Analysis of the paramagnetic shifts of haem carbon resonances in bovine ferricytochrome b 5

Recently published chemical shifts for haem ¹³C nuclei in bovine ferricytochrome b ₅₅ (Lee KB, Kweon J, Park H (1995) Assignment of hyperfine-shifted heme carbon resonances in ferricytochrome b ₅. FEBS Lett. 367:77–80) are analysed in terms of haem molecular orbitals with perturbed D_4h symmetry. Since a crystal structure of this protein is available, together with extensive ¹H assignments both in the oxidised and reduced forms, the paramagnetic shifts can be separated into dipolar and Fermi contact contributions by using an empirical magnetic susceptibility tensor. The results are compared with the orientation of the tensor and the geometry of the haem ligands. This comparison casts doubt on one of the ¹³C assignments and demonstrates that the asymmetry of the haem electronic structure is dominated by the influence of both of the His ligands. The ¹³C chemical shifts of two haem methyl groups in the minor form of the protein, in which the haem is approximately rotated by 180° about its 5CH15CH axis, are also evaluated.     

Sequential Main-Chain Proton and Carbon Resonance Assignments for Wild-Type Yeast Iso-1 Ferrocytochrome c and Identification of Secondary Structural Elements

Yeast iso-1 cytochrome c is a naturally occurring protein that possesses an unusually reactive Cysl02 that imbues iso-1 with a complicated solution chemistry which includes spontaneous dimerization and poorly characterized redox reactions. For this reason previous studies of this typical member of the c-type cytochromes have been relegated to variant proteins in which the 102 position has been mutated, with most common changes involving serine and threonine. However, we have determined sequential ¹H resonance assignments for the wild-type native protein because it is the actual participant in yeast mitochondrial electron transfer processes and because the wild-type native protein should be the fundamental assignment basis. In addition to ¹H resonance assignments for 97 of 106 amino acids, we have also provided an extensive database of long-range NOEs. Comparison of these NOEs and a chemical shift index based upon -H resonances has lead to identification of solution secondary structural elements that are consistent with the solid-state crystal structure. Although there is currently no efficient expression system that would facilitate isotope labeling of iso-1 cytochrome c, we tried to assess the usefulness of future heteronuclear experiments by using natural-abundance ¹H/¹³C HMQC experiments to unambiguously assign 35 -C resonances.     

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].     

A C-detection NMR approach for large glycoproteins

NMR spectroscopy has great potential to provide us with information on structure and dynamics at atomic resolution of glycoproteins in solution. In larger glycoproteins, however, the detrimental fast ¹H transverse relaxation renders the conventional ¹H-detected NMR experiments difficult. ¹³C direct detection potentially offers a valuable alternative to ¹H detection to overcome the fast T₂ relaxation. Here, we applied ¹³C-detected NMR methods to observe the NMR signals of ¹³C-labeled glycans attached to the Fc fragment of immunoglobulin G with a molecular mass of 56 kDa. Spectral analysis revealed that a ¹³C-detected ¹³C-¹³C NOESY experiment is highly useful for spectral assignments of the glycans of large glycoproteins. This approach would be, in part, complementary to ¹³C-¹³C TOCSY and ¹H-detection experiments.     

A13C NMR study of the hinge region of a mouse monoclonal antibody

Summary A¹³C NMR study is reported of the hinge region of an intact mouse monoclonal antibody with a molecular weight of 150 K. Cys, Ile, and Pro analogs of the antibody labeled with¹³C at the carbonyl carbon were prepared by growing hybridoma cells in the serum-free media. Resonance assignments have been performed as described previously [Kato, K., Matsunaga, C., Igarashi, T., Kim, H., Odaka, A., Shimada, I. and Arata, Y. (1991)Biochemistry,30, 270–278]. The spectral data obtained show that¹³C NMR can give detailed information about the structure of the hinge region of the intact antibody molecule. Prospects for the future role of¹³C NMR in the structural analyses of larger proteins are briefly discussed.Dedicated to the memory of Professor V.F. Bystrov     

Effect of disulfide bridge formation on the NMR spectrum of a protein: Studies on oxidized and reduced Escherichia coli thioredoxin

