NMR with 13C, 15N-doubly-labeled DNA: The shape Antennapedia homeodomain complex with a 14-mer DNA duplex

Nearly complete ¹H, ¹³C and¹⁵ N NMR assignments have been obtained for a doubly labeled 14-base pair DNA duplex in solution both in the free state and complexed with the uniformly ¹⁵N-labeled Antennapedia homeodomain. The DNA was either fully ¹³C,¹⁵N-labeled or contained uniformly ¹³C, ¹⁵N-labeled nucleotides only at those positions which form the protein–DNA interface in the previously determined NMR solution structure of the Antennapedia homeodomain–DNA complex. The resonance assignments were obtained in three steps: (i) identification of the deoxyribose spin systems via scalar couplings using 2D and 3D HCCH-COSY and soft-relayed HCCH-COSY; (ii) sequential assignment of the nucleotides via¹ H–¹H NOEs observed in 3D¹³ C-resolved NOESY; and (iii) assignment of the imino and amino groups via ¹H–¹H NOEs and¹⁵ N–¹H correlation spectroscopy. The assignment of the duplex in the 17 kDa protein–DNA complex was greatly facilitated by the fact that ¹H signals of the protein were filtered out in ¹³C-resolved spectroscopy and by the excellent carbon chemical shift dispersion of the DNA duplex. Comparison of corresponding ¹³C chemical shifts of the free and the protein-bound DNA indicates conformational changes in the DNA upon complex formation.     

Overexpression, on-column refolding and isotopic labeling of Hahellin from Hahella chejuensis, a putative member of the betagamma-crystallin superfamily

A gene which encodes a hypothetical protein of 40 kDa has been identified in the genome of a marine bacterium Hahella chejuensis, as a putative member of βγ-crystallin superfamily. This hypothetical protein contains a putative βγ-crystallin-like domain, along with other domains for carbohydrate binding regions. It is named as Hahellin. A PCR amplified stretch of 92-amino acid residue long protein was cloned into pET21a vector and overexpressed in Escherichia coli strain BL21(DE3)pLysS cells. The recombinant Hahellin, produced as inclusion bodies, was estimated to be around 50% of the total cellular protein content which was solubilized in 8 M urea. The protein was purified and refolded using an anion exchange column. The MALDI-TOF mass spectrometry revealed the purity and monomeric nature of the protein. Further, a method to prepare isotopically (¹⁵N/¹³C) labeled protein with high yield for NMR studies is reported. The uniformly ¹⁵N-labeled Hahellin thus produced has been characterized by recording a sensitivity enhanced 2D [¹⁵N]–[¹H] HSQC spectrum. The well, dispersed peaks in the spectrum confirm that the protein is indeed well folded and suitable for further studies by NMR.     

Orientation and Order of the Amide Group of Sphingomyelin in Bilayers Determined by Solid-State NMR

Sphingomyelin (SM) and cholesterol (Chol) are considered essential for the formation of lipid rafts; however, the types of molecular interactions involved in this process, such as intermolecular hydrogen bonding, are not well understood. Since, unlike other phospholipids, SM is characterized by the presence of an amide group, it is essential to determine the orientation of the amide and its order in the lipid bilayers to understand the nature of the hydrogen bonds in lipid rafts. For this study, 1′-¹³C-2-¹⁵N-labeled and 2′-¹³C-2-¹⁵N-labeled SMs were prepared, and the rotational-axis direction and order parameters of the SM amide in bilayers were determined based on ¹³C and ¹⁵N chemical-shift anisotropies and intramolecular ¹³C-¹⁵N dipole coupling constants. Results revealed that the amide orientation was minimally affected by Chol, whereas the order was enhanced significantly in its presence. Thus, Chol likely promotes the formation of an intermolecular hydrogen-bond network involving the SM amide without significantly changing its orientation, providing a higher order to the SM amide. To our knowledge, this study offers new insight into the significance of the SM amide orientation with regard to molecular recognition in lipid rafts, and therefore provides a deeper understanding of the mechanism of their formation.     

