Support of1H NMR assignments in proteins by biosynthetically directed fractional13C-labeling

Summary Biosynthetically directed fractional incorporation of¹³C into proteins results in nonrandom¹³C-labeling patterns that can be investigated by analysis of the¹³C–¹³C scalar coupling fine structures in heteronuclear¹³C–¹H or homonuclear¹³C–¹³C correlation experiments. Previously this approach was used for obtaining stereospecific¹H and¹³C assignments of the diastereotopic methyl groups of valine and leucine. In the present paper we investigate to what extent the labeling patterns are characteristic for other individual amino acids or groups of amino acids, and can thus be used to support the¹H spin-system identifications. Studies of the hydrolysates of fractionally¹³C-labeled proteins showed that the 59 aliphatic carbon positions in the 20 proteinogenic amino acids exhibit 16 different types of¹³C–¹³C coupling fine structures. These provide support for the assignment of the resonances of all methyl groups in a protein, which are otherwise often poorly resolved in homonuclear¹H NMR spectra. In particular, besides the individual methyl assignments in Val and Leu, unambiguous distinctions are obtained between the methyl groups of Ala and Thr, and between the - and -methyl groups of Ile. In addition to the methyl resonances, the CH₂ groups of Glu and Gln can be uniquely assigned because of the large coupling constant with the -carbon, and the identification of most of the other spin systems can be supported on the basis of coupling patterns that are common to small groups of amino acid residues.Abbreviations NOE nuclear Overhauser effect - fractional¹³C labeling biosynthetically directed fractional¹³C-labeling - TOCSY total correlation spectroscopy - ROESY rotating frame Overhauser enhancement spectroscopy - [¹³C,¹H]-COSY two-dimensional¹³C–¹H correlation spectroscopy - isotopomer isotope isomer - P22 c2 repressor c2 repressor of the salmonella phage P22 consisting of a polypeptide chain with 216 residues - P22 c2(1-76) N-terminal domain of the P22 c2 repressor with residues 1–76     

Proton nuclear magnetic resonance studies on huwentoxin-I from the venom of the spiderSelenocosmia huwena: 1. Sequence-specific1H-NMR assignments

The complete sequence-specific assignments of resonances in the¹H-NMR spectrum of huwentoxin-I from the Chinese bird spider,Selenocosmia huwena, is described. A combination of two-dimensional NMR experiments including 2D-COSY, 2D-NOESY, and 2D-TOCSY has been employed on samples of the toxin dissolved in D₂O and in H₂O for assignment purposes. Protons belonging to spin systems for each of the 33 amino acids were identified. The sequence-specific assignments were facilitated by the identification ofd _N connectivities on the fingerprint regions of the COSY and NOESY spectra and were supported by the identification ofd _NN andd _N connectivities in the TOCSY and NOESY spectra. These studies provide a basis for the determination of the solution-phase conformation of this toxin.Abbreviations HWTX-I huwentoxin-I - 2D two-dimensional - COSY 2D homonuclear correlation spectroscopy - NOE nuclear Overhauser enhancement - NOESY 2D nuclear Overhauser enhancement spectroscopy - TOCSY 2D total correlation spectroscopy - TPPI time-proportional phase incrementation - TSP sodium 3-(trimethyl-silyl)propionate-d4 - EDTA ethylenediaminetetraacetic acid     

Time-saving methods for heteronuclear multidimensional NMR of (13C, 15N) doubly labeled proteins

Summary 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.Abbreviations HMQC heteronuclear multiple-quantum coherence spectroscopy - HSQC heteronuclear single-quantum coherence spectroscopy - HSMQC heteronuclear single- and multiple-quantum coherence spectroscopy - NOESY nuclear Overhauser enhancement spectroscopy     

The program XEASY for computer-supported NMR spectral analysis of biological macromolecules

