in HSQC-type experiments on a sample of a decapeptide in H2O. The novel pulse sequence elements can be incorporated into multidimensional experiments.
744 citations
TL;DR: NMR methods which exploit the chemical shift dispersion of the 15N resonances of unfolded states and pulsed field gradient water suppression approaches for avoiding saturation and dephasing of amide protons which rapidly exchange with solvent were utilized for the assignment.
Abstract: The backbone 1H and 15N resonances of the N-terminal SH3 domain of the Drosophila signaling adapter protein, drk, have been assigned. This domain is in slow exchange on the NMR timescale between folded and predominantly unfolded states. Data were collected on both states simultaneously, on samples of the SH3 in near physiological buffer exhibiting an approximately 1:1 ratio of the two states. NMR methods which exploit the chemical shift dispersion of the 15N resonances of unfolded states and pulsed field gradient water suppression approaches for avoiding saturation and dephasing of amide protons which rapidly exchange with solvent were utilized for the assignment.
625 citations
TL;DR: A heteronuclear correlation experiment is described which permits simultaneous characterization of both 15N longitudinal decay rates and slow conformational exchange rates, enabling the extraction of accurate rate constants.
Abstract: A heteronuclear correlation experiment is described which permits simultaneous characterization of both 15N longitudinal decay rates and slow conformational exchange rates. Data pertaining to the exchange between folded and unfolded forms of an SH3 domain is used to illustrate the technique. Because the unfolded form of the molecule, on average, shows significantly higher NH exchange rates than the folded form, and approach which minimizes the degree of water saturation is employed, enabling the extraction of accurate rate constants.
418 citations
TL;DR: Two new methods are described for the measurement of three-bond JHNHαcouplings in proteins isotopically enriched with 15N, suggesting that a break in the ‘central helix’ occurs at the same position as previously observed in solution NMR studies of Ca2+-ligated calmodulin.
Abstract: Two new methods are described for the measurement of three-bond JHNHαcouplings in proteins isotopically enriched with 15N. Both methods leave the water magnetization in an unsaturated state, parallel to the z-axis, and therefore offer significant enhancements in sensitivity for rapidly exchanging backbone amide protons. The J couplings can be measured either from a set of constant-time 2D 1H-15N HMQC spectra, which are modulated in intensity by JHNHα, or from a water-flip-back version of the 3D HNHA experiment. The method is demonstrated for a sample of calcium-free calmodulin. Residues Lys75-Asp80 have JHNHαvalues in the 6–7 Hz range, suggesting that a break in the ‘central helix’ occurs at the same position as previously observed in solution NMR studies of Ca2+-ligated calmodulin.
336 citations
TL;DR: Calculations support the notion that the empirical constant used in earlier studies arises from neighboring peptide contributions in the reference state, and suggest that special values be used for glycine and proline residues, which differ significantly from other residues in their allowed ϕ,ψ-ranges.
Abstract: The contribution of peptide groups to Hα and Hβ proton chemical shifts can be modeled with empirical equations that represent magnetic anisotropy and electrostatic interactions [Osapay, K. and Case, D.A. (1991) J. Am. Chem. Soc., 113, 9436–9444]. Using these, a model for the ‘random coil’ reference state can be generated by averaging a dipeptide over energetically allowed regions of torsion-angle space. Such calculations support the notion that the empirical constant used in earlier studies arises from neighboring peptide contributions in the reference state, and suggest that special values be used for glycine and proline residues, which differ significantly from other residues in their allowed ϕ,ψ-ranges. New constants for these residues are reported that provide significant improvements in predicted backbone shifts. To illustrate how secondary structure affects backbone chemical shifts we report calculations on oligopeptide models for helices, sheets and turns. In addition to suggesting a physical mechanism for the widely recognized average difference between α and β secondary structures, these models suggest several additional regularities that should be expected: (a) Hα protons at the edges of β-sheets will have a two-residue periodicity; (b) the Hα2 and Hα3 protons of glycine residues will exhibit different shifts, particularly in sheets; (c) Hβ protons will also be sensitive to local secondary structure, but in different directions and to a smaller extent than Hα protons; (d) Hα protons in turns will generally be shifted upfield, except those in position 3 of type I turns. Examples of observed shift patterns in several proteins illustrate the application of these ideas.
