RESUMEN
Nuclear magnetic resonance spectroscopy (NMR) probes using thin-film high temperature superconducting (HTS) resonators provide exceptional mass sensitivity in small-sample NMR experiments for natural products chemistry and metabolomics. We report improvements in sensitivity to our 1.5 mm 13C-optimized NMR probe based on HTS resonators. The probe has a sample volume of 35 microliters and operates in a 14.1 T magnet. The probe also features HTS resonators for 1H transmission and detection and the 2H lock. The probe utilizes a 13C resonator design that provides greater efficiency than our previous design. The quality factor of the new resonator in the 14.1 T background field was measured to be 4,300, which is over 3x the value of the previous design. To effectively implement the improved quality factor, we demonstrate the effect of adding a shorted transmission line stub to increase the bandwidth and reduce the rise/fall time of 13C irradiation pulses. Initial NMR measurements verify 13C NMR sensitivity is significantly improved while preserving detection bandwidth. The probe will be used for applications in metabolomics.
RESUMEN
We present the design of a novel high-temperature superconductor double-sided racetrack resonator for a 13C optimized nuclear magnetic resonance (NMR) transmitter/receiver coil. The coils operate in a 21.1 T magnet and accommodate a 3 mm × 6.2 mm cross-section rectangular sample tube. The design includes the incorporation of revised finger lengths to improve the homogeneity of current density across the fingers, a new laser trimming approach for adjusting the resonance frequency, and improved ability to shift higher-order modes for suitability in 1H/13C NMR probes. Resonator design methodology, simulations and experimental results are presented.
RESUMEN
Nuclear magnetic resonance (NMR) probes using thin-film HTS coils offer high sensitivity and are particularly suitable for small-sample applications. Typically, HTS probes are optimized for the detection of multiple nuclei and require several coils to be located within a small volume near the sample. Coupling between the coils shifts coil resonances and complicates coil trimming when tuning HTS probes. We have modeled the magnetic coupling between the coils of a 1.5-mm all-HTS NMR probe with 13C, 1H, and 2H channels. By measuring the magnetic coupling coefficients between individual coils, we solve the general coupling matrix given by KVL for six coupled resonators. Our results indicate that required trims can be accurately predicted by applying single coil trimming simulations to this magnetic coupling model. Use of the magnetic coupling model significantly improves the efficiency of tuning HTS probes.
RESUMEN
Source attribution studies report that the consumption of contaminated poultry is the primary source for acquiring human campylobacteriosis. Oral administration of an engineered Escherichia coli strain expressing the Campylobacter jejuni N-glycan reduces bacterial colonization in specific-pathogen-free leghorn chickens, but only a fraction of birds respond to vaccination. Optimization of the vaccine for commercial broiler chickens has great potential to prevent the entry of the pathogen into the food chain. Here, we tested the same vaccination approach in broiler chickens and observed similar efficacies in pathogen load reduction, stimulation of the host IgY response, the lack of C. jejuni resistance development, uniformity in microbial gut composition, and the bimodal response to treatment. Gut microbiota analysis of leghorn and broiler vaccine responders identified one member of Clostridiales cluster XIVa, Anaerosporobacter mobilis, that was significantly more abundant in responder birds. In broiler chickens, coadministration of the live vaccine with A. mobilis or Lactobacillus reuteri, a commonly used probiotic, resulted in increased vaccine efficacy, antibody responses, and weight gain. To investigate whether the responder-nonresponder effect was due to the selection of a C. jejuni "supercolonizer mutant" with altered phase-variable genes, we analyzed all poly(G)-containing loci of the input strain compared to nonresponder colony isolates and found no evidence of phase state selection. However, untargeted nuclear magnetic resonance (NMR)-based metabolomics identified a potential biomarker negatively correlated with C. jejuni colonization levels that is possibly linked to increased microbial diversity in this subgroup. The comprehensive methods used to examine the bimodality of the vaccine response provide several opportunities to improve the C. jejuni vaccine and the efficacy of any vaccination strategy.IMPORTANCECampylobacter jejuni is a common cause of human diarrheal disease worldwide and is listed by the World Health Organization as a high-priority pathogen. C. jejuni infection typically occurs through the ingestion of contaminated chicken meat, so many efforts are targeted at reducing C. jejuni levels at the source. We previously developed a vaccine that reduces C. jejuni levels in egg-laying chickens. In this study, we improved vaccine performance in meat birds by supplementing the vaccine with probiotics. In addition, we demonstrated that C. jejuni colonization levels in chickens are negatively correlated with the abundance of clostridia, another group of common gut microbes. We describe new methods for vaccine optimization that will assist in improving the C. jejuni vaccine and other vaccines under development.