Efficient assignment of methyl resonances: Enhanced sensitivity by gradient selection in a DE-MQ–(H)CC<Superscript><Emphasis Type="Italic">m</Emphasis></Superscript>Ht <Superscript><Emphasis Type="Italic">m</Emphasis></Superscript>–TOCSY experiment

We present a gradient selected and doubly sensitivity-enhanced DE-MQ–(H)CC ^m H ^m –TOCSY experiment for the sequence-specific assignment of methyl resonances in ¹³C,¹⁵N labeled proteins. The proposed experiment provides improved sensitivity and artifact suppression relative to the phase-cycled experiments. One part of the ¹³Cchemical shift evolution takes place under heteronuclear multiple quantum coherence, whereas the other part occurs under ¹³C single quantum coherence in a semi-constant time fashion. The feasibility of the experiment was assessed using ¹⁵N,¹³C labeled Mus musculus coactosin (16 kDa), having a rotational correlation time of 14.5 ns at 15 °C in D₂O. A 16-h experiment on 600 MHz ¹H yielded good quality data and enabled the assignment of 70 out of 72 methyl groups in coactosin. As well as being an improved approach for methyl resonance assignment, this experiment can also be highly valuable for the rapid assignment of methyl resonances in SAR by NMR studies.     

Does Solid-state <Superscript>15</Superscript>N NMR Spectroscopy Detect all Soil Organic Nitrogen?

Virtually all of the N detected by ¹⁵N cross polarization (CP) NMR spectra of four HF-treated soil clay fractions is amide N. However, the intensity of this ¹⁵N CP NMR signal (per unit N) is 27–57% lower than detected for a wheat protein, gliadin. There are two possible explanations – either the amide N in the soil clay fractions produces proportionately less NMR signal than does the amide N in gliadin, or part of the N in the soil clay fractions produces little or no NMR signal. The cross polarization dynamics of the gliadin amide resonance and amide resonances detected for the soil clay fractions are very similar and thus should produce similar amounts of signal, ruling out the first possibility. Therefore up to half or even more of the organic N in these soil clay fractions must be in a form that is insensitive to NMR detection. For a model compound (caffeine), non-protonated heterocyclic N produced less than 20% of the signal of an equivalent amount of amide N in gliadin. Results from several ¹³C NMR techniques provide further evidence that much of the undetected N in the soil clay fractions may be heterocyclic.     

Acceleration of protein backbone NMR assignment by combinatorial labeling: Application to a small molecule binding study

Selective labeling with stable isotopes has long been recognized as a valuable tool in protein NMR to alleviate signal overlap and sensitivity limitations. In this study, combinatorial ¹⁵N‐, ¹³C^α‐, and ¹³C'‐selective labeling has been used during the backbone assignment of human cyclophilin D to explore binding of an inhibitor molecule. Using a cell‐free expression system, a scheme that involves ¹⁵N, 1‐¹³C, 2‐¹³C, fully ¹⁵N/¹³C, and unlabeled amino acids was optimized to gain a maximum of assignment information from three samples. This scheme was combined with time‐shared triple‐resonance NMR experiments, which allows a fast and efficient backbone assignment by giving the unambiguous assignment of unique amino acid pairs in the protein, the identity of ambiguous pairs and information about all 19 non‐proline amino acid types. It is therefore well suited for binding studies where de novo assignments of amide ¹H and ¹⁵N resonances need to be obtained, even in cases where sensitivity is the limiting factor.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N backbone resonance assignment of the lamin C-terminal region specific to prelamin A

Lamins are the main components of the nucleoskeleton. They form a protein meshwork that underlies the inner nuclear membrane. Mutations in the LMNA gene coding for A-type lamins (lamins A and C) cause a large panel of human diseases, referred to as laminopathies. These diseases include muscular dystrophies, lipodystrophies and premature aging diseases. Lamin A exhibits a C-terminal region that is different from lamin C and is post-translationally modified. It is produced as prelamin A and it is then farnesylated, cleaved, carboxymethylated and cleaved again in order to become mature lamin A. In patients with the severe Hutchinson–Gilford progeria syndrome, a specific single point mutation in LMNA leads to an aberrant splicing of the LMNA gene preventing the post-translational processing of prelamin A. This leads to the accumulation of a permanently farnesylated lamin A mutant lacking 50 amino acids named progerin. We here report the NMR ¹H, ¹⁵N, ¹³CO, ¹³Cα and ¹³Cβ chemical shift assignment of the C-terminal region that is specific to prelamin A, from amino acid 567 to amino acid 664. We also report the NMR ¹H, ¹⁵N, ¹³CO, ¹³Cα and ¹³Cβ chemical shift assignment of the C-terminal region of the progerin variant, from amino acid 567 to amino acid 614. Analysis of these chemical shift data confirms that both prelamin A and progerin C-terminal domains are largely disordered and identifies a common partially populated α-helix from amino acid 576 to amino acid 585. This helix is well conserved from fishes to mammals.     

