15N-labeled Protein Research Articles

The bioavailability and postprandial utilisation of sweet lupin (Lupinus albus)-flour protein is similar to that of purified soyabean protein in human subjects: a study using intrinsically15N-labelled proteins

Sweet lupin (Lupinus albus), a protein-rich legume devoid of anti-nutritional factors, is considered to have a high potential for protein nutrition in man. Results concerning the nutritional value of lupin protein are, however, conflicting in animals and very scarce in human subjects. Furthermore, where fibre-rich protein sources are concerned, the long-term nutritional results are often obscured, particularly since fibre-promoted colonic fermentation may bias the energy supply and redistribute N flux. We therefore studied, during the postprandial phase, the bioavailability and utilisation of lupin-flour protein in nine healthy men who had ingested a mixed meal containing intrinsically15N-labelled lupin flour as the protein source (Expt 1). The real ileal digestibility (RID) and ileal endogenous N losses (IENL) were assessed using a perfusion technique at the terminal ileum, and the N content and15N enrichment of ileal samples. Lupin flour exhibited a high RID of 91 (SD 3) % AND LOW IENL (5·4 (sd 1·3) mmol N/h). Postprandial dietary deamination was also assessed from body dietary urea and urinary dietary N excretion, and compared with results in nine healthy men following an isoenergetic meal containing a15N-soyabean-protein isolate with a similar RID, as a control (Expt 2). Postprandial dietary deamination was similar after lupin and soyabean meals (17 (sd 2) and 18 (sd 4) % ingested N respectively). We therefore conclude that lupin protein is highly bioavailable, even if included in fibre-rich flour, and that it can be used with the same efficiency as soyabean protein to achieve postprandial protein gain in healthy human subjects.

Solution structure of a phage-derived peptide antagonist in complex with vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a potent endothelial cell-specific mediator of angiogenesis and vasculogenesis. VEGF is involved pathologically in cancer, proliferative retinopathy and rheumatoid arthritis, and as such represents an important therapeutic target. Three classes of disulfide-constrained peptides that antagonize binding of the VEGF dimer to its receptors, KDR and Flt-1, were identified previously using phage display methods. NMR studies of a representative peptide from the most potent class of these peptide antagonists, v107 (GGNECDAIRMWEWECFERL), were undertaken to characterize its interactions with VEGF. v107 has no defined structure free in solution, but binding to VEGF induces folding of the peptide. The solution structure of the VEGF receptor-binding domain-v107 complex was determined using 3940 (1970 per VEGF monomer) internuclear distance and 476 (238 per VEGF monomer) dihedral angle restraints derived from NMR data obtained using samples containing either 13C/ 15N-labeled protein plus excess unlabeled peptide or 13C/ 15N-labeled peptide plus excess unlabeled protein. Residual dipolar coupling restraints supplemented the structure determination of the complex and were found to increase significantly both the global precision of VEGF in the complex and the agreement with available crystal structures of VEGF. The calculated ensemble of structures is of high precision and is in excellent agreement with the experimental restraints. v107 has a turn-helix conformation with hydrophobic residues partitioned to one face of the peptide and polar or charged residues at the other face. Contacts between two v107 peptides and the VEGF dimer are mediated by primarily hydrophobic side-chain interactions. The v107-binding site on VEGF overlaps partially with the binding site of KDR and is similar to that for domain 2 of Flt-1. The structure of the VEGF-v107 complex provides new insight into how binding to VEGF can be achieved that may be useful for the design of small molecule antagonists.

Measurement of conformational constraints in an elastin-mimetic protein by residue-pair selected solid-state NMR.