117 citations
TL;DR: The microdyramical parameters accessible by the ‘model-free’ approach clearly indicate a correlation between the structural flexibility and the tertiary structure of ribonuclease T1, as well as restricted mobility of certain regions of the protein backbone upon binding of the inhibitor.
Abstract: The backbone dynamics of free ribonuclease T1 and its complex with the competitive inhibitor 2′GMP have been studied by 15N longitudinal and transverse relaxation experiments, combined with {1H, 15H} NOE measurements. The intensity decay of individual amide cross peaks in a series of (1H, 15N)-HSQC spectra with appropriate relaxation periods (Kay, L.E. et al. (1989) Biochemistry, 28, 8972–8979; Kay, L.E. et al. (1992) J. Magn. Reson., 97, 359–375) was fitted to a single exponential by using a simplex algorithm in order to obtain 15N T1 and T2 relaxation times. These experimentally obtained values were analysed in terms of the ‘model-free’ approach introduced by Lipari and Szabo (Lipari, G. and Szabo, A. (1982) J. Am. Chem. Soc., 104, 4546–4559; 4559–4570). The microdyramical parameters accessible by this approach clearly indicate a correlation between the structural flexibility and the tertiary structure of ribonuclease T1, as well as restricted mobility of certain regions of the protein backbone upon binding of the inhibitor. The results obtained by NMR are compared to X-ray crystallographic data and to observations made in molecular dynamics simulations.
105 citations
TL;DR: It is shown that the nonlinearly sampled spectrum has a higher resolution, although it was recorded in less time, where the gain in experiment time obtained from nonlinear sampling is used to increase the resolution in the carbonyl dimension.
Abstract: Nonlinear sampling along the constant-time dimension is applied to the constant-time HNCO spectrum of the dimerization domain of Gal4. Nonlinear sampling was used for the nitrogen dimension, while the carbon and proton dimensions were sampled linearly. A conventional ct-HNCO spectrum is compared with a nonlinearly sampled spectrum, where the gain in experiment time obtained from nonlinear sampling is used to increase the resolution in the carbonyl dimension. Nonlinearly sampled data are processed by maximum entropy reconstruction. It is shown that the nonlinearly sampled spectrum has a higher resolution, although it was recorded in less time. The constant intensity of the signal in the constant-time dimension allows for a variety of sampling schedules. A schedule of randomly distributed sampling points yields the best results. This general method can be used to significantly increase the quality of heteronuclear constant-time spectra.
93 citations
TL;DR: There is a significant correlation between 15N chemical shift and secondary structure, and the backbone torsion angles φi, ψi−1 is reported, by using two-dimensional shielding surfaces Δ(φiψ1−1).
Abstract: An empirical correlation between the peptide 15N chemical shift, δ15Ni, and the backbone torsion angles φi, ψi−1 is reported. By using two-dimensional shielding surfaces Δ(φiψ1−1), it is possible in many cases to make reasonably accurate predictions of 15N chemical shifts for a given structure. On average, the rms error between experiment and prediction is about 3.5 ppm. Results for threonine, valine and isoleucine are worse (∼4.8 ppm), due presumably to χ1-distribution/γ-gauche effects. The rms errors for the other amino acids are ∼3 ppm, for a typical maximal chemical shift range of ∼15–20 ppm. Thus, there is a significant correlation between 15N chemical shift and secondary structure.
82 citations
TL;DR: Three experiments are introduced to determine a complete set of coupling constants in RNA oligomers by measuring vicinal proton-proton and carbon-phosphorus couplings that depend on the phosphodiester backbone torsion angles β and ε.
Abstract: Three experiments are introduced to determine a complete set of coupling constants in RNA oligomers. In the HCCH-E.COSY experiment, the vicinal proton-proton coupling constants can be measured with high accuracy. In the P-FIDS-CT-HSQC experiment, vicinal proton-phosphorus and carbon-phosphorus couplings are measured that depend on the phosphodiester backbone torsion angles beta and epsilon. In the refocussed HMBC experiment, vicinal carbon-proton couplings are measured that depend on the glycosidic torsion angle chi.
TL;DR: The results of this study illustrate that, although the difference between FMA and 3′-TFMA linkages is merely in the substitution of the T6(O3′) in the former by a sulfur atom in the latter, the stereoelectronic difference in a single atom can induce significant local structural distortion in an otherwise well-structured oligonucleotide duplex.
