3d haro-Noesy-ch3nh and caro-Noesy-ch3nh Experiments for Double Labeled Proteins

Precision in the determination of the 3D structures of proteins by NMR depends on obtaining an adequate number of NOE restraints. Ambiguity in the assignment of NOE cross peaks between aromatic and other protons is an impediment to high quality structure determination. Two pulse sequences, 3D H_aro-NOESY-CH₃NH and 3D C_aro-NOESY-CH₃NH, based on a modification of a technique for simultaneous detection of ¹³C-¹H (of CH₃) and ¹⁵N-¹H correlations in one measurement, are proposed in the present work. These 3D experiments, which are optimized for resolution in the ¹³C and ¹⁵N dimensions, provide NOE information between aromatic protons and methyl or amide protons. CH₂ moieties are filtered out and the CH groups in aromatic rings are selected, allowing their NOE cross peaks to be unambiguously assigned. Unambiguous NOEs connecting aromatic and methyl or amide protons will provide important restraints for protein structure calculations.     

Heteronuclear 1H-15N nuclear magnetic resonance studies of the c subunit of the Escherichia coli F1F0 ATP synthase: assignment and secondary structure.

Nuclear magnetic resonance (NMR) studies of the c subunit of F1F0 ATP synthase from Escherichia coli are presented. A combination of homonuclear (1H-1H) and heteronuclear (1H-15N) 2D and 3D methods was applied to the 79-residue protein, dissolved in trifluoroethanol. Resonance assignment for all the backbone amide groups and many C alpha H side-chain protons was achieved. Analysis of inter- and intraresidue 1H-1H nuclear Overhauser effect (NOE) data and scalar coupling constant information indicates that this protein contains two extended regions of predominant alpha-helical character (residues 10-40 and 48-77) separated by an eight-residue segment which displays little evidence of ordered secondary structure. This model is consistent with information about the molecular motion of the protein deduced from 15N-1H heteronuclear NOE data and observed pKa values of carboxylic acid groups.     

Untenability of the heteronomous DNA model for poly(dA).poly(dT) in solution. This DNA adopts a right-handed B-DNA duplex in which the two strands are conformationally equivalent. A 500 MHz NMR study using one dimensional NOE

1D NOE 1H NMR spectroscopy at 500 MHz was employed to examine the structure of poly(dA).poly(dT) in solution. NOE experiments were conducted as a function of presaturation pulse length (50, 30, 20 and 10 msec) and power (19 and 20 db) to distinguish the primary NOEs from spin diffusion. The 10 msec NOE experiments took 49 hrs and over 55,000 scans for each case and the difference spectra were almost free from diffusion. The spin diffused NOE difference spectra as well as difference NOE spectra in 90% H2O + 10% D2O in which TNH3 was presaturated enabled to make a complete assignment of the base and sugar protons. It is shown that poly(dA).poly(dT) melts in a fashion in which single stranded bubbles are formed with increasing temperature.     

Heteronuclear three-dimensional NMR spectroscopy of the inflammatory protein C5a

The utility of three-dimensional heteronuclear NMR spectroscopy for the assignment of 1H and 15N resonances of the inflammatory protein C5a (MW 8500), uniformly labeled with 15N, is demonstrated at a protein concentration of 0.7 mM. It is shown that dramatic simplification of the 2D nuclear Overhauser effect spectrum (NOESY) is obtained by editing with respect to the frequency of the 15N heteronucleus in a third dimension. The improved resolution in the 3D experiment largely facilitates the assignment of protein NMR spectra and allows for the determination of distance constraints from otherwise overlapping NOE cross peaks for purposes of 3D structure determination. The results show that 15N heteronuclear 3D NMR can facilitate the structure determination of small proteins and promises to be a useful tool for the study of larger systems that cannot be studied by conventional 2D NMR techniques.     