We introduce a solid-state NMR technique for selective detection of a residue pair in multiply labeled proteins to obtain site-specific structural constraints. The method exploits the frequency-offset dependence of cross polarization to achieve 13COi-->15Ni-->13Calphai transfer between two residues. A 13C, 15N-labeled elastin mimetic protein (VPGVG)n, is used to demonstrate the method. The technique selected the Gly3 Calpha signal while suppressing the Gly5 Calpha signal, and allowed the measurement of the Gly3 Calpha chemical shift anisotropy to derive information on the protein conformation. This residue-pair selection technique should simplify the study of protein structure at specific residues.

Enhanced diagonal peak suppression in three‐dimensional TROSY‐type 15N‐resolved 1HN1HN NOESY spectra

AbstractA new method for enhanced diagonal peak suppression in the three‐dimensional TROSY–NOESY experiment is presented. The main source of residual diagonal signals in the conventional technique is spin flips of the passive 15N spins during the 1H1H NOESY mixing period. The new pulse sequence does not suppress this pathway, but it generates peaks at the positions of the relaxation‐induced artifacts with opposite phase of the artifacts so that cancellation occurs. The new technique is demonstrated using an 15N‐labeled protein sample, Neural Cell Adhesion Molecule (NCAM) 213–308 in H2O at 500 MHz. © 2002 John Wiley & Sons, Inc. Concepts Magn Reson 14: 1–8, 2002

Cost-effective and uniform (13)C- and (15)N-labeling of the 24-kDa N-terminal domain of the Escherichia coli gyrase B by overexpression in the photoautotrophic cyanobacterium Anabaena sp. PCC 7120.

Structural studies of biomolecules using nuclear magnetic resonance (NMR) rely on the availability of samples enriched in (13)C and (15)N isotopes. While (13)C/(15)N-labeled proteins are generally obtained by overexpression in transformed Escherichia coli cells cultured in the presence of an expensive mixture of labeled precursors, those of the photoautotrophic cyanobacterium Anabaena sp. PCC 7120 can be uniformly labeled by growing them in medium containing Na(15)NO(3) and NaH(13)CO(3) as the sole nitrogen and carbon sources. We report here a novel vector-host system suitable for the efficient preparation of uniformly (13)C/(15)N-labeled proteins in Anabaena sp. PCC 7120. The 24-kDa N-terminal domain of the E. coli gyrase B subunit, used as a test protein, was cloned into the pRL25C shuttle vector under the control of the tac promoter. The transformed Anabaena cells were grown in the presence of the labeled mineral salts and culture conditions were optimized to obtain over 90% of (13)C and (15)N enrichment in the constitutively expressed 24-kDa polypeptide. The yield of purified 24-kDa protein after dual isotope labeling under anaerobic conditions was similar to that obtained with E. coli cells bearing a comparable expression vector and cultured in parallel in a commercially available labeling medium. Furthermore, as probed by NMR spectroscopy and mass spectrometry, the 24-kDa N-terminal domain expressed in Anabaena was identical to the E. coli sample, demonstrating that it was of sufficient quality for 3D-structure determination. Because the Anabaena system was far more advantageous taking into consideration the expense for the labels that were necessary, these results indicate that Anabaena sp. PCC 7120 is an economic alternative for the (13)C/(15)N-labeling of soluble recombinant proteins destined for structural studies.

Structure of a Conjugating Enzyme-Ubiquitin Thiolester Intermediate Reveals a Novel Role for the Ubiquitin Tail