Abstract: The DNA·DNA duplex \({\text{d}}\left( {{\text{CGCGTT}}_{{\text{SCH}}_{\text{2}} {\text{O}}} {\text{TTGCGC}}} \right)\)·d(GCGCAAAACGCG) (designated duplex III) containing a 3′-thioformacetal (3′-TFMA) linkage in the center of the sequence was characterized in detail by two- and three-dimensional homonuclear NMR spectroscopy. The NMR results were analyzed and compared with those of two duplexes of the same sequence: One is an unmodified reference sequence and the other contains a formacetal (OCH2O) linkage at the central T^T step (designated duplex I and duplex II, respectively). In general, the NMR spectra of duplex III closely resemble those of the analogous duplexes I and II, suggesting an overall B-type structure adopted by the 3′-TFMA-modified duplex III. Nonetheless, the detection of several distinct spectral features originating from the protons at the \({\text{T6}}_{{\text{3' - SCH}}_{\text{2}} {\text{O}}} {\text{T7}}\) modification site is indicative of a local conformation that is clearly different from the corresponding region in duplexes I and II. The 3′-thioformacetal linker, in contrast to the formacetal (FMA) linkage, cannot be accommodated in a conformation usually found in natural nucleic acid duplexes. As a consequence, the 3′-TFMA-modified T6 sugar adopts an O4′-endo form (an intermediate structure between the usual C2′-endo and C3′-endo forms). This change is accompanied by a change in the e (C4′−C3′−S3′−CH2) dihedral angle and by subsequent adjustments of other torsion angles along the backbone. Notably, this conformational readjustment at the T6–T7 backbone linkage is localized; its collective result has negligible effect on base-base stacking of the T6 and T7 residues. A close examination of the COSY data in all three duplexes reveals a subtle variation in sugar geometry, with more S-type character adopted by the modified duplexes II and III. The results of this study illustrate that, although the difference between FMA and 3′-TFMA linkages is merely in the substitution of the T6(O3′) in the former by a sulfur atom in the latter, the stereoelectronic difference in a single atom can induce significant local structural distortion in an otherwise well-structured oligonucleotide duplex.
TL;DR: It is found that their values at the N-terminal end of a helix provide a good predictor for the presence of a capping box, and these values are mainly associated with the unusually large value of ψ.
Abstract: We have examined the 13Cα and 13Cβ chemical shifts of a number of proteins and found that their values at the N-terminal end of a helix provide a good predictor for the presence of a capping box. A capping box consists of a hydrogen-bonded cycle of four amino acids in which the side chain of the N-cap residue forms a hydrogen bond with the backbone amide of the N3 residue, whose side chain in turn may accept a hydrogen bond from the amide of the N-cap residue. The N-cap residue exhibits characteristic values for its backbone torsion angles, with ϕ and ψ clustering around 94±15° and 167±5°, respectively. This is manifested by a 1–2 ppm upfield shift of the 13Cα resonance and a 1–4 ppm downfield shift of the 13Cβ resonance, relative to their random coil values, and is mainly associated with the unusually large value of ψ. The residues following the N-cap residue exhibit downfield shifts of 1–3 ppm for the 13Cα resonances and small upfield shifts for the 13Cβ ones, typical of an α-helix.
TL;DR: Time and ensemble averaging is extended to the direct refinement with experimental NOE data, and structure refinement with both time- and ensemble-averaged NOE restraints results in lower R-factors, indicating a better fit of the experimental data.
Abstract: NMR data are collected as time- and ensemble-averaged quantities. Yet, in commonly used methods for structure determination of biomolecules, structures are required to satisfy simultaneously a large number of constrainsts. Recently, however, methods have been developed that allow a better fit of the experimental data by the use of time- or ensemble-averaged restraints. Thus far, these methods have been applied to structure refinement using distance and J-coupling restraints. In this paper, time and ensemble averaging is extended to the direct refinement with experimental NOE data. The implementation of time- and ensemble-averaged NOE restraints in DINOSAUR is described and illustrated with experimental NMR data for crambin, a 46-residue protein. Structure refinement with both time- and ensemble-averaged NOE restraints results in lower R-factors, indicating a better fit of the experimental NOE data.