Summary A new program package, XEASY, was written for interactive computer support of the analysis of NMR spectra for three-dimensional structure determination of biological macromolecules. XEASY was developed for work with 2D, 3D and 4D NMR data sets. It includes all the functions performed by the precursor program EASY, which was designed for the analysis of 2D NMR spectra, i.e., peak picking and support of sequence-specific resonance assignments, cross-peak assignments, cross-peak integration and rate constant determination for dynamic processes. Since the program utilizes the X-window system and the Motif widget set, it is portable on a wide range of UNIX workstations. The design objective was to provide maximal computer support for the analysis of spectra, while providing the user with complete control over the final resonance assignments. Technically important features of XEASY are the use and flexible visual display of strips, i.e., two-dimensional spectral regions that contain the relevant parts of 3D or 4D NMR spectra, automated sorting routines to narrow down the selection of strips that need to be interactively considered in a particular assignment step, a protocol of resonance assignments that can be used for reliable bookkeeping, independent of the assignment strategy used, and capabilities for proper treatment of spectral folding and efficient transfer of resonance assignments between spectra of different types and different dimensionality, including projected, reduced-dimensionality triple-resonance experiments.Abbreviations 1D, 2D, 3D, 4D one-, two-, three-, four-dimensional - NOE nuclear Overhauser enhancement - NOESY nuclear Overhauser enhancement spectroscopy - TOCSY total correlation spectroscopy - COSY correlation spectroscopy - TPPI time-proportional phase incrementation     

A high-resolution 1H NMR approach for structure determination of membrane peptides and proteins in non-deuterated detergent: Application to mastoparan X solubilized in n-octylglucoside

Summary Application of ¹H 2D NMR methods to solubilized membrane proteins and peptides has up to now required the use of selectively deuterated detergents. The unavailability of any of the common biochemical detergents in deuterated form has therefore limited to some extent the scope of this approach. Here a ¹H NMR method is described which allows structure determination of membrane peptides and small membrane proteins by ¹H 2D NMR in any type of non-deuterated detergent. The approach is based on regioselective excitation of protein resonances with DANTE-Z or spin-pinging pulse trains. It is shown that regioselective excitation of the amide-aromatic region of solubilized membrane proteins and peptides leads to an almost complete suppression of the two orders of magnitude higher contribution of the protonated detergent to the ¹H NMR spectrum. Consistently TOCSY, COSY and NOESY sequences incorporating such regioselective excitation in the F2 dimension yield protein ¹H 2D NMR spectra of quality comparable to those obtained in deuterated detergents. Regioselective TOCSY and NOESY spectra display all through-bond and through-space correlations within amide-aromatic protons and between these protons and aliphatic and -protons. Regioselective COSY spectra provide scalar coupling constants between amide and -protons. Application of the method to the membrane-active peptide mastoparan X, solubilized in n-octylglucoside, yields complete sequence-specific assignments and extensive secondary structure-related spatial proximities and coupling constants. It is shown that mastoparan adopts an -helical conformation when bound to nonionic detergent micelles. The present method is expected to increase the applicability of ¹H solution NMR methods to membrane proteins and peptides.Abbreviations 2D NMR two-dimensional NMR - COSY correlated spectroscopy - DANTE delays alternating nutations for tailored excitation - NOESY nuclear Overhauser enhancement spectroscopy - TOCSY total correlation spectroscopy     

Resonance assignment and structural analysis of acid denatured E. coli [U-15N]-glutaredoxin 3

Virtually complete sequence specific ¹H and ¹⁵N resonance assignments are presented for acid denatured reduced E. coli glutaredoxin 3. The sequential resonance assignments of the backbone rely on the combined use of 3D F₁-decoupled ROESY-¹⁵N-HSQC and 3D ¹⁵N-HSQC-(TOCSY-NOESY)-¹⁵N-HSQC using a single uniformly ¹⁵N labelled protein sample. The sidechain resonances were assigned from a 3D TOCSY-¹⁵N-HSQC and a homonouclear TOCSY spectrum. The presented assignment strategy works in the absence of chemical exchange peaks with signals from the native conformation and without ¹³C/¹⁵N double labelling. Chemical shifts, ³J(H, NH) coupling constants and NOEs indicate extensive conformational averaging of both backbone and side chains in agreement with a random coil conformation. The only secondary structure element persisting at pH 3.5 appears to be a short helical segment comprising residues 37 to 40.Abbreviations HSQC heteronuclear single quantum coherence - NMR nuclear magnetic resonance - NOE nuclear Overhauser effect - NOESY two-dimensional NOE spectroscopy - ROE nuclear Overhauser effect in the rotating frame - ROESY two-dimensional ROE spectroscopy - TOCSY total correlation spectroscopy - TPPI time proportional phase incrementation Correspondence to: G. Otting     