Asunto(s)
Vacunas Bacterianas/farmacología , Infecciones por Campylobacter/veterinaria , Campylobacter jejuni/inmunología , Pollos , Polisacáridos/inmunología , Enfermedades de las Aves de Corral/prevención & control , Probióticos/farmacología , Administración Oral , Animales , Vacunas Bacterianas/administración & dosificación , Infecciones por Campylobacter/prevención & control , Escherichia coli/genética , Microorganismos Modificados Genéticamente , Polisacáridos/administración & dosificación , Probióticos/administración & dosificación , Organismos Libres de Patógenos EspecíficosRESUMEN
In order to increase the throughput of high-resolution nuclear magnetic resonance spectroscopy a multiple-coil probe, which enables the simultaneous analysis of eight different samples, was designed. The probe, consisting of eight identical solenoidal coils, was constructed for operation at 600 MHz. By using four receivers and radiofrequency switches, spectra from eight different chemical solutions were acquired in the time normally required for one. Two-dimensional COSY, gradient COSY, and TOCSY data have been acquired. Intercoil electrical isolation was between 25 and 45 dB, with signal cross-talk between approximately 1 and 5% measured by NMR. The spectral linewidths for the eight coils were between 3 and 6Hz for a single optimized shim setting.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular/instrumentación , Diseño de Equipo , Procesamiento de Señales Asistido por Computador/instrumentaciónRESUMEN
The melanocortin receptors are G-protein coupled receptors (GPCRs) that activate the cAMP signal transduction pathway and are stimulated by the melanocortin agonist alpha-melanocyte stimulating hormone (alpha-MSH). Members of these melanocortin receptors are antagonized by agouti (ASP) and agouti-related protein (AGRP), which are the only known endogenous antagonists of GPCRs identified to date. Structure-function studies of the hAGRP(109-118) decapeptide, Tyr-c[Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys]-Tyr-NH(2), by replacing the 26-membered disulfide Cys(2)-Cys(9) ring with lactam bridges resulted in the identification of a novel peripheral skin melanocortin-1 receptor (MC1R) antagonist. This antagonist, Tyr-c[Glu-Arg-Phe-Phe-Asn-Ala-Phe-Dpr]-Tyr-NH(2), possesses a 27-membered ring with the lactam bridge being formed from the Calpha-carboxyl moiety of Glu (instead of the typical side chain carboxyl moiety) with the amine of the diaminopropionic acid (Dpr) residue. This mouse MC1 receptor antagonist (pA(2) = 5.9) is also an antagonist at the brain melanocortin-4 receptor (pA(2) = 6.9), with no observable pharmacology at the melanocortin-3 or -5 receptors. This MC1R hAGRP(109-118) based decapeptide is novel in that AGRP(83-132) itself does not bind to, agonize, or antagonize the skin MC1R. Structural analysis has been performed using two-dimensional (1)H NMR and computer-assisted molecular modeling (CAMM) techniques in attempts to identify structural features of this Tyr-c[Glu-Arg-Phe-Phe-Asn-Ala-Phe-Dpr]-Tyr-NH(2) (cyclo Glu alphaCOOH-Dpr betaNH) peptide that can differentially result in antagonist versus agonist properties at the mMC1R.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular , Lactamas/síntesis química , Péptidos Cíclicos/síntesis química , Proteínas/química , Receptor de Melanocortina Tipo 3 , Receptores de Corticotropina/antagonistas & inhibidores , Proteína de Señalización Agouti , Proteína Relacionada con Agouti , Secuencia de Aminoácidos , Animales , Línea Celular , Humanos , Lactamas/química , Lactamas/farmacología , Espectroscopía de Resonancia Magnética , Ratones , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Receptores de Corticotropina/agonistas , Receptores de Melanocortina , Piel/química , Relación Estructura-Actividad , TransfecciónRESUMEN
The snail Lymnaea stagnalis produces a neuropeptide precursor protein that contains seven Arg-Gly-Asp (RGD) sites. These sites are recognized and cleaved by one or more prohormone convertases in the first processing step to yield mature neuropeptides in the secretory pathway. Conformations of two synthetic RGD-containing peptides derived from the L. stagnalis precursor protein were determined by NMR spectroscopy. The peptides were tested in a platelet aggregation assay for RGD activity and were processed in vitro by PC2 and furin. The native peptide with a proline following the RGD site has minimal structure around the RGD region, does not inhibit platelet aggregation, and is properly processed by the enzymes PC2 and furin. A variant of the native fragment with a serine following the RGD sequence has a significant amount of a reverse turn around the RGD region, is a potent inhibitor of platelet aggregation, and is processed with the same specificity as the native fragment. The large conformational differences between the two peptides provide a molecular mechanism for effects of proline residues following the RGD site and suggest that precursor processing is influenced more by flexibility than by the conformation of the processing site.