Hydrogen exchange of monomeric alpha-synuclein shows unfolded structure persists at physiological temperature and is independent of molecular crowding in Escherichia coli

Amide proton NMR signals from the N-terminal domain of monomeric α-synuclein (αS) are lost when the sample temperature is raised from 10°C to 35°C at pH 7.4. Although the temperature-induced effects have been attributed to conformational exchange caused by an increase in α-helix structure, we show that the loss of signals is due to fast amide proton exchange. At low ionic strength, hydrogen exchange rates are faster for the N-terminal segment of αS than for the acidic C-terminal domain. When the salt concentration is raised to 300 mM, exchange rates increase throughout the protein and become similar for the N- and C-terminal domains. This indicates that the enhanced protection of amide protons from the C-terminal domain at low salt is electrostatic in nature. Cα chemical shift data point to <10% residual α-helix structure at 10°C and 35°C. Conformational exchange contributions to R2 are negligible at both temperatures. In contrast to the situation in vitro, the majority of amide protons are observed at 37°C in ¹H-¹⁵N HSQC spectra of αS encapsulated within living Escherichia coli cells. Our finding that temperature effects on αS NMR spectra can be explained by hydrogen exchange obviates the need to invoke special cellular factors. The retention of signals is likely due to slowed hydrogen exchange caused by the lowered intracellular pH of high-density E. coli cultures. Taken together, our results emphasize that αS remains predominantly unfolded at physiological temperature and pH—an important conclusion for mechanistic models of the association of αS with membranes and fibrils.     

Production of amino acids by Azotobacter vinelandii and Azotobacter chroococcum with phenolic compounds as sole carbon source under diazotrophic and adiazotrophic conditions

Summary. Azotobacter vinelandii strain ATCC 12837 and Azotobacter chroococcum strain H23 (CECT4435) were tested to grow in N-free or NH₄Cl amended chemically defined media, with protocatechuic acid or sodium p-hydroxybenzoate as sole carbon (C) sources at a concentration of 2 mmol/L. Both substrates supported grow at similar rates than bacteria grown in control media amended with 2 mmol/L sodium succinate as C source. The two strains produced aspartic acid, serine, glutamic acid, glycine, hystidine, threonine, arginine, alanine, proline, cysteine, tyrosine, valine, methionine, lysine, isoleucine, leucine and phenylalanine after 72 h of growth in chemically defined media with 2 mmol/L of phenolic compounds or sodium succinate as sole C source amended or unamended with 0.1% (w/v) NH₄Cl. Qualitative and quantitative production of all amino acids was not affected by the use of different C and N substrates.     

Resource use by salmonids in riverine,lacustrine and marine environments: Evidence from stable isotope analysis

Synopsis We measured stable isotope ratios (δ¹³C and δ¹⁵N) of invertebrates, Atlantic salmon, Salmo salar, and brook trout, Salvelinus fontinalis, in three distinct freshwater environments (headwater tributary, ultra-oligotrophic lake, and main-stem river) in the Western Brook system, Newfoundland, Canada. Large differences in the stable carbon signatures of invertebrates allowed the identification of organic matter assimilation from each environment by resident parr and migrating smolts. Brook trout captured in the headwater tributary in June had a carbon signature characteristic of the tributary, while those collected in August had enriched ¹³C (maximum = −15.6‰) and ¹⁵N (maximum = 12.8‰) values. These enriched carbon and nitrogen signatures were indicative of foraging at sea. There was a low correlation between δ¹³C and δ¹⁵N (r² = 0.198) for individual fish that was likely due to the confounding influence of trout feeding in the lake and the lower main-stem of the river, where δ¹³C of food sources was high but δ¹⁵N was low. Smolts emigrating from Western Brook Pond where they had been foraging (based on lacustrine carbon signatures) were significantly larger than those emigrating from a nursery brook and the main river in the same basin, despite having the same median age. These results suggest better growth opportunities in the lake environment. Trout fork length was positively correlated with δ¹³C and δ¹⁵N, demonstrating that larger individuals had been feeding outside the brook. These results support previous studies that found increased growth potential for salmonids in lacustrine and marine environments, and further, indicate possible adaptive advantages for salmonid movement away from natal brooks.     