Solution studies of the SH2 domain from the fyn tyrosine kinase: secondary structure,backbone dynamics and protein association

The SH2 domain from Fyn tyrosine kinase, corresponding to residues 155–270 of the human enzyme, was expressed as a GST-fusion protein in a pGEX-E. coli system. After thrombin cleavage and removal of GST, the protein was studied by heteronuclear NMR. Two different phosphotyrosyl-peptides were synthesized and added to the SH2 domain. One peptide corresponded to the regulatory C-terminal tail region of Fyn. Sequence-specific assignment of NMR spectra was achieved using a combination of¹H-¹⁵N-correlated 2D HSQC,¹⁵N-edited 3D TOCSY-HMQC, and¹⁵N-edited 3D NOESY-HMQC spectra. By analysis of the -proton chemical shifts and NOE intensities, the positions of secondary structural elements were determined and found to correspond closely to that seen in the crystal structure of the, homologous, Src-SH2 domain.To investigate the internal dynamics of the protein backbone, T₁ and T₂ relaxation parameters were measured on the free protein, as well as on both peptide complexes. Analytical ultracentrifugation and dynamic light scattering were employed to measure the effect of concentration and peptide-binding on self-association. The results suggest that, at NMR-sample concentrations, the free protein is present in at least dimeric form. Phosphopeptide binding and lower concentration significantly, but not completely, shift the equilibrium towards monomers. The possible role of this protein association in the regulation of the Src-family tyrosine kinases is discussed.Abbreviations SH Src homology - GST glutathione-S-transferase - IPTG isopropyl--D-galactopyranoside - DTT dithiothreitol - PMSF phenyl-methyl-sulphonyl-fluoride - TBS 50 mM Tris, 150 mM NaCl, 5 mM DTT, pH 8.0 - MWCO molecular weight cut off - NMR nuclear magnetic resonance - HSQC heteronuclear single-quantum correlation - NOESY nuclear Overhauser effect spectroscopy     

Application of 1D and 2D NMR techniques to the structure elucidation of the O-polysaccharide from Proteus mirabilis O: 57

Summary The LPS O-polysaccharide (O-PS) produced by Proteus mirabilis serotype O: 57 (ATCC 49995) was shown by NMR spectroscopy and chemical analysis to be a high-molecular-weight acidic branched polymer of pentasaccharide repeating units, composed of d-glucose, d-galactose, 2-acetamido-2-deoxy-d-galactose and glycerophosphate residues (1:2:2:2:1). Application of one-and two-dimensional NMR methods allowed the complete assignment of notoriously crowded ¹H and ¹³C spectra of the O-PS, leading to the determination of its structure. Several of the NMR techniques used were applied for the first time to the structure elucidation of polysaccharides. The resonances of galactose H5, H6 and H6 were identified by a 1D analog of 3D NOESY-TOCSY and 2D {¹H, ¹H} triple-quantum experiments. The position and the nature of the phosphate group were determined from 2D ³¹P (₁)-half-filtered COSY and 2D ³¹P-relayed COSY spectra. 2D HMQC-TOCSY and 2D single-quantum proton-carbon long-range correlation techniques were used to overcome the difficulties of severe overlap and higher order effects in the ¹H NMR spectrum of the O-PS. The latter technique, together with 2D NOESY, enabled us to identify the substitution positions, the anomeric configurations and the sequence of the component glycose residues in the O-PS.     