Background: Ubiquitin-conjugating enzymes (E2s) are central enzymes involved in ubiquitin-mediated protein degradation. During this process, ubiquitin (Ub) and the E2 protein form an unstable E2-Ub thiolester intermediate prior to the transfer of ubiquitin to an E3-ligase protein and the labeling of a substrate for degradation. A series of complex interactions occur among the target substrate, ubiquitin, E2, and E3 in order to efficiently facilitate the transfer of the ubiquitin molecule. However, due to the inherent instability of the E2-Ub thiolester, the structural details of this complex intermediate are not known. Results: A three-dimensional model of the E2-Ub thiolester intermediate has been determined for the catalytic domain of the E2 protein Ubc1 (Ubc1 Δ450) and ubiquitin from S. cerevisiae. The interface of the E2-Ub intermediate was determined by kinetically monitoring thiolester formation by 1H- 15N HSQC spectra by using combinations of 15N-labeled and unlabeled Ubc1 Δ450 and Ub proteins. By using the surface interface as a guide and the X-ray structures of Ub and the 1.9 Å structure of Ubc1 Δ450 determined here, docking simulations followed by energy minimization were used to produce the first model of a E2-Ub thiolester intermediate. Conclusions: Complementary surfaces were found on the E2 and Ub proteins whereby the C terminus of Ub wraps around the E2 protein terminating in the thiolester between C88 (Ubc1 Δ450) and G76 (Ub). The model supports in vivo and in vitro experiments of E2 derivatives carrying surface residue substitutions. Furthermore, the model provides insights into the arrangement of Ub, E2, and E3 within a ternary targeting complex.

Guar gum does not impair the absorption and utilization of dietary nitrogen but affects early endogenous urea kinetics in humans

Viscous gums enhance viscosity in the upper gastrointestinal lumen, quickly disturbing motility and promoting fluid secretion. We sought to determine whether guar gum could acutely affect the absorption and utilization of dietary nitrogen and whether these luminal effects could also perturb the kinetics of urea. We studied the short-term effect of adding 1% of highly viscous guar gum to a (15)N-labeled protein meal (30 g soy protein isolate in 500 mL water) during the postprandial phase in humans. The effects on bioavailability were studied by using the [(13)C]glycine breath test (to assess gastric emptying) and (15)N enrichment in plasma amino acids (for systemic amino acid bioavailability). The kinetics of dietary and endogenous urea were assessed in plasma and urine. Guar gum modulated the gastric emptying kinetics of the liquid phase of the meal slightly (P < 0.05), but had no significant effect on either the systemic appearance of dietary amino acids or plasma and urinary dietary urea kinetics. Without significantly affecting plasma urea concentrations, guar gum reduced by approximately 40% the urinary excretion of endogenous urea for the first 2-h period after the meal (P < 0.01), although endogenous urinary excretion was similar at later stages. Guar gum did not significantly affect the bioavailability or utilization of dietary protein. We showed an early effect of guar gum on endogenous urea kinetics, which most probably arose from very early, short-term stimulation of the intestinal disposal of endogenous urea, at the expense of its urinary excretion.

NMR monitoring of accumulation and folding of 15N-labeled protein overexpressed in Pichia pastoris.

Postgenomic studies have led to an increasing demand for isotope-labeled proteins. We present a method for producing large quantities of truly native (15)N-labeled protein. Based on the secretion capabilities of the yeast Pichia pastoris, the recombinant protein is easily purified in a single step as it is secreted. Control of all nitrogen sources permits very high labeling yields. As a result, accumulation and folding of the recombinant protein can be monitored by heteronuclear NMR without purification. Comparison of sample spectra with the spectrum of the purified recombinant protein allows detection of the secreted protein in the culture and monitoring of its folding, from the start of the induction phase. The detection limit for a (15)N-labeled protein is estimated as 20 microM and corresponds, for a 10-kDa protein, to a load of 40 mg/liter in the fermentor. This concentration is reached by most reported preparations in P. pastoris. Further concentration by ultrafiltration would compensate for lower production. This procedure may be useful in many structural genomics and combinatorial chemistry screening projects where most protein productions meet the requirements for this method.

A comprehensive analysis of multifield 15N relaxation parameters in proteins: determination of 15N chemical shift anisotropies.

