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.     

Complete 1H, 13C, and 15N NMR resonance assignments and secondary structure of human glutaredoxin in the fully reduced form.

Human glutaredoxin is a member of the glutaredoxin family, which is characterized by a glutathione binding site and a redox-active dithiol/disulfide in the active site. Unlike Escherichia coli glutaredoxin-1, this protein has additional cysteine residues that have been suggested to play a regulatory role in its activity. Human glutaredoxin (106 amino acid residues, M(r) = 12,000) has been purified from a pET expression vector with both uniform 15N labeling and 13C/15N double labeling. The combination of three-dimensional 15N-edited TOCSY, 15N-edited NOESY, HNCA, HN(CO)CA, and gradient sensitivity-enhanced HNCACB and HNCO spectra were used to obtain sequential assignments for residues 2-106 of the protein. The gradient-enhanced version of the HCCH-TOCSY pulse sequence and HCCH-COSY were used to obtain side chain 1H and 13C assignments. The secondary structural elements in the reduced protein were identified based on NOE information, amide proton exchange data, and chemical shift index data. Human glutaredoxin contains five helices extending approximately from residues 4-10, 24-36, 53-64, 83-92, and 94-104. The secondary structure also shows four beta-strands comprised of residues 15-19, 43-48, 71-75, 78-80, which form a beta-sheet almost identical to that found in E. coli glutaredoxin-1. Complete 1H, 13C, and 15N assignments and the secondary structure of fully reduced human glutaredoxin are presented. Comparison to the structures of other glutaredoxins is presented and differences in the secondary structure elements are discussed.     

Dihydrofolate reductase: sequential resonance assignments using 2D and 3D NMR and secondary structure determination in solution

Three-dimensional (3D) heteronuclear NMR techniques have been used to make sequential 1H and 15N resonance assignments for most of the residues of Lactobacillus casei dihydrofolate reductase (DHFR), a monomeric protein of molecular mass 18,300 Da. A uniformly 15N-labeled sample of the protein was prepared and its complex with methotrexate (MTX) studied by 3D 15N/1H nuclear Overhauser-heteronuclear multiple quantum coherence (NOESY-HMQC), Hartmann-Hahn-heteronuclear multiple quantum coherence (HOHAHA-HMQC), and HMQC-NOESY-HMQC experiments. These experiments overcame most of the spectral overlap problems caused by chemical shift degeneracies in 2D spectra and allowed the 1H-1H through-space and through-bond connectivities to be identified unambiguously, leading to the resonance assignments. The novel HMQC-NOESY-HMQC experiment allows NOE cross peaks to be detected between NH protons even when their 1H chemical shifts are degenerate as long as the amide 15N chemical shifts are nondegenerate. The 3D experiments, in combination with conventional 2D NOESY, COSY, and HOHAHA experiments on unlabelled and selectively deuterated DHFR, provide backbone assignments for 146 of the 162 residues and side-chain assignments for 104 residues of the protein. Data from the NOE-based experiments and identification of the slowly exchanging amide protons provide detailed information about the secondary structure of the binary complex of the protein with methotrexate. Sequential NHi-NHi+1 NOEs define four regions with helical structure. Two of these regions, residues 44-49 and 79-89, correspond to within one amino acid to helices C and E in the crystal structure of the DHFR.methotrexate.NADPH complex [Bolin et al. (1982) J. Biol. Chem. 257, 13650-13662], while the NMR-determined helix formed by residues 26-35 is about one turn shorter at the N-terminus than helix B in the crystal structure, which spans residues 23-34. Similarly, the NMR-determined helical region comprising residues 102-110 is somewhat offset from the crystal structure's helix F, which encompasses residues 97-107. Regions of beta-sheet structure were characterized in the binary complex by strong alpha CHi-NHi+1 NOEs and by slowly exchanging amide protons. In addition, several long-range NOEs were identified linking together these stretches to form a beta-sheet. These elements align perfectly with corresponding elements in the crystal structure of the DHFR.methotrexate.NADPH complex, which contains an eight-stranded beta-sheet, indicating that the main body of the beta-sheet is preserved in the binary complex in solution.     