The Principal Neutralizing Determinant of HIV-1IIIB: Conformation of the Peptide Bound to a Neutralizing Antibody Studied by 2D-NMR

RP135 is a 24-residue peptide corresponding to the principal neutralizing determinant of the envelope glycoprotein gp120 of the human immunodeficiency virus type 1. We have studied the conformation of RP135 in complex with a neutralizing antibody 0.5 raised against gp120 by 2D NMR spectroscopy. The antigenic determinant recognized by this antibody was mapped using a combination of HOHAHA and ROESY measurements, in which resonances of the Fab and the tightly bound peptide residues are eliminated and the mobile residues of the bound peptide are sequentially assigned. We found that residues Ser⁶ - Thr¹⁹ are part of the epitope, while Lys⁵ and Ile²⁰ are at its boundaries. Difference spectroscopy was applied to study the conformation of the bound peptide representing the epitope within the 52 kDa of the Fab complex. Specific residues of the peptide were deuterated or replaced and the difference between the NOESY spectrum of the complex with the unlabeled residue and the NOESY spectrum of the complex with the modified residue revealed the interactions of the labeled residue both within the peptide and with the Fab fragment. A total of 122 distance restraints derived from the difference spectra enabled the calculation of the structure of the bound peptide. The peptide forms a 10-residue loop, while the two segments flanking this loop interact extensively with each other and possibly form anti-parallel -strands. The loop conformation could be observed due to the unusual large size (17 residues) of the antigenic determinant recognized by 0.5.     

Stereospecific assignment of β-methylene protons in larger proteins using 3D15N-separated Hartmann-Hahn and13C-separated rotating frame Overhauser spectroscopy

Summary ³J _x coupling constants and complementary nuclear Overhauser data on the intraresidue C ^x H–CH distances form an essential part of the data needed to obtain stereospecific assignments of -methylene protons in proteins. In this paper we show that information regarding the magnitude of the³J _x coupling constants can be extracted from a semi-quantitative interpretation of relative peak intensities in a 3D¹⁵N-separated¹H–¹H Hartmann-Hahn¹H–¹⁵N multiple quantum coherence (HOHAHA-HMQC) spectrum. In addition, we demonstrate that reliable information on the intraresidue C ^x H–CH distances, free of systematic errors arising from spin diffusion, can be obtained from a 3D¹³C-separated¹H–¹H rotating frame Overhauser effect¹H–¹³C multiple quantum coherence (ROESY-HMQC) spectrum. The applicability of these experiments to larger proteins is illustrated with respect to interleukin-1, a protein of 153 residues and 17.4 kDa molecular weight.Abbreviations 1L-1 interleukin-1 - NOE nuclear Overhauser effect - ROE rotating frame Overhauser effect - HOHAHA homonuclear Hartmann-Hahn spectroscopy - NOESY nuclear Overhauser enhancement spectroscopy - ROESY rotating frame Overhauser spectroscopy - HMQC heteronuclear multiple quantum coherence spectroscopy     

Two-dimensional NMR study of the conformation of (34–65)bacterioopsin polypeptide in SDS micelles

Summary The conformation of the synthetic 32-residue polypeptide, an analog of the membrane spanning segment B (residues 34-65) ofHalobacterium halobium bacteriobpsin, incorporated into perdeuterated sodium dodecyl sulfate micelles in the presence of trifluoroethanol was investigated by¹H NMR spectroscopy. The spectrum resonances were assigned by means of phase-sensitive DQF-COSY, TOCSY and NOESY techniques. Interproton nuclear Overhauser effects and deuterium exchange rates of individual NH groups were derived from two-dimensional NMR spectra. Analysis of the obtained data showed that segment B has a right-handed a-helical stretch from Lys⁴¹ to Leu⁶² with a kink at Pros⁵⁰. The-helix in the C-terminal part is terminated at Gly⁶³, which adopts a conformation typical of amino acid residues in a left-handed helix. The N-terminal part (residues 34–40) has no ordered conformation. NMR data are provided for comparison of the segment B conformation in the isotropic system of an organic solvent, in SDS micelles and in the purple membrane bacterioopsin. Factors affecting the conformation of membrane spanning segment B in various milieus are discussed.Dedicated to the memory of Professor V.F. Bystrov     