This study deals with the exploitation of the three classical 15N relaxation parameters (the longitudinal relaxation rate, R1, the transverse relaxation rate, R2, and the 1H-15N cross-relaxation rate, sigmaNH) measured at several magnetic fields in uniformly 15N-labeled proteins. Spectral densities involved in R1, R2 and sigmaNH are analyzed according to the functional form A + B/(1 + omega(2) taus(2)), where taus is the correlation time associated with slow motions sensed by the NH vector at the level of the residue to which it belongs. The coefficient B provides a realistic view of the backbone dynamics, whereas A is associated with fast local motions. According to the "model free approach", B can be identified with 2tausS(2) where S is the generalized order parameter. The correlation time taus is determined from the field dependency of the relaxation parameters while A and B are determined through linear equations. This simple data processing is needed for obtaining realistic error bars based on a statistical approach. This proved to be the key point for validating an extended analysis aiming at the determination of nitrogen chemical shift anisotropy. The protein C12A-p8(MTCP1) has been chosen as a model for this study. It will be shown that all data (obtained at five magnetic field strengths corresponding to proton resonance of 400, 500, 600, 700, and 800 MHz) are very consistently fitted provided that a specific effective correlation time associated with slow motions is defined for each residue. This is assessed by small deviations between experimental and recalculated values, which, in all cases, remain within experimental uncertainty. This strategy makes needless elaborate approaches based on the combination of several slow motions or their possible anisotropy. Within the core of the protein taus fluctuates in a relatively narrow range (with a mean value of 6.15 ns and a root-mean-square deviation of 0.36 ns) while it is considerably reduced at the protein extremities (down to approximately 3 ns). To a certain extent, these fluctuations are correlated with the protein structure. A is not obtained with sufficient accuracy to be valuably discussed. Conversely, order parameters derived from B exhibit a significant correlation with the protein structure. Finally, the multi-field analysis of the evolution of longitudinal and transverse relaxation rates has been refined by allowing the 15N chemical shift anisotropy (csa) to vary residue by residue. Within uncertainties (derived here on a statistical basis) an almost constant value is obtained. This strongly indicates an absence of correlation between the experimental value of this parameter obtained for a given residue in the protein, the nature of this residue, and the possible involvement of this residue in a structured area of the protein.

NMR structure of human apolipoprotein C-II in the presence of sodium dodecyl sulfate.

The structure and protein-detergent interactions of apolipoprotein C-II (apoC-II) in the presence of SDS micelles have been investigated using circular dichroism and heteronuclear NMR techniques applied to (15)N-labeled protein. Micellar SDS, a commonly used mimetic of the lipoprotein surface, inhibits the aggregation of apoC-II and induces a stable structure containing approximately 60% alpha-helix as determined by circular dichroism. NMR reveals the first 12 residues of apoC-II to be structurally heterogeneous and largely disordered, with the rest of the protein forming a predominantly helical structure. Three regions of helical conformation, residues 16-36, 50-56, and 63-77, are well-defined by NMR-derived constraints, with the intervening regions showing more loosely defined helical conformation. The structure of apoC-II is compared to that determined for other apolipoproteins in a similar environment. Our results shed light on the lipid interactions of apoC-II and its mechanism of lipoprotein lipase activation.

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Three-dimensional structure topology of the calreticulin P-domain based on NMR assignment

Calreticulin (CRT) is an abundant molecular chaperone of the endoplasmic reticulum. Its central, proline-rich P-domain, comprising residues 189–288, contains three copies of each of two repeat sequences (types 1 and 2), which are arranged in a characteristic ‘111222’ pattern. Here we show that the three-dimensional structure of CRT(189–288) contains a single hairpin fold formed by the entire polypeptide chain. The loop at the bottom of the hairpin consists of residues 227–247, and is closed by an anti-parallel β-sheet of residues 224–226 and 248–250. Two additional β-sheets contain residues 207–209 and 262–264, and 190–192 and 276–278. The 17-residue spacing of the β-strands in the N-terminal part of the hairpin and the 14-residue spacing in the C-terminal part reflect the length of the type 1 and type 2 sequence repeats. As a consequence of this topology the peptide segments separating the β-strands in the N-terminal part of the hairpin are likely to form bulges to accommodate the extra residues. These results are based on nearly complete sequence-specific NMR assignments for CRT(189–288), which were obtained using standard NMR techniques with the 13C/ 15N-labeled protein, and collection of nuclear Overhauser enhancement upper distance constraints.