Determination of the secondary structure of the DNA binding protein Ner from phage Mu using 1H homonuclear and 15N-1H heteronuclear NMR spectroscopy

The sequential resonance assignment of the 1H and 15N NMR spectra of the DNA binding protein Ner from phage Mu is presented. This is carried out by using a combination of 1H-1H and 1H-15N two-dimensional experiments. The availability of completely labeled 15N protein enabled us to record a variety of relayed heteronuclear multiple quantum coherence experiments, thereby enabling the correlation of proton-proton through-space and through-bond connectivities with the chemical shift of the directly bonded 15N atom. These heteronuclear experiments were crucial for the sequential assignment as the proton chemical shift dispersion of the Ner protein is limited and substantial overlap precluded unambiguous assignment of the homonuclear spectra in several cases. From a qualitative interpretation of the NOE data involving the NH, C alpha H, and C beta H protons, it is shown that Ner is composed of five helices extending from residues 11 to 22, 27 to 34, 38 to 45, 50 to 60, and 63 to 73.     

Polypeptide backbone resonance assignments and secondary structure of Bacillus subtilis enzyme IIIglc determined by two-dimensional and three-dimensional heteronuclear NMR spectroscopy

The enzyme IIIglc-like domain of Bacillus subtilis IIglc (IIIglc, 162 residues, 17.4 kDa) has been cloned and overexpressed in Escherichia coli. Sequence-specific assignment of the backbone 1H and 15N resonances has been carried out with a combination of homonuclear and heteronuclear two-dimensional and heteronuclear three-dimensional (3D) NMR spectroscopy. Amide proton solvent exchange rate constants have been determined from a series of 1H-15N heteronuclear single-quantum coherence (HSQC) spectra acquired following dissolution of the protein in D2O. Major structural features of IIIglc have been inferred from the pattern of short-, medium- and long-range NOEs in 3D heteronuclear 1H nuclear Overhauser effect 1H-15N multiple-quantum coherence (3D NOESY-HMQC) spectra, together with the exchange rate constants. IIIglc contains three antiparallel beta-sheets comprised of eight, three, and two beta-strands. In addition, five beta-bulges were identified. No evidence of regular helical structure was found. The N-terminal 15 residues of the protein appear disordered, which is consistent with their being part of the Q-linker that connects the C-terminal enzyme IIIglc-like domain to the membrane-bound IIglc domain. Significantly, two histidine residues, His 68 and His 83, which are important for phosphotransferase function, are found from NOE measurements to be in close proximity at the ends of adjacent strands in the major beta-sheet.     

Graph-theoretical assignment of secondary structure in multidimensional protein NMR spectra: Application to the lac repressor headpiece

Summary A novel procedure is presented for the automatic identification of secondary structures in proteins from their corresponding NOE data. The method uses a branch of mathematics known as graph theory to identify prescribed NOE connectivity patterns characteristic of the regular secondary structures. Resonance assignment is achieved by connecting these patterns of secondary structure together, thereby matching the connected spin systems to specific segments of the protein sequence. The method known as SERENDIPITY refers to a set of routines developed in a modular fashion, where each program has one or several well-defined tasks. NOE templates for several secondary structure motifs have been developed and the method has been successfully applied to data obtained from NOESY-type spectra. The present report describes the application of the SERENDIPITY protocol to a 3D NOESY-HMQC spectrum of the ¹⁵N-labelled lac repressor headpiece protein. The application demonstrates that, under favourable conditions, fully automated identification of secondary structures and semi-automated assignment are feasible.Abbreviations 2D, 3D two-, three-dimensional - NOESY nuclear Overhauser enhancement spectroscopy - HMQC heteronuclear multiple quantum coherence - SSE secondary structure element - SERENDIPITY SEcondary structuRE ideNtification in multiDImensional ProteIn specTra analYsis Supplementary Material available from the authors: Two tables containing the total number of mappings resulting from the graph search procedure for simulated and experimental NOE data.     

Assignments of backbone 1H, 13C, and 15N resonances and secondary structure of ribonuclease H from Escherichia coli by heteronuclear three-dimensional NMR spectroscopy.