NMR signal assignments of amide protons in the alpha-helical domains of staphylococcal nuclease

We report complete assignments of the amide proton signals in the three long dNN connectivity sequences observed in the NOESY spectrum of deuteriated staphylococcal nuclease (Nase) complexed with thymidine 3',5'-bisphosphate (pdTp) and Ca2+, Mr 18K. The assignments are made by comparing NOESY spectra with 1H-15N and 1H-13C heteronuclear multiple-quantum shift correlation (HMQC) spectra of Nase samples containing 15N- and 13C-labeled amino acids. The assignments show that the residues which are linked by the dNN connectivity sequences are located in three alpha-helical domains of Nase. Our results indicate that by combining NOESY and HMQC spectra of appropriately labeled samples it should be possible to delineate and study alpha-helical domains in soluble proteins having molecular weights that are greater than 18K.     

Complete 1H, 13C and 15N NMR assignments and secondary structure of the 269-residue serine protease PB92 from Bacillus alcalophilus

Summary The ¹H, ¹³C and ¹⁵N NMR resonances of serine protease PB92 have been assigned using 3D tripleresonance NMR techniques. With a molecular weight of 27 kDa (269 residues) this protein is one of the largest monomeric proteins assigned so far. The side-chain assignments were based mainly on 3D H(C)CH and 3D (H)CCH COSY and TOCSY experiments. The set of assignments encompasses all backbone carbonyl and CH_n carbons, all amide (NH and NH₂) nitrogens and 99.2% of the amide and CH_n protons. The secondary structure and general topology appear to be identical to those found in the crystal structure of serine protease PB92 [Van der Laan et al. (1992) Protein Eng., 5, 405–411], as judged by chemical shift deviations from random coil values, NH exchange data and analysis of NOEs between backbone NH groups.Abbreviations 2D/3D/4D two-/three-/four-dimensional - HSQC heteronuclear single-quantum coherence - HMQC heteronuclear multiple-quantum coherence - COSY correlation spectroscopy - TOCSY total correlation spectroscopy - NOE nuclear Overhauser enhancement (connectivity) - NOESY 2D NOE spectroscopy Experiment nomenclature (H(C)CH, etc.) follows the conventions used elsewhere [e.g. Ikura et al. (1990) Biochemistry, 29, 4659–4667].     

1H, 13C, and 15N NMR assignments and global folding pattern of the RNA-binding domain of the human hnRNP C proteins.

The hnRNP C1 and C2 proteins are abundant nuclear proteins that bind avidly to heterogeneous nuclear RNAs (hnRNAs) and appear to be involved with pre-mRNA processing. The RNA-binding activity of the hnRNP C proteins is contained in the amino-terminal 94 amino acid RNA-binding domain (RBD) that is identical for these two proteins. We have obtained the 1H, 13C, and 15N NMR assignments for the RBD of the human hnRNP C proteins. The assignment process was facilitated by extensive utilization of three- and four-dimensional heteronuclear-edited spectra. Sequential assignments of the backbone resonances were made using a combination of 15N-edited 3D NOESY-HMQC, 3D TOCSY-HMQC, and 3D TOCSY-NOESY-HSQC as well as 3D HNCA, HNCO, and HCACO spectra. Side-chain resonances were assigned using 3D HCCH-COSY and 3D HCH-TOCSY spectra. Four-dimensional 13C/13C-edited NOESY and 13C/15N-edited NOESY experiments were used to unambigously resolve NOEs. The overall global folding pattern was established by calculating a set of preliminary structures using constraints derived from the sequential NOEs and a small number of long-range NOEs. The beta alpha beta-beta alpha beta domain structure exhibits an antiparallel beta-sheet with the conserved RNP 1 and RNP 2 sequences [Dreyfuss et al. (1988) Trends Biochem. Sci. 13, 86-91] located adjacent to one another as the two inner strands of the beta-sheet.     

Sequence-specific 1H NMR assignments and secondary structure in solution of Escherichia coli trp repressor