Efficient enzymatic synthesis of 13 C,15N-labeled DNA for NMR studies

The power of heteronuclear NMR spectroscopy to study macromoleculesand their complexes has been amply demonstrated over the last decade. Theobstacle to routinely applying these techniques to the study of DNA has beenthe synthesis of ¹³C,¹⁵N-labeled DNA. Here wepresent a simple and efficient method to generate isotope-labeled DNA forNMR studies that is as easy as that for isotope labeling of RNA. The methodwas used to synthesize a uniformly¹³ C,¹⁵N-labeled 32-nucleotide DNA that binds tohuman basic fibroblast growth factor with high affinity and specificity.Isotope-edited experiments were applied to the¹³ C,¹⁵N-labeled DNA bound to unlabeled protein,and the ¹³ C,¹⁵N-labeled DNA was also examined incomplex with ¹⁵N-labeled protein. The NMR experiments showthat the DNA adopts a well-defined stable structure when bound to theprotein, and illustrate the potential of¹³ C,¹⁵N-labeled DNA for structural studies ofDNA–protein complexes.     

New 13C-detected experiments for the assignment of intrinsically disordered proteins

NMR assignment of intrinsically disordered proteins (IDPs) by conventional HN-detected methods is hampered by the small dispersion of the amide protons chemical shifts and exchange broadening of amide proton signals. Therefore several alternative assignment strategies have been proposed in the last years. Attempting to seize that dispersion of ¹³C′ and ¹⁵N chemical shifts holds even in IDPs, we recently proposed two ¹³C-detected experiments to directly correlate the chemical shifts of two consecutive ¹³C′–¹⁵N groups in proteins, i.e. without mediation of other nuclei. Main drawback of these experiments is the interruption of the connection at prolines. Here we present new ¹³C-detected experiments to correlate consecutive ¹³C′–¹⁵N groups in IDPs, hacacoNcaNCO and hacaCOncaNCO, that overcome this limitation. Moreover, the experiments provide recognition of glycine residues, thereby facilitating the assignment process.     

Subunit-specific backbone NMR assignments of a 64 kDa trp repressor/DNA complex: A role for N-terminal residues in tandem binding

Deuterium decoupled, triple resonance NMR spectroscopy was used to analyze complexes of ²H,¹⁵N,¹³C labelled intact and (des2–7) trp repressor (2–7 trpR) from E. coli bound in tandem to an idealized 22 basepair trp operator DNA fragment and the corepressor 5-methyltryptophan. The DNA sequence used here binds two trpR dimers in tandem resulting in chemically nonequivalent environments for the two subunits of each dimer. Sequence- and subunit-specific NMR resonance assignments were made for backbone ¹HN, ¹⁵N, ¹³C positions in both forms of the protein and for¹³ C in the intact repressor. The differences in backbone chemical shifts between the two subunits within each dimer of 2–7 trpR reflect dimer-dimer contacts involving the helix-turn-helix domains and N-terminal residues consistent with a previously determined crystal structure [Lawson and Carey (1993) Nature, 366, 178–182]. Comparison of the backbone chemical shifts of DNA-bound 2–7 trpR with those of DNA-bound intact trpR reveals significant changes for those residues involved in N-terminal-mediated interactions observed in the crystal structure. In addition, our solution NMR data contain three sets of resonances for residues 2–12 in intact trpR suggesting that the N-terminus has multiple conformations in the tandem complex. Analysis of C chemical shifts using a chemical shift index (CSI) modified for deuterium isotope effects has allowed a comparison of the secondary structure of intact and 2–7 tprR. Overall these data demonstrate that NMR backbone chemical shift data can be readily used to study specific structural details of large protein complexes.     