The assignments of individual magnetic resonances of backbone nuclei of a larger protein, ribonuclease H from Escherichia coli, which consists of 155 amino acid residues and has a molecular mass of 17.6 kDa are presented. To remove the problem of degenerate chemical shifts, which is inevitable in proteins of this size, three-dimensional NMR was applied. The strategy for the sequential assignment was, first, resonance peaks of amides were classified into 15 amino acid types by 1H-15N HMQC experiments with samples in which specific amino acids were labeled with 15N. Second, the amide 1H-15N peaks were connected along the amino acid sequence by tracing intraresidue and sequential NOE cross peaks. In order to obtain unambiguous NOE connectivities, four types of heteronuclear 3D NMR techniques, 1H-15N-1H 3D NOESY-HMQC, 1H-15N-1H 3D TOCSY-HMQC, 13C-1H-1H 3D HMQC-NOESY, and 13C-1H-1H 3D HMQC-TOCSY, were applied to proteins uniformly labeled either with 15N or with 13C. This method gave a systematic way to assign backbone nuclei (N, NH, C alpha H, and C alpha) of larger proteins. Results of the sequential assignments and identification of secondary structure elements that were revealed by NOE cross peaks among backbone protons are reported.     

Main-chain-directed strategy for the assignment of 1H NMR spectra of proteins

A strategy for assigning the resonances in two-dimensional (2D) NMR spectra of proteins is described. The method emphasizes the analysis of through-space relationships between protons by use of the two-dimensional nuclear Overhauser effect (NOE) experiment. NOE patterns used in the algorithm were derived from a statistical analysis of the combinations of short proton-proton distances observed in the high-resolution crystal structures of 21 proteins. One starts with a search for authentic main-chain NH-C alpha H-C beta H J-coupled units, which can be found with high reliability. The many main-chain units of a protein are then placed in their proper juxtaposition by recognition of predefined NOE connectivity patterns. To discover these connectivities, the 2D NOE spectrum is examined, in a prescribed order, for the distinct NOE patterns characteristic of helices, sheets, turns, and extended chain. Finally, the recognition of a few amino acid side-chain types places the discovered secondary structure elements within the polypeptide sequence. Unlike the sequential assignment approach, the main-chain-directed strategy does not rely on the difficult task of recognizing many side-chain spin systems in J-correlated spectra, the assignment process is not in general sequential with the polypeptide chain, and the prescribed connectivity patterns are cyclic rather than linear. The latter characteristic avoids ambiguous branch points in the analysis and imposes an internally confirmatory property on each forward step.     

A novel approach for sequential assignment of 1H, 13C, and 15N spectra of proteins: heteronuclear triple-resonance three-dimensional NMR spectroscopy. Application to calmodulin

A novel approach is described for obtaining sequential assignment of the backbone 1H, 13C, and 15N resonances of larger proteins. The approach is demonstrated for the protein calmodulin (16.7 kDa), uniformly (approximately 95%) labeled with 15N and 13C. Sequential assignment of the backbone residues by standard methods was not possible because of the very narrow chemical shift distribution range of both NH and C alpha H protons in this largely alpha-helical protein. We demonstrate that the combined use of four new types of heteronuclear 3D NMR spectra together with the previously described HOHAHA-HMQC 3D experiment [Marion, D., et al. (1989) Biochemistry 28, 6150-6156] can provide unambiguous sequential assignment of protein backbone resonances. Sequential connectivity is derived from one-bond J couplings and the procedure is therefore independent of the backbone conformation. All the new 3D NMR experiments use 1H detection and rely on multiple-step magnetization transfers via well-resolved one-bond J couplings, offering high sensitivity and requiring a total of only 9 days for the recording of all five 3D spectra. Because the combination of 3D spectra offers at least two and often three independent pathways for determining sequential connectivity, the new assignment procedure is easily automated. Complete assignments are reported for the proton, carbon, and nitrogen backbone resonances of calmodulin, complexed with calcium.     