Sequence-specific 1H NMR assignments are reported for the active L-tryptophan-bound form of Escherichia coli trp repressor. The repressor is a symmetric dimer of 107 residues per monomer; thus at 25 kDa, this is the largest protein for which such detailed sequence-specific assignments have been made. At this molecular mass the broad line widths of the NMR resonances preclude the use of assignment methods based on 1H-1H scalar coupling. Our assignment strategy centers on two-dimensional nuclear Overhauser spectroscopy (NOESY) of a series of selectively deuterated repressor analogues. A new methodology was developed for analysis of the spectra on the basis of the effects of selective deuteration on cross-peak intensities in the NOESY spectra. A total of 90% of the backbone amide protons have been assigned, and 70% of the alpha and side-chain proton resonances are assigned. The local secondary structure was calculated from sequential and medium-range backbone NOEs with the double-iterated Kalman filter method [Altman, R. B., & Jardetzky, O. (1989) Methods Enzymol. 177, 218-246]. The secondary structure agrees with that of the crystal structure [Schevitz, R., Otwinowski, Z., Joachimiak, A., Lawson, C. L., & Sigler, P. B. (1985) Nature 317, 782], except that the solution state is somewhat more disordered in the DNA binding region and in the N-terminal region of the first alpha-helix. Since the repressor is a symmetric dimer, long-range intersubunit NOEs were distinguished from intrasubunit interactions by formation of heterodimers between two appropriate selectively deuterated proteins and comparison of the resulting NOESY spectrum with that of each selectively deuterated homodimer. Thus, from spectra of three heterodimers, long-range NOEs between eight pairs of residues were identified as intersubunit NOEs, and two additional long-range intrasubunits NOEs were assigned.     

1H NMR sequential assignments and secondary structure analysis of human fibrinogen gamma-chain C-terminal residues 385-411

The human fibrinogen gamma-chain, C-terminal fragment, residues 385-411, i.e., KIIPFNRLTIGEGQQHHLGGAKQAGDV, contains two biologically important functional domains: (1) fibrinogen gamma-chain polymerization center and (2) platelet receptor recognition domain. This peptide was isolated from cyanogen bromide degraded human fibrinogen and was investigated by 1H NMR (500 MHz) spectroscopy. Sequence-specific assignments of NMR resonances were obtained for backbone and side-chain protons via analysis of 2D NMR COSY, double quantum filtered COSY, HOHAHA, and NOESY spectra. The N-terminal segment from residues 385-403 seems to adopt a relatively fixed solution conformation. Strong sequential alpha CH-NH NOESY connectivities and a continuous run of NH-NH NOESY connectivities and several long-lived backbone NH protons strongly suggest the presence of multiple-turn or helix-like structure for residues 390 to about 402. The conformation of residues 403-411 seems to be much less constrained as evidenced by the presence of weaker and sequential alpha CH-NH NOEs, the absence of sequential NH-NH NOEs, and the lack of longer lived amides. Chemical shifts of resonances from backbone and side-chain protons of the C-terminal dodecapeptide, residues 400-411, differ significantly from those of the parent chain, suggesting that some preferred C-terminal conformation does exist.     

NMR solution structure of the catalytic fragment of human fibroblast collagenase complexed with a sulfonamide derivative of a hydroxamic acid compound.

The solution structure of the catalytic fragment of human fibroblast collagenase (MMP-1) complexed with a sulfonamide derivative of a hydroxamic acid compound (CGS-27023A) has been determined using two-dimensional and three-dimensional heteronuclear NMR spectroscopy. The solution structure of the complex was calculated by means of hybrid distance geometry-simulated annealing using a combination of experimental NMR restraints obtained from the previous refinement of the inhibitor-free MMP-1 (1) and recent restraints for the MMP-1:CGS-27023A complex. The hydroxamic acid moiety of CGS-27023A was found to chelate to the "right" of the catalytic zinc where the p-methoxyphenyl sits in the S1' active-site pocket, the isopropyl group is in contact with H83 and N80, and the pyridine ring is solvent exposed. The sulfonyl oxygens are in hydrogen-bonding distance to the backbone NHs of L81 and A82. This is similar to the conformation determined by NMR of the inhibitor bound to stromelysin (2, 3). A total of 48 distance restraints were observed between MMP-1 and CGS-27023A from 3D 13C-edited/12C-filtered NOESY and 3D 15N-edited NOESY experiments. An additional 18 intramolecular restraints were observed for CGS-27023A from a 2D 12C-filtered NOESY experiment. A minimal set of NMR experiments in combination with the free MMP-1 assignments were used to assign the MMP-1 (1)H, 13C, and 15N resonances in the MMP-1:CGS-27023A complex. The assignments of CGS-27023A in the complex were obtained from 2D 12C-filtered NOESY and 2D 12C-filtered TOCSY experiments.     