Intraspecific differences in metabolic rates shape carbon stable isotope trophic discrimination factors of muscle tissue in the common teleost Eurasian perch (Perca fluviatilis)

1. Stable isotopes represent a unique approach to provide insights into the ecology of organisms. δ¹³C and δ¹⁵N have specifically been used to obtain information on the trophic ecology and food‐web interactions. Trophic discrimination factors (TDF, Δ¹³C and Δ¹⁵N) describe the isotopic fractionation occurring from diet to consumer tissue, and these factors are critical for obtaining precise estimates within any application of δ¹³C and δ¹⁵N values. It is widely acknowledged that metabolism influences TDF, being responsible for different TDF between tissues of variable metabolic activity (e.g., liver vs. muscle tissue) or species body size (small vs. large). However, the connection between the variation of metabolism occurring within a single species during its ontogeny and TDF has rarely been considered.
2. Here, we conducted a 9‐month feeding experiment to report Δ¹³C and Δ¹⁵N of muscle and liver tissues for several weight classes of Eurasian perch (Perca fluviatilis), a widespread teleost often studied using stable isotopes, but without established TDF for feeding on a natural diet. In addition, we assessed the relationship between the standard metabolic rate (SMR) and TDF by measuring the oxygen consumption of the individuals.
3. Our results showed a significant negative relationship of SMR with Δ¹³C, and a significant positive relationship of SMR with Δ¹⁵N of muscle tissue, but not with TDF of liver tissue. SMR varies inversely with size, which translated into a significantly different TDF of muscle tissue between size classes.
4. In summary, our results emphasize the role of metabolism in shaping‐specific TDF (i.e., Δ¹³C and Δ¹⁵N of muscle tissue) and especially highlight the substantial differences between individuals of different ontogenetic stages within a species. Our findings thus have direct implications for the use of stable isotope data and the applications of stable isotopes in food‐web studies.

     

Annual variability in dentin δ15N and δ13C reveal sex differences in weaning age and feeding habits in Risso's dolphins (Grampus griseus)

Teeth of odontocetes accumulate annual dentinal growth layer groups (GLGs) that record isotope ratios, which reflect the time of their synthesis. Collectively, they provide lifetime records of individual feeding patterns from which life history traits can be inferred. We subsampled the prenatal dentin and postnatal GLGs in Risso's dolphins (Grampus griseus) (n = 65) that stranded or were collected as bycatch in Taiwan (1994–2014) and analyzed them for δ¹⁵N and δ¹³C. Age‐specific δ¹⁵N and δ¹³C values were corrected for effects of calendar year, stranding site, C/N, and sex. δ¹⁵N values were higher in prenatal layers (14.94‰ ± 0.74‰) than in adult female GLGs (12.58‰ ± 0.20‰), suggesting fetal enrichment during gestation. Decreasing δ¹⁵N values in early GLGs suggested changes in dietary protein sources during transition to complete weaning. Weaning age was earlier in males (1.09 yr) than in females (1.81 yr). Significant differences in δ¹⁵N values between weaned males and females suggest potential sexual segregation in feeding habits. δ¹³C values increased from the prenatal to the 4th GLG by ~1.0‰, indicative of a diet shift from ¹³C‐depleted milk to prey items. Our results provide novel insights into the sex‐specific ontogenetic changes in feeding patterns and some life history traits of Risso's dolphins.     