Epidermal growth factor from the mouse. Physical evidence for a tiered beta-sheet domain: two-dimensional NMR correlated spectroscopy and nuclear Overhauser experiments on backbone amide protons

When H2O-exchanged, lyophilized mouse epidermal growth factor (mEGF) is dissolved in deuterium oxide at low pH (i.e., below approximately 6.0), 13 well-resolved, amide proton resonances are observed in the downfield region of an NMR spectrum (500 MHz). Under the conditions of these experiments, the lifetimes of these amide protons in exchange for deuterons of the deuterium oxide solvent suggest that these amide protons are hydrogen-bonded, backbone amide protons. Several of these amide proton resonances show splittings (i.e., JNH alpha-CH) of approximately 8-10 Hz, indicating that their associated amide protons are in some type of beta-structure. Selective nuclear Overhauser effect (NOE) experiments performed on all amide proton resonances strongly suggest that all 13 of these backbone amide protons are part of a single-tiered beta-sheet structural domain in mEGF. Correlation of 2D NMR correlated spectroscopy data, identifying scaler coupled protons, with NOE data, identifying protons close to the irradiated amide protons, allows tentative assignment of some resonances in the NOE difference spectra to specific amino acid residues. These data allow a partial structural model of the tiered beta-sheet domain in mEGF to be postulated.     

1H NMR investigation of manganese peroxidase from Phanerochaete chrysosporium. A comparison with other peroxidases.

1H NMR spectra at 200- and 600-MHz of manganese peroxidase from Phanerochaete chrysosporium and of its cyanide derivative are reported. The spectrum of the native protein is very similar to that of other peroxidases. The assignment of the spectrum of the cyanide derivative has been performed through 1D NOE, 2D NOESY, and COSY experiments. This protein is very similar to lignin peroxidase, the only meaningful difference being the shift of H delta 2 of the proximal histidine. The spectra of the cyanide derivative of these two proteins are compared with those of horseradish peroxidase and cytochrome c peroxidase. The shift pattern of the protons of the proximal histidine is discussed relative to the structural properties which affect the Fe3+/Fe2+ redox potential.     

Nonnative isomers of proline-93 and -114 predominate in heat-unfolded ribonuclease A

The peptide bonds preceding both Pro-93 and Pro-114, which are in the cis conformation in native RNase A, are predominantly in the trans conformation in the heat-unfolded protein. The percentages are estimated to be 60% and 63%, respectively, with a standard deviation of +/- 7% in each quantity. These ratios are close to those found for corresponding sequences in X-Pro-Y peptides. The concentration of the trans proline species was determined from the integrated intensities of resonance peaks of the C alpha H protons of Tyr-92 and Asn-113, which are well resolved in the 1D proton NMR spectrum of heat-unfolded RNase A. The assignments of the resonances were deduced from 2D NOESY and DQF-COSY spectra of unfolded RNase A in D2O. Furthermore, the C alpha H protons of both Tyr-92 and Asn-113 had an intense NOE cross-peak with the C delta H and C delta' H of the respective following prolines. For both Pro-93 and Pro-114, these NOE cross-peaks would arise only if the X-Pro peptide bond were in the trans conformation. It is generally believed that the rate of refolding of RNase A is considerably reduced by nonnative proline isomers, such as trans Pro-93. Two models for folding RNase A, that are consistent with these new results and the work of previous investigators, are presented here.     

NMR spectroscopy for studying structure and intramolecular dynamics of modified analogs of steroid hormones

Special features of the use of homo- and heteronuclear correlation methods of NMR in one and two dimensions for studying the spatial structure and intramolecular dynamics of modified analogues of steroid hormones (MASH) are considered. The application of these methods to the assignment of resonances in the high-field 1H NMR region and to the determination of the most stereospecifically important parameters, such as the vicinal constants of spin-spin coupling (3JH-H) and nuclear Overhauser effects (NOE), are discussed using the example of NMR studies of some estrogens and androgens at 300 MHz and on the basis of literature data. The most efficient combination of the methods and the necessary modification of each of them may be chosen considering the spectral and relaxation parameters of MASH in liquid medium, including the anisotropy of the overall diffusive motion. The characteristics of MASH are the wide use of correlations through long-range couplings (COSY-45 and DQF-COSY), the application of the 4,5JH-H constants for the determination of spatial structure, and the advantage of heteronuclear HSQC methods with and without 13C decoupling over the corresponding HMQC methods in both resolution and sensitivity. In the conformationally rigid MASH molecules, the anisotropy of the MASH diffusive motion in liquid adversely affects the determination of interproton distances by the calibrating processing method for the NOE difference and NOESY spectra: it results in both overestimated and underestimated distance values depending on the polar angle ratios of the reference and the determined distances. Under certain conditions, conformationally mobile MASH demonstrate the additional contribution of the scalar relaxation mechanism between the indirectly (scalarly) bound protons. This mechanism is responsible for the underestimated values of NOE and the corresponding errors in the distance determination.     