Probing water-protein contacts in a MMP-12/CGS27023A complex by nuclear magnetic resonance spectroscopy

Using the case of the catalytic domain of MMP-12 in complex with the known inhibitor CGS27023A, a recently assembled 3D (15)N-edited/(14)N,(12)C-filtered ROESY experiment is used to monitor and distinguish protein amide protons in fast exchange with bulk water from amide protons close to water molecules with longer residence times, the latter possibly reflecting water molecules of structural or functional importance. The (15)N-edited/(14)N,(12)C-filtered ROESY spectra were compared to the original (15)N-edited/(14)N,(12)C-filtered NOESY and the conventional amide-water exchange experiment, CLEANEX. Three protein backbone amide protons experiencing direct dipolar cross relaxation with water in the (15)N-edited/(14)N,(12)C-filtered ROESY spectrum were assigned. In an ensemble of six crystal structures, two conserved water molecules within 3 ? of the three amide protons were identified. These two water molecules are buried into cavities in the protein surface and thus sufficiently slowed down by the protein topology to account for the observed dipolar interaction. Structural analysis of an ensemble of six crystal structures ruled out any exchange-relayed contributions for the amide-water interactions of interest.     

Proton NMR assignments and regular backbone structure of bovine pancreatic ribonuclease A in aqueous solution

Proton NMR assignments have been made for 121 of the 124 residues of bovine pancreatic ribonuclease A (RNase A). During the first stage of assignment, COSY and relayed COSY data were used to identify 40 amino acid spin systems belonging to alanine, valine, threonine, isoleucine, and serine residues. Approximately 60 other NH-alpha CH-beta CH systems were also identified but not assigned to specific amino acid type. NOESY data then were used to connect sequentially neighboring spin systems; approximately 475 of the possible 700 resonances in RNase A were assigned in this way. Our assignments agree with those for 20 residues assigned previously [Hahn, U., & Rüterjans, H. (1985) Eur. J. Biochem. 152, 481-491]. Additional NOESY correlations were used to identify regular backbone structure elements in RNase A, which are very similar to those observed in X-ray crystallographic studies [Wlodawer, A., Borkakoti, N., Moss, D. S., & Howlin, B. (1986) Acta Crystallogr. B42, 379-387].     

海南捕鸟蛛毒素-Ⅰ(HNTX-Ⅰ)的1H-NMR信号归属和二级结构分析

海南捕鸟蛛毒素-Ⅰ(HNTX-Ⅰ)是从海南捕鸟蛛(Ornithoctonus hainana)的粗毒中纯化的一种新型神经毒素.应用二维1H-NMR技术研究HNTX-Ⅰ的溶液结构特点,通过分析水和重水中的DQF-COSY、TOCSY和NOESY谱,识别出HNTX-Ⅰ全部33个氨基酸残基自旋体系;通过NOESY谱中的dαN、dβN、dNN和dαδ联系完成了序列专一的谱峰归属,从而确认了HNTX-Ⅰ所有的主链质子和大于96%的侧链质子的化学位移.并通过分析3JNH-CαH耦合常数、序列间的NOE联系以及慢氢交换质子等,确定HNTX-Ⅰ的二级结构主要是由三股反平行的β-折迭组成(Lys7-Cys9,Tyr20-Asn23和Trp28-Val31),这些结构特点与已经探明结构的其它蜘蛛毒素的基本相同.这些结果为完全解析HNTX-Ⅰ的溶液三维结构奠定了基础.     

Sequence-specific 1H NMR assignments and secondary structure of porcine motilin

The solution structure of the 22-residue peptide hormone motilin has been studied by circular dichroism and two-dimensional 1H nuclear magnetic resonance spectroscopy. Circular dichroism spectra indicate the presence of alpha-helical secondary structure in aqueous solution, and the secondary structure can be stabilized with hexafluoro-2-propanol. Sequence-specific assignments of the proton NMR spectrum of porcine motilin in 30% hexafluoro-2-propanol have been made by using two-dimensional NMR techniques. All backbone proton resonances (NH and alpha CH) and most of the side-chain resonances have been assigned by using double-quantum-filtered COSY, RELAYED-COSY, and NOESY experiments. Simulations of NOESY cross-peak intensities as a function of mixing time indicate that spin diffusion has a relatively small effect in peptides the size of motilin, thereby allowing the use of long mixing times to confidently make assignments and delineate secondary structure. Sequential alpha CH-NH and NH-NH NOESY connectivities were observed over a significant portion of the length of the peptide. A number of medium-range NOESY cross-peaks indicate that the peptide is folded into alpha-helix from Glu9 to Lys20, which agrees favorably with the 50% helical content determined from CD measurements. The intensities of selected NOESY cross-peaks relative to corresponding diagonal peaks were used to estimate a rotational correlation time of approximately 2.5 ns for the peptide, indicating that the peptide exists as a monomer in solution under the conditions used here.     