Comparative study of conformational behaviour of leucine and methionine enkephalinamides by1H-nuclear magnetic resonance spectroscopy

The conformational proclivity of leucine and methionine enkephalinamides in deuterated dimethyl sulphoxide has been investigated using proton magnetic resonance at 500 MHz. The resonances from the spin system of the various amino acid residues have been assigned from the 2-dimensional correlated spectroscopy spectra. The temperature variation of the amide proton shifts indicates that none of the amide proton is intramolecularly hydrogen-bonded or solvent-shielded. The analysis of vicinal coupling constants,³J_{HN.C 2}H,along with temperature coefficients and the absence of characteristic nuclear Overhauser effect cross peaks between the NH protons reveal that there is no evidence of the chain folding in these molecules. However, the observation of nuclear Overhauser effect cross peaks between the NH and the C^αH of the preceding residue indicates preference for extended backbone conformation with preferred side chain orientations particularly of Tyr and Phe in both [Leu⁵]- and [Met⁵]-enkephalinamides.     

Dipolar filtered 1H-13C heteronuclear correlation spectroscopy for resonance assignment of proteins

Resonance assignment is necessary for the comprehensive structure determination of insoluble proteins by solid-state NMR spectroscopy. While various 2D and 3D correlation techniques involving ¹³C and ¹⁵N spins have been developed for this purpose, ¹H chemical shift has not been exploited sufficiently. We demonstrate the combination of the regular ¹H-¹³C heteronuclear correlation (HETCOR) experiment and a dipolar filtered HETCOR technique to obtain better resolved ¹H chemical shift spectra. The dipolar filtered experiment, MELODI-HETCOR, simplifies the ¹H spectra by suppressing the directly bonded C-H correlation peaks and retaining only the medium- and long-range cross peaks. We apply this MELODI-HETCOR technique to several amino acids and proteins with various isotopic labeling patterns. The enhanced ¹H chemical shift resolution allows the assignment of overlapping H and H resonances in Ser, identifies the ¹H chemical shift differences between neutral and cationic imidazole rings of His, and permits the assignment of residues with side chain nitrogen atoms in ubiquitin. The potential utility of this dipolar filtered HETCOR technique to resonance assignment of extensively labeled proteins is discussed.     

Backbone assignment,secondary structure and Protein A binding of an isolated,human antibody VH domain

Summary Antibody heavy chain variable domains (VH) lacking their light chain domain (VL) partner are prime candidates for the design of minimum-size immunoreagents. To obtain structural information about isolated VH domains, a human VH was labelled with ¹⁵N or doubly labelled with both ¹⁵N and ¹³C and was studied by heteronuclear nuclear magnetic resonance spectroscopy. Most (90%) of the ¹H and ¹⁵N main-chain signals were assigned through two-dimensional TOCSY and NOESY experiments on the unlabelled VH and three-dimensional heteronuclear multiple quantum correlation TOCSY and NOESY experiments on the ¹⁵N-labelled VH. Four short stretches of the polypeptide chain could only be assigned on the basis of three-dimensional HNCA and HN(CO)CA experiments on the ¹³C-/¹⁵N-labelled protein. Long-range interstrand backbone NOEs suggest the presence of two adjacent -sheets formed by altogether nine antiparallel -strands. ³J_H ^NHC coupling constants and the location of slowly exchanging backbone amides support this interpretation. The secondary structure of the isolated VH is identical to that of heavy chain variable domains in intact antibodies, where VH domains are packed against a VL domain. The backbone assignments of the VH made it possible to locate its Protein A binding site. Chemical shift movements after complexing with the IgG binding fragment of Protein A indicate binding through one of the two -sheets of the VH. This -sheet is solvent exposed in intact antibodies. The Protein A binding site obviously differs from that on the Fc portion of immunoglobulins and is unique to members of the human VH_III gene subgroup.Abbreviations CDR complementarity determining region - CHAPS [(cholamidopropyl)-dimethylammonio]-1-propanesulfonate - DQF-COSY double-quantum-filtered correlated spectroscopy - Fab antigen binding antibody fragment - Fc crystallisable antibody fragment - Fv heterodimer of VH and VL - H1 (2, 3) hypervariable loop 1 (2, 3) - IgG immunoglobulin G - NOE nuclear Overhauser effect - NOESY nuclear Overhauser enhancement spectroscopy - HMQC heteronuclear multiple quantum correlation spectroscopy - HSQC heteronuclear single quantum correlation spectroscopy - scFv single chain Fv - TOCSY total correlation spectroscopy - TPPI time-proportional phase incrementation - VH antibody heavy chain variable region - VL antibody light chain variable region. Mutants are denoted by the wild-type amino acid (one-letter code), follwed by the residue number and the new amino acid     