Summary As a prelude to complete structure calculations of both the oxidized and reduced forms of Escherchia coli thioredoxin (M_r 11 700), we have analyzed the NMR data obtained for the two proteins under identical conditions. The complete aliphatic ¹³C assignments for both oxidized and reduced thioredoxin are reported. Correlations previously noted between ¹³C chemical shifts and secondary structure are confirmed in this work, and significant differences are observed in the C and C shifts between cis- and trans-proline, consistent with previous work that identifies this as a simple and unambiguous method of identifying cis-proline residues in proteins. Reduction of the disulfide bond in the active-site Cys³²-Gly-Pro-Cys³⁵ sequence causes changes in the ¹H, ¹⁵N and ¹³C chemical shifts of residues close to the active site, some of them quite far distant in the amino acid sequence. Coupling constants, both backbone and side chain, show some differences between the two proteins, and the NOE connectivities and chemical shifts are consistent with small changes in the positions of several side chains, including the two tryptophan rings (Trp²⁸ and Trp³¹). These results show that, consistent with the biochemical behavior of thioredoxin, there are minimal differences in backbone configuration between the oxidized and reduced forms of the protein.     

1H, 13C, and 15N resonance assignment of the C103S mutant of the N-terminal domain of DsbD from Neisseria meningitidis

We report the nearly complete ¹H, ¹³C, and ¹⁵N resonance assignments of the C103S mutant of the N-terminal domain of DsbD from Neisseria meningitides. Secondary structure determination using CSI method leads to the prediction of nine β-sheet parts.     

1H, 15N, 13C and 13CO assignments and secondary structure determination of basic fibroblast growth factor using 3D heteronuclear NMR spectroscopy

Summary The assignments of the ¹H, ¹⁵N, ¹³CO and ¹³C resonances of recombinant human basic fibroblast growth factor (FGF-2), a protein comprising 154 residues and with a molecular mass of 17.2 kDa, is presented based on a series of three-dimensional triple-resonance heteronuclear NMR experiments. These studies employ uniformly labeled ¹⁵N- and ¹⁵N-/¹³C-labeled FGF-2 with an isotope incorporation >95% for the protein expressed in E. coli. The sequence-specific backbone assignments were based primarily on the interresidue correlation of C, C and H to the backbone amide ¹H and ¹⁵N of the next residue in the CBCA(CO)NH and HBHA(CO)NH experiments and the intraresidue correlation of C, C and H to the backbone amide ¹H and ¹⁵N in the CBCANH and HNHA experiments. In addition, C and C chemical shift assignments were used to determine amino acid types. Sequential assignments were verified from carbonyl correlations observed in the HNCO and HCACO experiments and C correlations from the carbonyl correlations observed in the HNCO and HCACO experiments and C correlations from the HNCA experiment. Aliphatic side-chain spin systems were assigned primarily from H(CCO)NH and C(CO)NH experiments that correlate all the aliphatic ¹H and ¹³C resonances of a given residue with the amide resonance of the next residue. Additional side-chain assignments were made from HCCH-COSY and HCCH-TOCSY experiments. The secondary structure of FGF-2 is based on NOE data involving the NH, H and H protons as well as ³J_H n _H coupling constants, amide exchange and ¹³C and ¹³C secondary chemical shifts. It is shown that FGF-2 consists of 11 well-defined antiparallel -sheets (residues 30–34, 39–44, 48–53, 62–67, 71–76, 81–85, 91–94, 103–108, 113–118, 123–125 and 148–152) and a helix-like structure (residues 131–136), which are connected primarily by tight turns. This structure differs from the refined X-ray crystal structures of FGF-2, where residues 131–136 were defined as -strand XI. The discovery of the helix-like region in the primary heparin-binding site (residues 128–138) instead of the -strand conformation described in the X-ray structures may have important implications in understanding the nature of heparin-FGF-2 interactions. In addition, two distinct conformations exist in solution for the N-terminal residues 9–28. This is consistent with the X-ray structures of FGF-2, where the first 17–19 residues were ill defined.     

Deuterium isotope effects in carbohydrates revisited. Cryoprobe studies of the anomerization and NH to ND deuterium isotope induced 13C NMR chemical shifts of acetamidodeoxy and aminodeoxy sugars

Complete ¹H and ¹³C NMR chemical shift assignments have been generated from a series of acetamidodeoxy and aminodeoxy sugar derivatives. For free sugars, the enhanced sensitivity of an NMR cryoprobe allowed simple 1D and 2D NMR spectra to be obtained from essentially single anomers, before significant mutarotation had occurred. The NMR assignments have been used to characterize deuterium isotope effects on ¹³C chemical shifts measured under conditions of slow NH to ND exchange in single solutions. Within a range of 0 to −0.138 ppm, β, γ, δ, and ζ deuterium isotope effects have been observed, thus providing additional reference data for assignment of the ¹³C NMR spectra of nitrogenous saccharides.     