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Endopeptidasas/metabolismo , Neuropéptidos/química , Oligopéptidos/química , Oligopéptidos/metabolismo , Precursores de Proteínas/química , Secuencia de Aminoácidos , Animales , Furina , Humanos , Hidrólisis , Lymnaea , Datos de Secuencia Molecular , Neuropéptidos/metabolismo , Resonancia Magnética Nuclear Biomolecular , Inhibidores de Agregación Plaquetaria/química , Inhibidores de Agregación Plaquetaria/metabolismo , Conformación Proteica , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Relación Estructura-Actividad , Subtilisinas/metabolismoRESUMEN
We recently identified conformational changes that occur upon phosphorylation of myristoylated alanine-rich protein kinase C substrate (MARCKS) that preclude efficient cross-linking of actin filaments (Bubb, M. R., Lenox, R. H., and Edison, A. S. (1999) J. Biol. Chem. 274, 36472-36478). These results implied that the phosphorylation site domain of MARCKS has two actin-binding sites. We now present evidence for the existence of two actin-binding sites that not only mutually compete but also specifically compete with the actin-binding proteins thymosin beta(4) and actobindin to bind to actin. The effects of substitution of alanine for phenylalanine within a repeated hexapeptide segment suggest that the noncharged region of the domain contributes to binding affinity, but the binding affinity of peptides corresponding to each binding site has a steep dependence on salt concentration, consistent with presumed electrostatic interactions between these polycationic peptides and the polyanionic N terminus of actin. Phosphorylation decreases the site-specific affinity by no more than 0.7 kcal/mol, which is less than the effect of alanine substitution. However, phosphorylation has a much greater effect than alanine substitution on the loss of actin filament cross-linking activity. These results are consistent with the hypothesis that the compact structure resulting from conformational changes due to phosphorylation, in addition to modest decreases in site-specific affinity, explains the loss of cross-linking activity in phosphorylated MARCKS.
Asunto(s)
Actinas/metabolismo , Proteínas Contráctiles , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Unión Competitiva , Proteínas Portadoras/metabolismo , Proteínas de Microfilamentos/metabolismo , Datos de Secuencia Molecular , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fosforilación , Profilinas , Proteínas/química , Proteínas Protozoarias , Conejos , Timosina/metabolismoRESUMEN
The primary structure of the Streptococcus mutans lantibiotic mutacin 1140 was elucidated by NMR spectroscopy, mass spectrometry, and chemical sequencing. The structure is in agreement with other closely related lantibiotics, such as epidermin. A novel method was developed in which mutacin 1140 was chemically modified with sodium borohydride followed by ethanethiol, allowing the differentiation of the thioether-containing residues from the dehydrated residues. This double-labeling strategy provides a simple method to reliably identify all modified lantibiotic residues with a minimal amount of material. While NMR spectroscopy is still required to obtain thioether bridging patterns and thus the complete covalent structure, the double-labeling technique, along with mass spectrometry, provides most of the information in a fraction of the time required for a complete NMR analysis. Thus, with these new techniques lantibiotics can be rapidly characterized.