Solution structure of neuronal bungarotoxin determined by two-dimensional NMR spectroscopy: sequence-specific assignments, secondary structure, and dimer formation

The solution structure of neuronal bungarotoxin (nBgt) has been studied by using two-dimensional 1H NMR spectroscopy. Sequence-specific assignments for over 95% of the backbone resonances and 85% of the side-chain resonances have been made by using a series of two-dimensional spectra at four temperatures. From these assignments over 75% of the NOESY spectrum has been assigned, which has in turn provided 582 distance constraints. Twenty-seven coupling constants (NH-alpha CH) were determined from the COSY spectra, which have provided dihedral angle constraints. In addition, hydrogen exchange experiments have suggested the probable position of hydrogen bonds. The NOE constraints, dihedral angle constraints, and the rates of amide proton exchange suggest that a triple-stranded antiparallel beta sheet is the major component of secondary structure, which includes 25% of the amino acid residues. A number of NOE peaks were observed that were inconsistent with the antiparallel beta-sheet structure. Because we have confirmed by sedimentation equilibrium that nBgt exists as a dimer, we have reinterpreted these NOE constraints as intermolecular interactions. These constraints suggest that the dimer consists of a six-stranded antiparallel beta sheet (three from each monomer), with residues 55-59 forming the dimer interface.     

Backbone assignments and secondary structure of the Escherichia coli enzyme-II mannitol A domain determined by heteronuclear three-dimensional NMR spectroscopy.

This report presents the backbone assignments and the secondary structure determination of the A domain of the Escherichia coli mannitol transport protein, enzyme-IImtl. The backbone resonances were partially assigned using three-dimensional heteronuclear 1H NOE 1H-15N single-quantum coherence (15N NOESY-HSQC) spectroscopy and three-dimensional heteronuclear 1H total correlation 1H-15N single-quantum coherence (15N TOCSY-HSQC) spectroscopy on uniformly 15N enriched protein. Triple-resonance experiments on uniformly 15N/13C enriched protein were necessary to complete the backbone assignments, due to overlapping 1H and 15N frequencies. Data obtained from three-dimensional 1H-15N-13C alpha correlation experiments (HNCA and HN(CO)CA), a three-dimensional 1H-15N-13CO correlation experiment (HNCO), and a three-dimensional 1H alpha-13C alpha-13CO correlation experiment (COCAH) were combined using SNARF software, and yielded the assignments of virtually all observed backbone resonances. Determination of the secondary structure of IIAmtl is based upon NOE information from the 15N NOESY-HSQC and the 1H alpha and 13C alpha secondary chemical shifts. The resulting secondary structure is considerably different from that reported for IIAglc of E. coli and Bacillus subtilis determined by NMR and X-ray.     

Sequence-specific assignments of the 1H nuclear magnetic resonance spectra of reduced high-potential ferredoxin (HiPIP) from Chromatium vinosum.

The 1H resonances of the high-potential [4Fe-4S]2+ ferredoxin from Chromatium vinosum have been assigned through conventional sequential methodology applied to 2D NMR spectra. Almost 80% of the residues were identified using standard 2D COSY, HOHAHA, and NOESY pulse sequences. These residues correspond to four segments of the primary structure that do not interact strongly with the iron-sulfur cluster. A minor correction to the amino acid sequence is strongly suggested by these NMR data. Additional protons more sensitive to the proximity of the cluster were assigned by a combination of NOESY experiments with fast repetition rates and short mixing times and of HOHAHA spectra recorded with reduced spin-lock duration aimed at compensating for the short relaxation rates. Hence, the contributions of 79 residues out of 85 were identified in NMR spectra, among which the assignments of 64 residues were completed. Even the fastest relaxing protons, like those of the cysteine ligands, could be correlated, partly because the strong hyperfine shifts isolate them from the crowded diamagnetic region. However, other protons, in particular those involved in NH-S hydrogen bonds with the iron-sulfur cluster, were more difficult to identify, most probably because their relatively broad signals overlapped with those of protons not or less perturbed by the active site. The availability of the major part of the 1H NMR assignments has enabled the detection and identification of many interresidue NOESY cross peaks. These data are in full agreement with the elements of secondary structure previously revealed by X-ray crystallographic analysis of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

1H, 13C, and 15N backbone assignment and secondary structure of the receptor-binding domain of vascular endothelial growth factor.