Characterization of the complex formation of 1,6-anhydro-β-maltose and potassium ions using NMR spectroscopy and single-crystal X-ray crystallography

The formation of a complex between 1,6-anhydro-β-maltose and potassium ions was characterized using ¹H, ¹³C and ³⁹K NMR spectroscopy and single-crystal X-ray crystallography. In the NMR study, the spin-lattice relaxation times (T₁) of C1, C3, C5, C6, and C5′ significantly decreased in the presence of potassium ions, and ³⁹K-T₁ also decreased in the presence of 1,6-anhydro-β-maltose, indicating complex formation. In a crystal, both 8- and 9-coordination structures, corresponding to the distorted capped pentagonal bipyramidal structure and the capped hexagonal bipyramidal structure, respectively, were identified. A potassium ion was positioned in the center of each bipyramidal structure.     

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     

1H, 13C and 15N backbone and side chain resonance assignments of the C-terminal domain of CdnL from Myxococcus xanthus

CdnL, a 164-residue protein essential for Myxococcus xanthus viability, is a member of a large family of bacterial proteins of unknown structure and function. Here, we report the ¹H, ¹³C and ¹⁵N backbone and side chain assignments for the stable C-terminal domain of CdnL identified by limited proteolysis.     

Sequence specific 1H, 13C and 15N resonance assignments of a calmodulin-like calcium-binding protein from the protozoan parasite Entamoeba histolytica (EhCaM)

We report almost complete sequence specific ¹H, ¹³C and ¹⁵N NMR assignments of a 151-residue long calmodulin-like calcium-binding protein from Entamoeba histolytica (EhCaM).     

NMR assignment of reduced form of copper, zinc superoxide dismutase from Salmonella enterica

Almost complete assignment (97%) of NMR resonances was obtained for the reduced, Cu(I), form of prokaryotic CuZnSOD from Salmonella enterica. ¹³C direct detection was used to complement the standard bouquet of ¹H detected triple resonance experiments and contributed to the identification of proline backbone resonances and to side chains assignments of Asx, Glx and aromatic rings. This is the only complete assignment available for monomer SOD from prokaryotic organisms.     

The 13C Chemical-Shift Index: A simple method for the identification of protein secondary structure using 13C chemical-shift data

Summary A simple technique for identifying protein secondary structures through the analysis of backbone ¹³C chemical shifts is described. It is based on the Chemical-Shift Index [Wishart et al. (1992) Biochemistry, 31, 1647–1651] which was originally developed for the analysis of ¹H chemical shifts. By extending the Chemical-Shift Index to include ¹³C, ¹³C and carbonyl ¹³C chemical shifts, it is now possible to use four independent chemical-shift measurements to identify and locate protein secondary structures. It is shown that by combining both ¹H and ¹³C chemical-shift indices to produce a consensus estimate of secondary structure, it is possible to achieve a predictive accuracy in excess of 92%. This suggests that the secondary structure of peptides and proteins can be accurately obtained from ¹H and ¹³C chemical shifts, without recourse to NOE measurements.Supplementary material is available in the form of a 10-page table (Table S1) describing the exact location of secondary structures in all 20 proteins as determined using the methods described in this paper. Requests for Table S1 should be directed to the authors.     

C, N CP MAS and high resolution multinuclear NMR study of methyl

Simple pulse schemes are presented for the measurement of methyl ¹³C and ¹H CSA values from ¹H–¹³C dipole/¹³C CSA and ¹H–¹³C dipole/¹H CSA cross-correlated relaxation. The methodology is applied to protein L and malate synthase G. Average ¹³C CSA values are considerably smaller for Ile than Leu/Val (17 vs 25 ppm) and are in good agreement with previous solid state NMR studies of powders of amino acids and dipeptides and in reasonable agreement with quantum-chemical DFT calculations of methyl carbon CSA values in peptide fragments. Small averaged ¹H CSA values on the order of 1 ppm are measured, consistent with a solid state NMR determination of the methyl group ¹H CSA in dimethylmalonic acid.     