Backbone 1H and 15N resonance assignments of the N-terminal SH3 domain of drk in folded and unfolded states using enhanced-sensitivity pulsed field gradient NMR techniques

Summary 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.Abbreviations 2D, 3D two-, three-dimensional - drkN SH3 N-terminal SH3 domain of Drosophila drk - HSQC heteronuclear single-quantum spectroscopy - NOE nuclear Overhauser enhancement - SH3 Src homology domain 3 - TOCSY total correlation spectroscopy     

Application of 2D and 3D NMR experiments to the conformational study of a diantennary oligosaccharide

Summary By the application of homonuclear 3D NOE-HOHAHA and heteronuclear 3D HMQC-NOE experiments in studies of complex oligosaccharides. NOEs can be investigated which are hidden in conventional 2D NOE spectra. In the 3D NOE-HOHAHA spectrum ₃ cross sections were considered to be the most suitable for assignment of NOEs. Alternatively, these cross sections could be measured separately in selective 2D HOHAHA-NOE spectroscopy. The advantages and limitations of the 2D alternative are compared with those of the 3D NOE-HOHAHA approach. In 3D HMQC-NOE spectroscopy the larger chemical shift displacement of the carbon spectrum with respect to the proton spectrum can be used to unmask NOEs hidden in the bulk region. If the extra proton dimension is not needed, 2D HMQC-NOE is a good alternative.The suitability of 2D and 3D NOE-HOHAHA and HMQC-NOE experiments for the estimation of proton-proton distances is demonstrated by comparing the results of these experiments on a diantennary asparagine-linked oligosaccharide with those of a conventional 2D NOE experiment. NOEs identified in the 2D and 3D NOE-HOHAHA as well as HMQC-NOE experiments, so far not identified or not quantified in 2D NOE experiments, are discussed in relation to each glycosidic linkage. The flexibility of the Man(1-3)Man linkage is demonstrated, confirming the existence of an ensemble of conformations for this linkage.     

An unambiguous assignment method by 2D selective-TOCSY-HSQC and selective-TOCSY-DQFCOSY and structural analysis by selective-TOCSY-NOESY experiments of a biantennary undecasaccharide

In this paper we present a newly developed 2D selective-TOCSY-HSQC experiment for unambiguous assignment of individual sugar components of a biantennary undecasaccharide. The correlation signals between the protons and carbons of a selectively excited sugar component in the undecasaccharide were observed in a 2D selective-TOCSY-HSQC experiment. The network of proton signals of this sugar was observed in a 2D selective-TOCSY-DQFCOSY experiment. These two experiments enabled us to perform unambiguous sequential assignment of both protons and carbons of the individual sugar components in the undecasaccharide. In addition, a three-dimensional structural analysis was performed by a 2D selective-TOCSY-NOESY experiment, which clearly distinguishes between inter-sugar and intra-sugar ring NOE signals.     

1H and 15N NMR characterization of free and bound states of an amphiphilic peptide interacting with calmodulin.