Calmodulin tagging provides a general method of using lanthanide induced magnetic field orientation to observe residual dipolar couplings in proteins in solution.

A general method is presented for magnetic field alignment of proteins in solution. By tagging a target protein with calmodulin saturated with paramagnetic lanthanide ions it is possible to measure substantial residual dipolar couplings (RDC) whilst minimising the effects of pseudocontact shifts on the target protein. A construct was made consisting of a calmodulin-binding peptide (M13 from sk-MLCK) attached to a target protein, dihydrofolate reductase in this case. The engineered protein binds tightly to calmodulin saturated with terbium, a paramagnetic lanthanide ion. By using only a short linker region between the M13 and the target protein, some of the magnetic field alignment induced in the CaM(Tb3+)4 is effectively transmitted to the target protein (DHFR). 1H-15N HSQC IPAP experiments on the tagged complex containing 15N-labelled DHFR-M13 protein and unlabelled CaM(Tb3+)4 allow one to measure RDC contributions in the aligned complex. RDC values in the range +4.0 to -7.4 Hz were measured at 600 MHz. Comparisons of 1H-15N HSQC spectra of 15N-DHFR-M13 alone and its complexes with CaM(Ca2+)4 and CaM(Tb3+)4 indicated that (i) the structure of the target protein is not affected by the complex formation and (ii) the spectra of the target protein are not seriously perturbed by pseudocontact shifts. The use of a relatively large tagging group (CaM) allows us to use a lanthanide ion with a very high magnetic susceptibility anisotropy (such as Tb3+) to give large alignments while maintaining relatively long distances from the target protein nuclei (and hence giving only small pseudocontact shift contributions).

7] Nuclear magnetic resonance relaxation in determination of residue-specific 15N chemical shift tensors in proteins in solution: Protein dynamics, structure, and applications of transverse relaxation optimized spectroscopy

We developed several approaches to direct determination of the 15N CSA from relaxation measurements in uniformly 15N-labeled proteins in solution. These methods are based on multiple-field measurements and could be extended to other nuclei in proteins and other molecules. Combined with the isotropic chemical shift measurements, this provides an experimental approach to full characterization of chemical shift tensors in proteins in their native milieu, which is likely to provide valuable information on the nature of chemical shifts and their relation to protein structure. Knowledge of 15N CSA is essential for an accurate characterization of protein dynamics from relaxation measurements.

Rapid determination of protein folds using residual dipolar couplings

Over the next few years, various genome projects will sequence many new genes and yield many new gene products. Many of these products will have no known function and little, if any, sequence homology to existing proteins. There is reason to believe that a rapid determination of a protein fold, even at low resolution, can aid in the identification of function and expedite the determination of structure at higher resolution. Recently devised NMR methods of measuring residual dipolar couplings provide one route to the determination of a fold. They do this by allowing the alignment of previously identified secondary structural elements with respect to each other. When combined with constraints involving loops connecting elements or other short-range experimental distance information, a fold is produced. We illustrate this approach to protein fold determination on 15N-labeled Eschericia coli acyl carrier protein using a limited set of 15N- 1H and 1H- 1H dipolar couplings. We also illustrate an approach using a more extended set of heteronuclear couplings on a related protein, 13C, 15N-labeled NodF protein from Rhizobium leguminosarum.

Expression, purification, refolding, and characterization of recombinant human interleukin-13: utilization of intracellular processing.