Solution structure of a LewisX analogue by off-resonance 1H NMR spectroscopy without use of an internal distance reference

Summary A recent ¹H NMR method has been applied to the determination of the solution structure and internal dynamics of a synthetic mixed C/O trisaccharide related to sialyl Lewis^x. Varying the rf field offset in ROESY-type experiments enabled the measurement of longitudinal and transverse dipolar cross-relaxation rates with high accuracy. Assuming that for each proton pair the motion could be represented by a single exponential autocorrelation function, it was possible to derive geometrical parameters (r) and dynamic parameters _cp. With this assumption, 224 cross-relaxation rates have been transformed into 30 interproton distance constraints and 30 dipolar correlation times. The distance constraints have been used in a simulated-annealing procedure. This trisaccharide exhibits a structure close to the O-glycosidic analogue, but its flexibility seems highly reduced. On the basis of the determined structure and dynamics, it is shown that no conformational exchange occurs, the molecule existing in the form of a unique family in aqueous solution. In order to assess the quality of the resulting structures and to validate this new experimental procedure of distance extraction, we finally compare these solution structures to the ones obtained using three different sets of distances deduced from three choices of internal reference. It appears that this procedure allows the determination of the most precise and accurate solution.Abbreviations COSY correlation spectroscopy - NOE nuclear Overhauser enhancement - NOESY nuclear Overhauser enhancement spectroscopy; rmsd, root-mean-square deviation - ROESY rotating frame Overhauser enhancement spectroscopy - SLe^x sialyl Lewis^x - TOCSY total correlation spectroscopy     

Structure and Conformational Heterogeneity of a Weak Toxin from the Cobra Naja kaouthiaVenom

Resonances in the two-dimensional ¹H NMR spectra of a weak toxin (WTX) from the venom of cobra Naja kaouthiafor all 65 amino acid residues were assigned. The amino acid sequence of WTX, determined by the sequential assignment of spin systems, was found to be similar to that of the CM-9a toxin from the N. kaouthiavenom. Unlike CM-9a, WTX contains an additional Trp36 residue; Lys50 and Tyr52 are interchanged; and there is a Thr residue in place of Arg2. For some residues of WTX, the presence of two components of approximately equal intensities in the spectra was shown, which is explained by the conformational heterogeneity of the polypeptide owing to the cis–transisomerization of the peptide bond Arg32–Pro33. The data (contacts of the nuclear Overhauser effect, constants of spin–spin coupling of protons, and rates of exchange of amide protons for deuterium of the solvent) made it possible to determine the secondary structure of two forms of WTX, which is characterized by the presence of two antiparallel -sheets, one of which consists of two strands (regions 1–5 and 13–17) and the other, of three strands (regions 23–28, 38–43, and 55–59).     