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Antibacterianos/química , Bacteriocinas/química , Bioquímica/métodos , Péptidos , Secuencia de Aminoácidos , Borohidruros/química , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Datos de Secuencia Molecular , Compuestos de Sulfhidrilo/químicaRESUMEN
Conformational states and thermodynamic properties for two similar neuropeptides, GDPFLRF-NH(2) and GYPFLRF-NH(2), have been computed by Monte Carlo simulated annealing (MCSA) conformational searches and Metropolis Monte Carlo (MMC) calculations. These peptides were recently shown to have dramatically different conformations in solution by NMR [Edison et al., J Neuroscience 1999;19:6318-6326]. Final conformations of multiple independent MCSA runs were the starting points for MMC calculations, and conformations saved at intervals during MMC runs were characterized in terms of total energy, configuration entropy, side-chain fraction population, and ensemble average inter-nuclear distances. Without the use of any NMR data-generated pseudo-potentials, the present calculations were in excellent qualitative agreement with all previous NMR experimental data and provided a foundation by which to more quantitatively interpret the experimental NMR results. Proteins 2000;40:367-377.
Asunto(s)
FMRFamida/análogos & derivados , Neuropéptidos/química , Oligopéptidos/química , Simulación por Computador , Modelos Moleculares , Modelos Teóricos , Conformación Molecular , Resonancia Magnética Nuclear Biomolecular , TermodinámicaRESUMEN
FMRFamide-like peptide (FLP) amino acid sequences have been collected and statistically analyzed. FLP amino acid composition as a function of position in the peptide is graphically presented for several major phyla. Results of total amino acid composition and frequencies of pairs of FLP amino acids have been computed and compared with corresponding values from the entire GenBank protein sequence database. The data for pairwise distributions of amino acids should help in future structure-function studies of FLPs. To aid in future peptide discovery, a computer program and search protocol was developed to identify FLPs from the GenBank protein database without the use of keywords.
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Sistemas de Administración de Bases de Datos , FMRFamida/química , Análisis de Secuencia de Proteína/métodos , Análisis de Secuencia de Proteína/estadística & datos numéricos , Secuencia de Aminoácidos , Animales , Humanos , Datos de Secuencia Molecular , Neuropéptidos/químicaRESUMEN
Phosphorylation of myristoylated alanine-rich protein kinase C substrate (MARCKS) by protein kinase C eliminates actin filament cross-linking activity, but residual filament binding activity docks phosphorylated MARCKS on filamentous actin. The postulated actin-binding region of MARCKS, which includes a Ca(2+)-calmodulin-binding site, has been portrayed with alpha-helical structure, analogous to other calmodulin-binding domains. Previous speculation suggested that MARCKS may dimerize to form the two functional actin-binding sites requisite for cross-linking activity. Contrary to these hypotheses, we show that MARCKS peptide with actin-cross-linking activity has an extended structure in aqueous solution but assumes a more compact structure upon phosphorylation. We hypothesize that structural changes in the MARCKS peptide induced by phosphorylation create a dynamic structure that, on average, has only one actin-binding site. Moreover, independent of the state of phosphorylation, this peptide is monomeric rather than dimeric, implying that two distinct actin-binding sites are responsible for the actin-cross-linking activity of unphosphorylated MARCKS. These studies uniquely elucidate the mechanism by which phosphorylation of MARCKS induces structural changes and suggest how these structural changes determine biological activity.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Conformación Proteica , Proteína Quinasa C/metabolismo , Proteínas/química , Proteínas/metabolismo , Secuencia de Aminoácidos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Fosforilación , Unión Proteica , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Conformational properties of several similar FMRFamide-like neuropeptides from mollusks were investigated by nuclear magnetic resonance (NMR) spectroscopy. It was found that amino acid substitutions in the N-terminal variable regions of the peptides had dramatic effects on the populations of reverse turns in solution. The populations of turns, as measured by two independent NMR parameters, were found to be highly correlated (r(2) = 0.93 and 0. 82) with IC(50) values using receptor membrane preparations from Helix aspersa (Payza, 1987; Payza et al., 1989). These results suggest that the amount of turn in the free peptide can influence the receptor binding affinities of that peptide. On the basis of these observations, a model was developed in which only a single species from a conformational ensemble of an unbound peptide will bind to a particular receptor. Thus, the conformational ensemble reduces the effective concentration of a particular peptide with respect to a particular receptor.