Interleukin-13 (IL-13) is a pleiotropic cytokine that elicits both proinflammatory and anti-inflammatory immune responses. Recent studies underscore its role in several diseases, including asthma and cancer. Solution studies of IL-13 and its soluble receptors may facilitate the design of antagonists/agonists which would require milligram quantities of specifically labeled protein. A synthetic gene encoding human IL-13 (hIL-13) was inserted into the pMAL-c2 vector with a cleavage site for the tobacco etch virus (TEV) protease. Coexpression of the fusion protein and TEV protease led to in vivo cleavage, resulting in high levels of hIL-13 production. hIL-13, localized to inclusion bodies, was purified and refolded to yield approximately 2 mg per liter of bacteria grown in minimal media. Subsequent biochemical and biophysical analysis of both the unlabeled and (15)N-labeled protein revealed a bioactive helical monomer. In addition, the two disulfide bonds were unambiguously demonstrated to be Cys29-Cys57 and Cys45-Cys71 by a combined proteolytic digestion and mass spectrometric analysis.

Structural Characterization of the Cysteine-rich Domain of TFIIH p44 Subunit

In an effort to understand the structure function relationship of TFIIH, a transcription/repair factor, we focused our attention on the p44 subunit, which plays a central role in both mechanisms. The amino-terminal portion of p44 has been shown to be involved in the regulation of the XPD helicase activity; here we show that its carboxyl-terminal domain is essential for TFIIH transcription activity and that it binds three zinc atoms through two independent modules. The first contains a C4 zinc finger motif, whereas the second is characterized by a CX(2)CX(2-4)FCADCD motif, corresponding to interleaved zinc binding sites. The solution structure of this second module reveals an unexpected homology with the regulatory domain of protein kinase C and provides a framework to study its role at the molecular level.

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 [(15)N]NH(4)Cl and [(13)C]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 (13)C,(15)N-labeled protein secreted by approximately 10(10) D. 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 (1)H-(13)C HSQC spectrum confirms (13)C 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.

Encapsulation of protein in humic acid from a histosol as an explanation for the occurrence of organic nitrogen in soil and sediment

Recent work suggests that nitrogen in humic acids exists primarily as amide functional groups that mirror those in protein. However, the mode for the existence of such labile materials as protein still remains unclear. With the combined applications of NMR spectroscopy, tetramethylammonium hydroxide (TMAH) thermochemolysis, pyrolysis/GC/MS, and elemental analysis, we propose that the survival of proteins in humic acids is carried out by encapsulation into hydrophobic domains of humic acids. To test this hypothesis, we simulated encapsulation of 15N-labeled protein extracts into humic acids and demonstrated that complete hydrolysis of the protein is prevented by the encapsulating humic acid. Results from this study constitute evidence to support the encapsulation mechanism involved in the formation of refractory organic nitrogen during sediment diagenesis.

Molten globule structure of equine β-lactoglobulin probed by hydrogen exchange

The molten globule structure of equine β-lactoglobulin has been inferred from the hydrogen exchange protection of the backbone amide protons. In order to make it possible to measure the hydrogen exchange kinetics of the individual backbone amide protons, the uniformly 15N-labeled recombinant protein was expressed in Escherichia coli and the NMR peak assignment was obtained for most of the backbone protons. The chemical shift and NOE results obtained under the condition where the protein assumes the native structure are fully consistent with the known secondary structure of bovine β-lactoglobulin, indicating that the equine protein has a similar native conformation to that of the bovine protein. The hydrogen exchange rate of the individual backbone amide protons was measured under the conditions where the protein assumes the native and molten globule states. In the native state, strong protection was observed for the residues located in the eight (A to H) strands, which form a barrel structure, and residues of a major helix. In the molten globule state at acidic pH conditions, significant protection from the exchange has been observed for residues located in the A, F, G and H strands in the native structure. The pattern of protection is consistent with a native-like β-sheet formation by these strands. The residues located in a major helix of the native structure are also protected, suggesting that this helix is formed in the molten globule and is packed against the sheet as in the native structure. These results indicate that a native-like subdomain is formed in the molten globule state of equine β-lactoglobulin.