A peptide of 17 amino acid residues Ac-L-K-W-K-K-L-L-K-L-L-K-K-L-L-K-L-G-NH2, designed to form an amphiphilic basic alpha-helix [DeGrado, W.F., Prendergast, F. G., Wolfe, H. R., Jr., & Cox, J. A. (1985) J. Cell. Biochem. 29, 83-93], was labeled with 15N at positions 1, 7, 9, and 10. Homo- and heteronuclear NMR techniques were used to characterize the conformational changes of the peptide when it binds to calmodulin in the presence of Ca2+ ions. The spectrum of the free peptide in aqueous solution at pH 6.3 and 298 K was completely assigned by a combined application of several two-dimensional proton NMR methods. Analysis of the short- and medium-range NOE connectivities and of the secondary chemical shifts indicated that the peptide populates, to a significant extent, an alpha-helix conformational state, in agreement with circular dichroism measurements under similar physicochemical conditions. 15N-edited 1D spectra and 15N(omega 2)-half-filtered two-dimensional NMR experiments on the peptide in a 1:1 complex with calmodulin allowed assignment of half of the amide proton resonances and three C alpha H resonances of the bound peptide. The observed NOE connectivities between the peptide backbone protons are indicative of a stable helical secondary structure spanning at least the fragment L1-K11. The equilibrium and dynamic NMR parameters of the bound peptide are discussed in terms of a molecular interaction model.     

Assignment of the backbone 1H and 15N NMR resonances of bacteriophage T4 lysozyme

The proton and nitrogen (15NH-H alpha-H beta) resonances of bacteriophage T4 lysozyme were assigned by 15N-aided 1H NMR. The assignments were directed from the backbone amide 1H-15N nuclei, with the heteronuclear single-multiple-quantum coherence (HSMQC) spectrum of uniformly 15N enriched protein serving as the master template for this work. The main-chain amide 1H-15N resonances and H alpha resonances were resolved and classified into 18 amino acid types by using HMQC and 15N-edited COSY measurements, respectively, of T4 lysozymes selectively enriched with one or more of alpha-15N-labeled Ala, Arg, Asn, Asp, Gly, Gln, Glu, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val. The heteronuclear spectra were complemented by proton DQF-COSY and TOCSY spectra of unlabeled protein in H2O and D2O buffers, from which the H beta resonances of many residues were identified. The NOE cross peaks to almost every amide proton were resolved in 15N-edited NOESY spectra of the selectively 15N enriched protein samples. Residue specific assignments were determined by using NOE connectivities between protons in the 15NH-H alpha-H beta spin systems of known amino acid type. Additional assignments of the aromatic proton resonances were obtained from 1H NMR spectra of unlabeled and selectively deuterated protein samples. The secondary structure of T4 lysozyme indicated from a qualitative analysis of the NOESY data is consistent with the crystallographic model of the protein.     

Protein NMR structure determination with automated NOE-identification in the NOESY spectra using the new software ATNOS

Novel algorithms are presented for automated NOESY peak picking and NOE signal identification in homonuclear 2D and heteronuclear-resolved 3D [¹H,¹H]-NOESY spectra during de novoprotein structure determination by NMR, which have been implemented in the new software ATNOS (automated NOESY peak picking). The input for ATNOS consists of the amino acid sequence of the protein, chemical shift lists from the sequence-specific resonance assignment, and one or several 2D or 3D NOESY spectra. In the present implementation, ATNOS performs multiple cycles of NOE peak identification in concert with automated NOE assignment with the software CANDID and protein structure calculation with the program DYANA. In the second and subsequent cycles, the intermediate protein structures are used as an additional guide for the interpretation of the NOESY spectra. By incorporating the analysis of the raw NMR data into the process of automated de novoprotein NMR structure determination, ATNOS enables direct feedback between the protein structure, the NOE assignments and the experimental NOESY spectra. The main elements of the algorithms for NOESY spectral analysis are techniques for local baseline correction and evaluation of local noise level amplitudes, automated determination of spectrum-specific threshold parameters, the use of symmetry relations, and the inclusion of the chemical shift information and the intermediate protein structures in the process of distinguishing between NOE peaks and artifacts. The ATNOS procedure has been validated with experimental NMR data sets of three proteins, for which high-quality NMR structures had previously been obtained by interactive interpretation of the NOESY spectra. The ATNOS-based structures coincide closely with those obtained with interactive peak picking. Overall, we present the algorithms used in this paper as a further important step towards objective and efficient de novoprotein structure determination by NMR.