1H and 15N resonance assignments and secondary structure of the carbon monoxide complex of sperm whale myoglobin

Summary Sequence-specific backbone ¹H and ¹⁵N resonance assignments have been made for 95% of the amino acids in sperm whale myoglobin, complexed with carbon monoxide (MbCO). Many assignments for side-chain resonances have also been obtained. Assignments were made by analysis of an extensive series of homonuclear 2D spectra, measured with unlabeled protein, and both 2D and 3D ¹H-¹⁵N-correlated spectra obtained from uniformly ¹⁵N-labeled myoglobin. Patterns of medium-range NOE connectivities indicate the presence of eight helices in positions that are very similar to those found in the crystal structures of sperm whale myoglobin. The resonance assignments of MbCO form the basis for determination of the solution structure and for hydrogen-exchange measurements to probe the stability and folding pathways of myoglobin. They will also form a basis for assignment of the spectra of single-site mutants with altered ligand-binding properties.     

3D 13C-15N-heteronuclear two-spin coherence spectroscopy for polypeptide backbone assignments in 13C-15N-double-labeled proteins

Summary The pulse sequence of a new constant-time 3D triple-resonance experiment, ct-HA[CAN]HN, is presented. This experiment delineates exclusively scalar connectivities and uses ¹³C–¹⁵N heteronuclear two-spin coherence to overlay the chemical shift evolution periods of the ¹³C and ¹⁵N nuclei, thereby providing the four resonance frequencies of the -proton, the -carbon, the amide nitrogen, and the amide proton of a given amino acid residue in three dimensions. This experiment promises to be a valid alternative to 4D experiments, providing the same information on intraresidue polypeptide backbone connectivities in ¹³C-¹⁵N-double-labeled proteins.Abbreviations 3D, 4D three-dimensional, four-dimensional - TPPI time-proportional phase incrementation - ct constant-time - rf radiofrequency - NOE nuclear Overhauser enhancement - NOESY two-dimensional nuclear Overhauser enhancement spectroscopy - glutaredoxin(C14S) mutant E. coli glutaredoxin with the cysteine at position 14 replaced by serine     

Measurement of inter-glycosidic 13C-1H coupling constants in a cyclic β(1→2)-glucan by 13C-filtered 2D {1H,1H}ROESY

Summary A method for measuring three-bond ¹³C-¹H scalar coupling constants across glycosidic bonds in a cyclic (12)-glucan icosamer is presented. This oligosaccharide molecule, with its high degree of symmetry, represents a particular challenge for NMR spectroscopy to distinguish inter-residue from intra-residue heteronuclear coupling effects. Chemically equivalent H2 protons in adjacent glucosyl residues are distinguished on the basis of their different through-space, dipolar interactions with the anomeric protons (H1). The strong NOE contact between anomeric (H1) and aglyconic (H2) protons permits the selective observation of the inter-residue heteronuclear couplings ³J_C1H2 and ³J_C2H1 in a natural-abundance ¹³C-₁-half-filtered {¹H,¹H} ROESY experiment.Abbreviations COSY scalar correlated spectroscopy - NOE nuclear Overhauser effect - NOESY NOE spectroscopy - ROESY rotating-frame NOE spectroscopy     

A two-dimensional 1H NMR study on Megasphaera elsdenii flavodoxin in the reduced state. Sequential assignments

Assignments for the 137 amino acid residues of Megasphaera elsdenii flavodoxin in the reduced state have been made using the sequential resonance assignment procedure. Several hydroxyl and sulfhydryl protons were observed at 41 degrees C at pH 8.3. Spin systems were sequentially assigned using phase-sensitive two-dimensional-correlated spectroscopy and phase-sensitive nuclear Overhauser enhancement spectroscopy. Spectra of the protein in H2O and of protein preparations either completely or partly exchanged against 2H2O were obtained. Use of the fast electron shuttle between the paramagnetic semiquinone and the diamagnetic hydroquinone state greatly simplified the NMR spectra, making it possible to assign easily the 1H resonances of amino acid residues located in the immediate neighbourhood of the isoalloxazine ring. The majority of the nuclear Overhauser effect contracts between the flavin and the apoprotein correspond to the crystal structure of the flavin domain of Clostridium MP flavodoxin, but differences are also observed. The assignments provide the basis for the structure determination of M. elsdenii flavodoxin in the reduced state as well as for assigning the resonances of the oxidized flavodoxin.