Estimates of methyl 13C and 1H CSA values (Δσ) in proteins from cross-correlated spin relaxation

Simple pulse schemes are presented for the measurement of methyl ¹³C and ¹H CSA values from ¹H–¹³C dipole/¹³C CSA and ¹H–¹³C dipole/¹H CSA cross-correlated relaxation. The methodology is applied to protein L and malate synthase G. Average ¹³C CSA values are considerably smaller for Ile than Leu/Val (17 vs 25 ppm) and are in good agreement with previous solid state NMR studies of powders of amino acids and dipeptides and in reasonable agreement with quantum-chemical DFT calculations of methyl carbon CSA values in peptide fragments. Small averaged ¹H CSA values on the order of 1 ppm are measured, consistent with a solid state NMR determination of the methyl group ¹H CSA in dimethylmalonic acid.     

Dictyostelium discoideum as Expression Host: Isotopic Labeling of a Recombinant Glycoprotein for NMR Studies

The advantages of the organism Dictyostelium discoideum as an expression host for recombinant glycoproteins have been exploited for the production of an isotopically labeled cell surface protein for NMR structure studies. Growth medium containing [¹⁵N]NH₄Cl and [¹³C]glycerol was used to generate isotopically labeled Escherichia coli, which was subsequently introduced to D. discoideum cells in simple Mes buffer. A variety of growth conditions were screened to establish minimal amounts of nitrogen and carbon metabolites for a cost-effective protocol. Following single-step purification by anion-exchange chromatography, 8 mg of uniformly ¹³C,¹⁵N-labeled protein secreted by approximately 10¹⁰D. discoideum cells was isolated from 3.3 liters of supernatant. Mass spectrometry showed the recombinant protein of 16 kDa to have incorporated greater than 99.9% isotopic label. The two-dimensional ¹H-¹³C HSQC spectrum confirms ¹³C labeling of both glycan and amino acid residues of the glycoprotein. All heteronuclear NMR spectra showed a good dispersion of cross-peaks essential for high-quality structure determination.     

Biosynthetically directed fractional 13C labeling facilitates identification of Phe and Tyr aromatic signals in proteins

Analysis of 2D [¹³C,¹H]-HSQC spectra of biosynthetic fractionally ¹³C labeled proteins is a reliable, straightforward means to obtain stereospecific assignments of Val and Leu methyl sites in proteins. Herein we show that the same fractionally labeled protein sample facilitates observation and identification of Phe and Tyr aromatic signals. This is the case, in part, because the fractional ¹³C labeling yields aromatic rings in which some of the ¹³C-¹³C J-couplings, present in uniformly labeled samples, are absent. Also, the number of homonuclear J-coupling partners differs for the -, - and -carbons. This enabled us to vary their signal intensities in distinctly different ways by appropriately setting the ¹³C constant-time period in 2D [¹³C,¹H]-HSQC spectra. We illustrate the application of this approach to an 18 kDa protein, c-VIAF, a modulator of apoptosis. In addition, we show that cancellation of the aromatic ¹³C CSA and ¹³C-¹H dipolar interactions can be fruitfully utilized in the case of the fractionally labeled sample to obtain high resolution ¹³C constant-time spectra with good sensitivity.     

Novel three-dimensional 1H−13C−31P triple resonance experiments for sequential backbone correlations in nucleic acids

Summary Backbone-driven assignment methods that utilize covalent connectivities have greatly facilitated spectral assignments of proteins. In nucleic acids, ¹H–¹³C–³¹P correlations could play a similar role, and several related experiments (HCP) have recently been presented for backbone-driven sequential assignments in RNA. The three-dimensional extension of ¹H–³¹P Het-Cor (P,H-COSY-H,C-HMQC) and Het-TOCSY (P,H-TOCSY-H,C-HMQC) experiments presented here complements HCP experiments as tools for spectral assignments and extraction of dihydral angle constraints. By relying on ¹H–³¹P rather than ¹³C–³¹P couplings to generate cross peaks, the strongest connectivities are observed in different spectral regions, increasing the likelihood of resolving spectral overlap. In addition, semiquantitative estimates of ¹H–³¹P and ¹³C–³¹P couplings provide dihedral angle constraints for RNA structure determination.