TL;DR: It is shown that the two-bond 1H-15N HSQC experiment can be used on 15N-labeled RNA to correlate the intranucleotide H1′ and H8,H6,H5 resonances indirectly through the shared glycosidic nitrogen.
Abstract: The advent of methods for preparing 15N- and 13C-labeled RNA oligonucleotides holds promise for extending the size of RNA molecules that can be studies by NMR spectroscopy. A practical limitation is the expense of the 13C label. It may therefore sometimes be desirable to prepare a relatively inexpensive 15N-labeled sample only. Here we show that the two-bond 1H-15N HSQC experiment can be used on 15N-labeled RNA to correlate the intranucleotide H1′ and H8,H6,H5 resonances indirectly through the shared glycosidic nitrogen. The nonrefocused version of a standard HSQC experiment for 2D proton-detected 1H-15N chemical-shift correlation is applied in order to minimize the sensitivity loss due to the relatively fast spin-spin relaxation of RNA oligonucleotides. The experiment is applied to the 30-nucleotide RNA RBE3 which contains the high-affinity binding site of the RRE (rev response element) for the Rev protein of HIV. The results indicate that this simple experiment allows a straightforward identification of the base proton resonances CH5, CH6, UH5, UH6, purine H8, and AH2 as well as the intranucleotide H1′ and H8,H6,H5 connectivities. When combined with a NOESY experiment, complete sequential assignments can be obtained.
TL;DR: In this paper, the presence of four-bond sequential HN-Hα J-couplings in the protein staphylococcal nuclease was unambiguously established.
Abstract: Quantitative J-correlation and triple-resonance ECOSY-type experiments are used to unambiguously establish the presence of four-bond sequential HN-Hα J-couplings in the protein staphylococcal nuclease Substantially negative \({}^{\text{4}}{\text{J}}_{{\text{H}}^\alpha {\text{H}}^{\text{N}} } \) values, ranging from -08 to -23 Hz, are observed when the Ψ angle is near +120°, and the following ϕ angle near +60° For other conformations, the four-bond HN-Hα J-couplings fall between -05 and +05 Hz
TL;DR: Comparison of this structure with structures of Cyp-CsA and other Cyp-peptide complexes determined by different approaches shows extensive similarities.
Abstract: The three-dimensional NMR solution structure of the cyclophilin A (Cyp)-cyclosporin A (CsA) complex was determined, and here we provide a detailed description of the analysis of the NMR data and the structure calculation. Using 15N-and 13C-resolved three- and four-dimensional [1H, 1H]-nuclear Overhauser enhancement (NOE) spectroscopy with uniformly isotope-labeled Cyp in the complex, a final data set of 1810 intra-Cyp, 107 intra-CsA and 63 intermolecular NOE upper distance constraints was collected as input for the structure calculation with the program DIANA. A group of DIANA conformers, selected by a previously described analysis of the dependence of the maximal root-mean-square deviation (rmsd) among the individual conformers on the residual target function value, was subjected to energy refinement with the program FANTOM. The 22 best energy-refined conformers were then used to represent the solution structure. The average rmsd relative to the mean structure of these 22 conformers is 1.1 A for the backbone atoms of all residues of the complex. The molecular architecture of Cyp in the Cyp-CsA complex includes an eight-stranded antiparallel β-barrel, which is closed on each side by an amphipathic helix. CsA is bound in a cavity formed by part of the barrel surface and four loops with nonregular secondary structure. Comparison of this structure with structures of Cyp-CsA and other Cyp-peptide complexes determined by different approaches shows extensive similarities.
TL;DR: Model studies on the macrocyclic immunosuppressive agent FK506 challenge traditional approaches to defining a structure from data collected during a 2D NMR experiment, and it is clear that a greater emphasis is required on dynamics and on the fact that an NMR structure represents a time average.
Abstract: Model studies on the macrocyclic immunosuppressive agent FK506 challenge traditional approaches to defining a structure from data collected during a 2D NMR experiment. A variety of joint molecular dynamics/NMR-distance refinement methodologies are characterized. From the results it is clear that the traditional presentation of an NMR structure as a single representative minimized conformation or as a fairly tight envelope of conformers best meeting the imposed restraints can be misleading; a greater emphasis is required on dynamics and on the fact that an NMR structure represents a time average.