1H, 13C and 15N NMR assignments and secondary structure of the paramagnetic form of rat cytochrome b 5

Summary Modern multidimensional double- and triple-resonance NMR methods have been applied to assign the backbone and side-chain ¹³C resonances for both equilibrium conformers of the paramagnetic form of rat liver microsomal cytochrome b ₅. The assignment of backbone ¹³C resonances was used to confirm previous ¹H and ¹⁵N resonance assignments [Guiles, R.D. et al. (1993) Biochemistry, 32, 8329–8340]. On the basis of short- and medium-range NOEs and backbone ¹³C chemical shifts, the solution secondary structure of rat cytochrome b ₅ has been determined. The striking similarity of backbone ¹³C resonances for both equilibrium forms strongly suggests that the secondary structures of the two isomers are virtually identical. It has been found that the ¹³C chemical shifts of both backbone and side-chain atoms are relatively insensitive to paramagnetic effects. The reliability of such methods in anisotropic paramagnetic systems, where large pseudocontact shifts can be observed, is evaluated through calculations of the magnitude of such shifts.Abbreviations DANTE delays alternating with nutation for tailored excitation - DEAE diethylaminoethyl - DQF-COSY 2D double-quantum-filtered correlation spectroscopy - EDTA ethylenediaminetetraacetic acid - HCCH-TOCSY 3D proton-correlated carbon TOCSY experiment - HMQC 2D heteronuclear multiple-quantum correlation spectroscopy - HNCA 3D triple-resonance experiment correlating amide protons, amide nitrogens and alpha carbons - HNCO 3D triple-resonance experiment correlating amide protons, amide nitrogens and carbonyl carbons - HNCOCA 3D triple-resonance experiment correlating amide protons, amide nitrogens and alpha carbons via carbonyl carbons - HOHAHA 2D homonuclear Hartmann-Hahn spectroscopy - HOHAHA-HMQC 3D HOHAHA relayed HMQC - HSQC 2D heteronuclear single-quantum correlation spectroscopy - IPTG isopropyl thiogalactoside - NOESY 2D nuclear Overhauser enhancement spectroscopy - NOESY-HSQC 3D NOESY relayed HSQC - TOCSY 2D total correlation spectroscopy - TPPI time-proportional phase incrementation - TSP trimethyl silyl propionate     

1H, 15N and 13C NMR assignments of mouse methionine sulfoxide reductase B2

A recombinant mouse methionine-r-sulfoxide reductase 2 (MsrB2ΔS) isotopically labeled with ¹⁵N and ¹⁵N/¹³C was generated. We report here the ¹H, ¹⁵N, and ¹³C NMR assignments of the reduced form of this protein. An erratum to this article can be found at     

Measurement of the exchange rates of rapidly exchanging amide protons: Application to the study of calmodulin and its complex with a myosin light chain kinase fragment

Summary A technique is described for measuring the approximate exchange rates of the more labile amide protons in a protein. The technique relies on a comparison of the intensities in¹H–¹⁵N correlation spectra recorded with and without presaturation of the water resonance. To distinguish resonance attenuation caused by hydrogen exchange from attenuation caused by cross relation, the experiment is repeated at several different pH values and the difference in attenuation of any particular amide resonance upon presaturation is used for calculating its exchange rate. The technique is demonstrated for calmodulin and for calmodulin complexed with its binding domain of skeletal muscle myosin light chain kinase. Upon complexation, increased amide exchange rates are observed for residues Lys⁷⁵ through Thr⁷⁹ located in the central helix of calmodulin, and for the C-terminal residues Ser¹⁴⁷ and Lys¹⁴⁸. In contrast, a decrease in amide exchange rate is observed at the C-terminal end of the F helix, from residues Thr¹¹⁰ through Glu¹¹⁴.Istituto Guido Donegani, Novara, Italy     

Cyanoglycosylation products of 17-O-acetyl-testosterone

17-O-Acetyl testosterone, which has no susceptible hydroxyl or carboxyl group for glycosylation, was glycosylated with 2,3,4,6-tetra-O-acetyl-α- -glucopyranosyl bromide in the presence of a mixed catalyst, Hg(CN)₂ and HgBr₂, in benzene–nitromethane. Reaction occurred on the α,β-unsaturated ketone on the six–membered A-ring to give six 3-O-glycosides, each bearing a cyano group at the 3- or 5-position of the aglycon, and a 3-O-glycoside bearing a CONH₂ group at the 3-position. Structural analyses of these products were carried out by various NMR (¹H, ¹³C NMR, ¹H–¹H and ¹H–¹³C COSY, HMBC, and DEPT), FABMS and X-ray analyses. The mechanisms of the formations of the products are discussed. It was determined that mercuric cyanide was essential as a catalyst for the progress of the cyanoglycosylation.