Solid-state 15N Nuclear Magnetic Resonance Research Articles

Catalytic roles of βLys87 in tryptophan synthase: 15N solid state NMR studies

The proposed mechanism for tryptophan synthase shows βLys87 playing multiple catalytic roles: it bonds to the PLP cofactor, activates C4′ for nucleophilic attack via a protonated Schiff base nitrogen, and abstracts and returns protons to PLP-bound substrates (i.e. acid–base catalysis). ε-15N-lysine TS was prepared to access the protonation state of βLys87 using 15N solid-state nuclear magnetic resonance (SSNMR) spectroscopy for three quasi-stable intermediates along the reaction pathway. These experiments establish that the protonation state of the ε-amino group switches between protonated and neutral states as the β-site undergoes conversion from one intermediate to the next during catalysis, corresponding to mechanistic steps where this lysine residue has been anticipated to play alternating acid and base catalytic roles that help steer reaction specificity in tryptophan synthase catalysis. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications. Guest Editors: Andrea Mozzarelli and Loredano Pollegioni.

13C and 15N solid-state NMR studies on albendazole and cyclodextrin albendazole complexes

13C and 15N solid-state nuclear magnetic resonance (NMR) spectra were recorded from albendazole (ABZ) and from ABZ:β-cyclodextrin, ABZ:methyl-β-cyclodextrin, ABZ:hydroxypropyl-β-cyclodextrin and ABZ:citrate-β-cyclodextrin, which were prepared by the spray-drying technique. ABZ signals were typical of a crystalline solid for the pure drug and of an amorphous compound obtained from ABZ:cyclodextrin samples. Relevant spectral differences were correlated with chemical interaction between ABZ and cyclodextrins. The number and type of complexes revealed a strong dependence on the cyclodextrin group substituent. Solid-state NMR data were consistent with the presence of stable inclusion complexes.

H-bonds and Protons in Molecular Crystals: Insight from Combined XPS/ssNMR/XRD

Physicochemical properties of molecular crystals are significantly influenced by non-covalent interactions and proton transfer. A well known application is the tuning of solubility, bioavailability and stability of pharmaceutical actives through co-crystal (hydrogen-bonding) or salt (ionic, Brønsted acceptors) formation. X-ray Photoelectron Spectroscopy (XPS) is an intrinsically local structural probe, providing information on the chemical state and chemical environment of atoms in molecules and crystals through the photoemission of core level electrons. We have recently studied a wide range of acid-base complexes in molecular crystals and found that analyzing the chemical shifts of N1s core level binding energies provides a facile route for characterizing the chemical and structural changes at functional groups involved in hydrogen bonding and proton transfer [1]. Very importantly, XPS unequivocally distinguishes protonated (salt) from hydrogen-bonded (co-crystal) nitrogen moieties. We have complemented our results for nitrogen species with 15N Solid-State Nuclear Magnetic Resonance (ssNMR) chemical shifts, which reveal low frequency shifts with protonation, but the magnitude of these shifts is additionally influenced by the wider chemical environment [2]. When crystallographic structure information is available, ssNMR shifts can be computationally predicted and thereby related to H-bond lengths, giving a measure of H-bond strength (NMR crystallography). The wide variety of donor/acceptor systems we have investigated has covered a large range of pKa values and demonstrates the generic nature of taking an XPS/ssNMR/XRD approach to organic molecule crystallography (Fig 1). The excellent agreement between the conclusions drawn by XPS and combined ssNMR/CASTEP investigations opens up a reliable avenue for local structure characterization in molecular systems even in the absence of crystal structure information, for example with non-crystalline or amorphous matter.

Expression and purification of the aortic amyloid polypeptide medin

The 50-amino acid protein medin is the main fibrillar component of human aortic medial amyloid (AMA), the most common form of localised amyloid which affects 97% of Caucasians over the age of 50. Structural models for several amyloid assemblies, including the Alzheimer’s amyloid-β peptides, have been defined from solid-state nuclear magnetic resonance (SSNMR) measurements on 13C- and 15N-labelled protein fibrils. SSNMR-derived structural information on fibrillar medin is scant, however, because studies to date have been restricted to limited measurements on site-specifically labelled protein prepared by solid-phase synthesis. Here we report a procedure for the expression of a SUMO-medin fusion protein in Escherichia coli and IMAC purification yielding pure, uniformly 13C,15N-labelled medin in quantities required for SSNMR analysis. Thioflavin T fluorescence and dynamic light scattering measurements and transmission electron microscopy analysis confirm that recombinant medin assembles into amyloid-like fibrils over a 48-h period. The first 13C and 15N SSNMR spectra obtained for uniformly-labelled fibrils indicate that medin adopts a predominantly β-sheet conformation with some unstructured elements, and provide the basis for further, more detailed structural investigations.