Nearly complete sequence-specific 1H, 13C, and 15N resonance assignments are reported for the backbone atoms of the receptor-binding domain of vascular endothelial growth factor (VEGF), a 23-kDa homodimeric protein that is a major regulator of both normal and pathological angiogenesis. The assignment strategy relied on the use of seven 3D triple-resonance experiments [HN(CO)CA, HNCA, HNCO, (HCA)CONH, HN(COCA)HA, HN(CA)HA, and CBCA-(CO)NH] and a 3D 15N-TOCSY-HSQC experiment recorded on a 0.5 mM (12 mg/mL) sample at 500 MHz, pH 7.0, 45 degrees C. Under these conditions, 15N relaxation data show that the protein has a rotational correlation time of 15.0 ns. Despite this unusually long correlation time, assignments were obtained for 94 of the 99 residues; 8 residues lack amide 1H and 15N assignments, presumably due to rapid exchange of the amide 1H with solvent under the experimental conditions used. The secondary structure of the protein was deduced from the chemical shift indices of the 1H alpha, 13C alpha, 13C beta, and 13CO nuclei, and from analysis of backbone NOEs observed in a 3D 15N-NOESY-HSQC spectrum. Two helices and a significant amount of beta-sheet structure were identified, in general agreement with the secondary structure found in a recently determined crystal structure of a similar VEGF construct [Muller YA et al., 1997, Proc Natl Acad Sci USA 94:7192-7197].     

Identification of the internal axial ligand of HO2-cobalt(III)-bleomycin: 1H[15N] HSQC NMR investigation of bleomycin,deglycobleomycin, and their hydroperoxide-cobalt(III) complexes

The identity of the axial ligand contributed by the drug in hydroperoxide-Co(III)-bleomycin and hydroperoxide-Co(III)-deglycobleomycin has been in doubt. With each structure, a combination of (1)H[(15)N] HSQC and HMBC and (1)H COSY and NOESY NMR spectroscopy was used to observe and completely assign the nonaromatic (15)N chemical shifts of natural abundance bleomycin in the two hydroperoxide-Co(III) structures. Together with the (15)N assignments from a published 1D (15)N spectrum, the results permitted the assignment of the primary amine nitrogen to an axial ligand position in both structures.     

Assignment of the 15N NMR spectra of reduced and oxidized Escherichia coli thioredoxin

As a necessary first step in the use of heteronuclear correlated spectra to obtain high resolution solution structures of the protein, assignment of the 15N NMR spectra of reduced and oxidized Escherichia coli thioredoxin (Mr 12,000) uniformly labeled with 15N has been performed. The 15N chemical shifts of backbone amide nitrogen atoms have been determined for both oxidation states of thioredoxin using 15N-1H correlated and two-dimensional heteronuclear single-quantum coherence (HSQC) TOCSY and NOESY spectra. The backbone assignments are complete, except for the proline imide nitrogen resonances and include Gly33, whose amide proton resonance is difficult to observe in homonuclear 1H spectra. The differences in the 15N chemical shift between oxidized and reduced thioredoxin, which occur mainly in the vicinity of the two active site cysteines, including residues distant in the amino acid sequence which form a hydrophobic surface close to the active site, are consistent with the differences observed for proton chemical shifts in earlier work on thioredoxin.     

Determination of the local structure of the protein insectotoxin I5A from the scorpion Buthus eupeus from 1H-NMR spectroscopy data

The local structure (torsion angles phi, psi and chi 1 of amino acid residues) of insectotoxin I5A (35 residues) of scorpion Buthus eupeus has been determined from cross-peak integral intensities in two-dimensional nuclear Overhauser enhancement (NOESY) spectra and spin coupling constants of vicinal H--NC alpha--H and H--C alpha C beta--H protons. The local structure determination was carried out by fitting complete relaxation matrix of peptide unit protons (protons of a given residue and NH proton of the next residue in the amino acid sequence) with experimental NOESY cross-peak intensities. The obtained intervals of backbone torsional angles phi and psi consistent with NMR data were determined for all but Gly residues. The predominant C alpha--C beta rotamer of the side chain has been unambiguously determined for 42% of the insectotoxin amino acid residues whereas for another 46% residues experimental data are fitted equally well with two rotamers. Stereospecific assignments were obtained for 38% of beta-methylene groups. The determined torsional angles phi, psi and chi 1 correspond to the sterically allowed conformations of the amino acid residues and agree with the insectotoxin secondary structure established earlier by 1H NMR spectroscopy.