TL;DR: Sensitive three-dimensional NMR experiments, based on the E.COSY principle, are presented for the measurement of the 3J(HN,Hα) and3J( HN,C′) coupling constants in uniformly 13C- and 15N-labeled proteins.
Abstract: Sensitive three-dimensional NMR experiments, based on the E.COSY principle, are presented for the measurement of the 3J(HN,Hα) and 3J(HN,C′) coupling constants in uniformly 13C- and 15N-labeled proteins. They employ gradient coherence selection in combination with the sensitivity enhancement method in HSQC-type spectra (Cavanagh et al., 1991; Palmer et al., 1991). In most cases, the two measured coupling constants unambiguously define the ϕ-angle for protein structure determination. The method is applied to uniformly 13C, 15N-labeled ribonuclease T1.
TL;DR: A protocol based on the computer-based assignment of the NMR signals, identification of distance restraints from nuclear Overhauser effects, and generation of 3D structures by using the N MR-derived restraints may yield an approximate order of magnitude reduction in the time required for the generation of3D structures of proteins from NMR data.
Abstract: A strategy is presented for the semiautomated assignment and 3D structure determination of proteins from heteronuclear multidimensional Nuclear Magnetic Resonance (NMR) data. This approach involves the computer-based assignment of the NMR signals, identification of distance restraints from nuclear Overhauser effects, and generation of 3D structures by using the NMR-derived restraints. The protocol is described in detail and illustrated on a resonance assignment and structure determination of the FK506 binding protein (FKBP, 107 amino acids) complexed to the immunosuppressant, ascomycin. The 3D structures produced from this automated protocol attained backbone and heavy atom rmsd of 1.17 and 1.69 A, respectively. Although more highly resolved structures of the complex have been obtained by standard interpretation of NMR data (Meadows et al. (1993) Biochemistry, 32, 754–765), the structures generated with this automated protocol required minimal manual intervention during the spectral assignment and 3D structure calculations stages. Thus, the protocol may yield an approximate order of magnitude reduction in the time required for the generation of 3D structures of proteins from NMR data.
TL;DR: Two-dimensional versions of HNCA and HNCO experiments are described, which provide essentially the same information as the 3D sequence, but with different information content.
Abstract: Two-dimensional versions of HNCA and HNCO experiments are described, which provide essentially the same information as the 3D sequence. A multiple-quantum coherence involving either 15N and 13C alpha or 15N and 13CO is created. One of the two frequencies is given by the middle point between the two cross peaks (zero- and double-quantum) and the other by their separation. Quadrature detection can be performed on either nucleus, modifying only the appearance of the 2D spectrum, but not the information content. These experiments, named MQ-HNCA and MQ-HNCO, are illustrated on a (15N, 13C) doubly labelled cytochrome c2 from Rhodobacter capsulatus (116 amino acids).
TL;DR: Comparison of the different referencing methods for 13C chemical shifts in organic cosolvent mixtures showed that an external standard (either TMS or TSP capillary) was the most appropriate.
Abstract: The 13C chemical shifts for all of the protonated carbons of the 20 common amino acid residues in the protected linear pentapeptide Gly-Gly-X-Gly-Gly have been obtained in water at low pH as well as in aqueous solution containing 10, 20 and 30% acetonitrile or trifluoroethanol. Dioxane was used as an internal reference and its carbon chemical shift value was found to be 66.6 ppm relative to external TMS in water. Comparison of the different referencing methods for 13C chemical shifts in organic cosolvent mixtures showed that an external standard (either TMS or TSP capillary) was the most appropriate. In the present study, external TSP was chosen to define the 0 ppm of the 13C chemical shift scale. When the difference in referencing the dioxane carbon resonance is taken into account, the carbon chemical shift values of the amino acids in aqueous solution are similar to those previously reported (Richarz and Wuthrich (1978) Biopolymers, 17, 2133–2141; Howarth and Lilley (1979) Prog. NMR Spectrosc., 12, 1–40). The pentapeptides studied were assumed to be in a random coil conformation and the measured 13C chemical shifts were used as reference values to correlate carbon chemical shifts with the secondary structure of two well-characterized peptides, bombesin and the 1–29 amino acid fragment of Nle27 human growth hormone-releasing factor. In both cases, the Cα chemical shifts exhibited a characteristic positive deviation from the random coil values, which indicates the presence of α-helices.