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Neuropéptidos/química , Neuropéptidos/fisiología , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos/genética , Sustitución de Aminoácidos , Animales , FMRFamida/química , Caracoles Helix/metabolismo , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Modelos Químicos , Conformación MolecularRESUMEN
In earlier work, we [McCormick, K. A., et al. (1993) J. Biol. Chem. 268, 24683-24691] observed that mutations at Ala-79 of the b subunit affect assembly of F1F0 ATP synthase. Polypeptides modeled on the soluble portion of the b subunit (bsol) with substitutions at the position corresponding to Ala-79 have been used to investigate secondary structure and dimerization of the b subunit. Circular dichroism spectra and chymotrypsin digestion experiments suggested that the recombinant polypeptides with Ala-79 substitutions assumed conformations similar to the bsol polypeptide. However, cross-linking studies of the Ala-79 substitution bsol polypeptides revealed defects in dimerization. The efficiency of dimer formation appeared to be related to the capacity of the altered bsol polypeptides for competing with F1-ATPase for binding to F1-depleted membrane vesicles. Ala-79 substitution polypeptides displaying limited dimerization, such as bsol Ala-79-->Leu, were shown to elute with F1-ATPase during size exclusion chromatography, suggesting a specific interaction. Sedimentation equilibrium studies indicated that 8% of the bsol Ala-79-->Leu polypeptide was in the form of a 30.6 kDa dimer and 92% a 15.3 kDa monomer. When the dimer concentration of bsol Ala-79-->Leu was normalized to the concentration of bsol, both had virtually identical capacities for competing with F1-depleted membrane vesicles for binding F1-ATPase. The result indicated that the amount of dimer formed is directly proportional to its ability to bind F1-ATPase. This suggests that formation of the b subunit dimer may be a necessary step preceding F1-ATPase binding in the assembly of the enzyme complex.
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Escherichia coli/enzimología , ATPasas de Translocación de Protón/metabolismo , Sustitución de Aminoácidos/genética , Dimerización , Escherichia coli/genética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Peso Molecular , Péptidos/síntesis química , Péptidos/química , Péptidos/genética , Unión Proteica/genética , ATPasas de Translocación de Protón/antagonistas & inhibidores , ATPasas de Translocación de Protón/genética , Proteínas Recombinantes/síntesis química , Proteínas Recombinantes/químicaRESUMEN
We have identified a gene, afp-1, that encodes a new subfamily of six FMRFamide-like neuropeptides in the nematode Ascaris suum. The predicted peptides share the C-terminal sequence PGVLRF-NH2 but have different N-terminal extensions. We discuss possible functional roles of these different peptides based upon experiments with Ascaris as well as results from other organisms. Three of the peptides were previously isolated from extracts of A. suum (4) and three other are novel sequences. The translated product of afp-1 is a precursor protein containing two main halves: a C-terminal region containing a series of putative peptides separated by characteristic processing sites and a relatively hydrophobic N-terminal region with no obvious peptides. Although the overall structure of the translated product of afp-1 is similar to flp-1 from C. elegans (18), there is little evidence for homology between the two nematode neuropeptide genes. At least four different transcripts of afp-1 have been identified. These transcripts differ in their 3' and 5' untranslated regions, and one of the transcripts predicts a truncated precursor protein which contains only the C-terminal peptide-containing region.
Asunto(s)
Ascaris suum/genética , FMRFamida/análogos & derivados , FMRFamida/genética , Genes de Helminto , Neuropéptidos/genética , Secuencia de Aminoácidos , Animales , Ascaris suum/fisiología , Secuencia de Bases , Caenorhabditis/genética , Cartilla de ADN/genética , ADN Complementario/genética , ADN de Helmintos/genética , Evolución Molecular , FMRFamida/fisiología , Datos de Secuencia Molecular , Neuropéptidos/clasificación , Neuropéptidos/fisiología , Reacción en Cadena de la Polimerasa , Especificidad de la Especie , Transcripción GenéticaRESUMEN
4(S)-Hydroxyproline (Hyp) residues constitute about 10% of most forms of collagen, the most abundant protein in vertebrates. X-Ray diffraction analysis was used to ascertain how the structure of proline residues is affected by the inductive effect elicited by the hydroxyl group of Hyp residues. N-Acetylproline methylester (1), N-acetyl-4(S)-hydroxyproline methylester (2) and N-acetyl-4(S)- fluoroproline methylester (3) were synthesized, and their crystalline structures were determined at high resolution. The amide bond of crystalline 1 was in the cis conformation, which is the minor isomer in solution, and the pyrrolidine ring of 1 had C gamma-endo pucker. In crystalline 2 and 3 the amide bonds were in the trans conformation, and the pyrrolidine rings had C-exo pucker. The lengths of the bonds between sp3-hybridized carbon atoms in the pyrrolidine ring were significantly shorter in 2 and 3 than in 1, as was predicted by ab initio molecular orbital calculations at the RHF/3-21G level of theory. No significant change in bond length was observed in the other bonds of 1,2 or 3. The pyramidylization of the nitrogen atom increased dramatically in the order: 1 < 2 < 3. Together, these results indicate that electron-withdrawing substituents in the 4-position of proline residues can have a significant influence on the structure of these residues. In particular, the change in pyramidylization suggests that such substituents increase the sp3-character of the prolyl nitrogen atom and could thereby alter the rate of prolyl peptide bond isomerization.
Asunto(s)
Prolina/análogos & derivados , Prolina/química , Animales , Hidroxiprolina/análogos & derivados , Hidroxiprolina/química , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Molecular , Prolina/síntesis química , Vertebrados , Difracción de Rayos XRESUMEN
We present ab initio calculations of the Fermi contact term and experimental correlations of six coupling constants, 3JHNH alpha, 1JC alpha H alpha, 2JC'H alpha, 1JC alpha N, 2JC alpha N and 1JC'N, in a peptide as functions of the backbone dihedral angles, phi and psi. Given estimates of experimental uncertainties, we find semiquantitative experimental correlations for 3JHNH alpha, 1JC alpha N and 2JC alpha N, qualitative correlations for 1JC alpha H alpha and 2JC'H alpha, but no experimental correlations of practical utility for 1JC'N, owing to its complex dependence on at least four dihedral angles. Errors in the estimation of dihedral angles from X-ray crystallographic data for proteins, which result from uncertainties in atom-to-atom distances, place substantial limitations on the quantitative reliability of coupling constant calculations fitted to such data. In the accompanying paper [Edison, A.S. et al., J. Biomol. NMR, 4, 543-551] we apply the results of the coupling constant calculations presented here to the estimation of phi and psi angles in staphylococcal nuclease from experimental coupling constants.
Asunto(s)
Alanina/análogos & derivados , Espectroscopía de Resonancia Magnética , Alanina/química , Cristalografía por Rayos X , Análisis de Fourier , Programas InformáticosRESUMEN
Calculated coupling constants (3JHNH alpha, 1JC alpha H alpha, 2JC'H alpha, 1JC alpha N and 2JC alpha N) from our accompanying paper [Edison, A.S. et al. (1994) J. Biomol. NMR, 4, 519-542] have been used to generate error surfaces that can provide estimates of the phi and psi angles in proteins. We have used experimental coupling data [3JHNH alpha: Kay, L.E. et al. (1989) J. Am. Chem. Soc., 111, 5488-5490; 1JC alpha H alpha: Vuister, G. W. et al. (1993) J. Biomol. NMR, 3, 67-80; 2JC'H alpha: Vuister, G.W. and Bax, A. (1992) J. Biomol. NMR, 2, 401-405; 1JC alpha N and 2JC alpha N: Delaglio, F. et al. (1991) J. Biomol. NMR, 1, 439-446] to create error surfaces for selected residues of the protein staphylococcal nuclease. The residues were chosen to include all those with five experimental couplings, as well as some with four experimental couplings, to demonstrate the relative importance of 3JHNH alpha and 1JC alpha H alpha. For most of the cases, we obtained good agreement between the X-ray structure [Loll, P.J. and Lattman, E.E. (1989) Protein Struct. Funct. Genet., 5, 183-201] and the NMR data.