TL;DR: In this paper, simple 3D experiments are used to provide J correlations between methionine Ce methyl carbons and either the CγH2 protons or Cβ and Cγ.
Abstract: Several simple 3D experiments are used to provide J correlations between methionine Ce methyl carbons and either the CγH2 protons or Cβ and Cγ. The intensity of the J correlations provides information on the size of the three-bond J couplings and thereby on the χ3 torsion angle. In addition, a simple 3D version of the HMBC experiment provides a sensitive link between the CeH3 methyl protons and Cγ. The methods are demonstrated for a 20 kDa complex between calmodulin and a 26-residue peptide fragment of skeletal muscle myosin light chain kinase.
TL;DR: Simulated neural networks are described which aid the assignment of protein NMR spectra by being trained to recognize amino acid type from TOCSY data and tested on spin systems from spinach ACP, which has a 37% sequence homology with E. coli ACP.
Abstract: Simulated neural networks are described which aid the assignment of protein NMR spectra. A network trained to recognize amino acid type from TOCSY data was trained on 148 assigned spin systems from E. coli acyl carrier proteins (ACPs) and tested on spin systems from spinach ACP, which has a 37% sequence homology with E. coli ACP and a similar secondary structure. The output unit corresponding to the correct amino acid is one of the four most activated units in 83% of the spin systems tested. The utility of this information is illustrated by a second network which uses a constraint satisfaction algorithm to find the best fit of the spin systems to the amino acid sequence. Application to a stretch of 20 amino acids in spinach ACP results in 75% correct sequential assignment. Since the output of the amino acid type identification network can be coupled with a variety of sequential assignment strategies, the approach offers substantial potential for expediting assignment of protein NMR spectra.
TL;DR: An automated approach for determining the sequential order of amino acid spin systems in small proteins through the analysis of multidimensional HCC(CO)NH-TOCSY spectra, which provides sufficient constraints to assign most proton and nitrogen resonances of small proteins.
Abstract: We have developed an automated approach for determining the sequential order of amino acid spin systems in small proteins. A key step in this procedure is the analysis of multidimensional HCC(CO)NH-TOCSY spectra that provide connections from the aliphatic resonances of residue i to the amide resonances of residue i+1. These data, combined with information about the amino acid spin systems, provide sufficient constraints to assign most proton and nitrogen resonances of small proteins. Constraint propagation methods progressively narrow the set of possible assignments of amino acid spin systems to sequence-specific positions in the process of NMR data analysis. The constraint satisfaction paradigm provides a framework in which the necessary constraint-based reasoning can be expressed, while an object-oriented representation structures and facilitates the extensive list processing and indexing involved in matching. A prototype expert system, AUTOASSIGN, provides correct and nearly complete resonance assignments with one real and 31 simulated 3D NMR data sets for a 72-amino acid domain, derived from the Protein A of Staphylococcus aureus, and with 31 simulated NMR data sets for the 50-amino acid human type-α transforming growth factor.
TL;DR: Heteronuclear 2D 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.
Abstract: 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.
TL;DR: In this article, a suite of macros that run under a modified version of the FELIX 1.0 program is used to determine the 1HN, 15N, 13Cα and 13Cβ chemical shift assignments of protein residues with minimal human intervention.
Abstract: A computer algorithm that determines the 1HN, 15N, 13Cα, 1Hα, 13Cβ chemical-shift assignments of protein residues with minimal human intervention is described. The algorithm is implemented as a suite of macros that run under a modified version of the FELIX 1.0 program (Hare Research, Bothell, WA). The input to the algorithm is obtained from six multidimensional, triple-resonance experiments: 3D HNCACB, 3D CBCA(CO)HN, 4D HNCAHA, 4D HN(CO)CAHA, 3D HBHA(CO)NH and 3D HNHA(Gly). For small proteins, the two 4D spectra can be replaced by either the 3D HN(CA)HA, 3D H(CA)NNH, or the 15N-edited TOCSY-HSQC experiments. The algorithm begins by identifying and collecting the intraresidue and sequential resonances of the backbone and 13Cβ atoms into groups. These groups are sequentially linked and then assigned to residues by matching the 13Cα and 13Cβ chemical-shift profiles of the linked groups to that of the protein's primary structure. A major strength of the algorithm is its ability to overcome imperfect data, e.g., missing or overlapping peaks. The viability of the procedure is demonstrated with two test cases. In the first, NMR data from the six experiments listed above were used to reassign the backbone resonances of the 93-residue human hnRNP C RNA-binding domain. In the second, a simulated cross-peak list, generated from the published NMR assignments of calmodulin, was used to test the ability of the algorithm to assign the backbone resonances of proteins containing internally homologous segments. Finally, the automated method was used to assign the backbone resonances of apokedarcidin, a previously unassigned, 114-residue protein.
TL;DR: A marked dependence of the 1H resonances of TSP and DSS (internal standards) on the concentration of proteins in the molten globule state has been found and indicates that TSPand DSS interact with these proteins.
Abstract: A marked dependence of the (1)H resonances of TSP and DSS (internal standards) on the concentration of proteins in the molten globule state has been found. This result indicates that TSP and DSS interact with these proteins. Therefore, when the chemical shift is used as an indicator of the residual structure of proteins in the molten globule state, great care must be taken in using TSP and DSS.
TL;DR: The nucleocapsid protein of Moloney murine leukemia virus (NCp10) is a 56-amino acid protein which contains one zinc finger of the CysX2CysX4HisX4Cys form, a highly conserved motif present in most retroviruses and retroelements.
Abstract: The nucleocapsid protein of Moloney murine leukemia virus (NCp10) is a 56-amino acid protein which contains one zinc finger of the CysX2CysX4HisX4Cys form, a highly conserved motif present in most retroviruses and retroelements. At pH≥5, NCp10 binds one zinc atom and the complexation induces a folding of the CysX2CysX4HisX4Cys box, similar to that observed for the zinc-binding domains of HIV-1 NC protein. The three-dimensional structure of NCp10 has been determined in aqueous solution by 600 MHz 1H NMR spectroscopy. The proton resonances could be almost completely assigned by means of phase-sensitive double-quantum-filtered COSY, TOCSY and NOESY techniques. NOESY spectra yielded 597 relevant structural constraints, which were used as input for distance geometry calculations with DIANA. Further refinement was performed by minimization with the program AMBER, which was modified by introducing a zinc force field. The solution structure is characterized by a well-defined central zinc finger (rmsd of 0.747±0.209 A for backbone atoms and 1.709±0.187 A when all atoms are considered), surrounded by flexible N- and C-terminal domains. The Tyr28, Trp35, Lys37, Lys41 and Lys42 residues, which are essential for activity, lie on the same face of the zinc finger, forming a bulge structure probably involved in viral RNA binding. The significance of these structural characteristics for the various biological functions of the protein is discussed, taking into account the results obtained with various mutants.
TL;DR: A ‘best first’ approach, in which high-scoring local assignments are made before and in preference to lower scoring assignments, is shown to be superior (in terms of the current set of CONTRAST scoring routines) to approaches such as simulated annealing that seek to maximize the combined scores of the individual assignments.
Abstract: The peptide sequential assignment algorithm presented here was implemented as a macro within the CONnectivity TRacing ASsignment Tools (CONTRAST) computer software package. The algorithm provides a semi- or fully automated global means of sequentially assigning the NMR backbone resonances of proteins. The program's performance is demonstrated here by its analysis of realistic computer-generated data for IIIGlc, a 168-residue signal-transducing protein of Escherichia coli [Pelton et al. (1991) Biochemistry, 30, 10043–10057]. Missing experimental data (19 resonances) were generated so that a complete assignment set could be tested. The algorithm produces sequential assignments from appropriate peak lists of nD NMR data. It quantifies the ambiguity of each assignment and provides ranked alternatives. A ‘best first’ approach, in which high-scoring local assignments are made before and in preference to lower scoring assignments, is shown to be superior (in terms of the current set of CONTRAST scoring routines) to approaches such as simulated annealing that seek to maximize the combined scores of the individual assignments. The robustness of the algorithm was tested by evaluating the effects of imposed frequency imprecision (scatter), added false signals (noise), missing peaks (incomplete data), and variation in userdefined tolerances on the performance of the algorithm.