New insights into the structure and chemistry of Titan’s tholins via13C and 15N solid state nuclear magnetic resonance spectroscopy

Tholins are complex C,N-containing organic compounds produced in the laboratory. They are considered to provide materials that are analogous to those responsible for the haze observed in Titan’s atmosphere. These compounds present an astrobiological interest due to their ability to release amino acids upon hydrolysis. Their chemical structure has been investigated using a large number of techniques. However, to date no detailed nuclear magnetic resonance (NMR) study has been performed on these materials despite the high potential of this technique for investigating the environment of given nuclei. Here 13C and 15N solid state NMR spectroscopy was applied to obtain new insights into the chemical structure of tholins produced through plasma discharge in gaseous N2CH4 mixtures designed to simulate the atmosphere of Titan. Due to the low natural abundance of these isotopes, a 13C and 15N-enriched tholin sample was synthesized using isotopically enriched gas precursors. Various pulse sequences including 13C and 15N single pulse, 1H13C and 1H15N cross-polarisation and 1H15N13C double cross-polarisation were used. These techniques allowed complete characterisation of the chemical and structural environments of the carbon and nitrogen atoms. The NMR assignments were supplemented and confirmed by ab initio electronic structure calculations for model structures and molecular fragments.

Structural analysis of microbial poly(ɛ-L-lysine)/poly(acrylic acid) complex by FT-IR, DSC, and solid-state 13C and 15N NMR

Interpolymer complexes of microbial poly(ɛ-L-lysine) (ɛ-PL) and poly(acrylic acid) (PAA) were studied by Fourier transform infrared spectroscopy, differential scanning calorimetry and solid-state 13C and 15N nuclear magnetic resonance (NMR) experiments. These measurements suggested the poly-ionic complex formation between ɛ-PL and PAA, ɛ-PL-H3N+···−OOC-PAA. By analyzing 13C NMR spectra of the complex, we concluded that there exist three forms of the carboxyl group of PAA, namely: (1) the ionized form, interpolymer ion-complex between ɛ-PL and PAA, (2) the dimeric form, intrapolymer hydrogen bonding among PAA molecules and (3) the free form, no particular form of hydrogen bonding.

High Impact Properties of Polyketone/Polyamide-6 Alloys Induced by Characteristic Morphology and Water Absorption

Nagata et al. recently developed high-impact polyketone/polyamide-6 (PK/PA) alloys and investigated their mechanical properties (Nagata et al. Polym. Prep. Jpn. 2006, 55, 4286−4287). This paper presents the results of investigations of their morphology, such as the domain size of PA and PK, features of crystalline phase of PK (the long period and thickness of lamellae are deduced), and the change in crystalline form of PA in the PK/PA alloys, using solid-state nuclear magnetic resonance (NMR), small-angle X-ray scattering (SAXS), and transmission electron microscopy (TEM). Solid-state 13C and 15N NMR results revealed that the γ crystalline phase of PA changes into a thermodynamically stable α crystalline phase after alloying with PK. The difference in the long period of lamellae of PK/PA alloys between dry and wet conditions is discussed. We conclude that the characteristic morphology of PK and humidity of the amorphous phase of PA in the wet condition are very important factors enabling the PK/PA alloys to exhibit high impact resistance, high-strength, and high modulus.

Fate of the Amino Acid in Glucose−Glycine Melanoidins Investigated by Solid-State Nuclear Magnetic Resonance (NMR)

The fate of the amino acid in the model Maillard reaction between glucose and glycine in a 1:1 molar ratio has been investigated by applying advanced 13C and 15N solid-state nuclear magnetic resonance (NMR) techniques to 13C- and 15N-labeled melanoidins formed in dry and solution reactions. Quantitative 13C NMR shows that approximately 23% of carbon is from glycine; the approximately 2% loss compared to the 25% glycine C in the reactants is due to the COO moiety being liberated as CO2 (Strecker degradation). 13C J-modulation experiments on melanoidins made from doubly 13C-labeled glycine show that the C-C backbone bond of about two-thirds of the incorporated amino acid stays intact, and about half of all glycine is incorporated as N-CH2-COO without fragmentation. Degradation processes without CO2 loss affect about one-eighth of glycine in dry reaction and about one-fourth in solution. These results indicate that Strecker degradation affects about one-fourth (dry reaction) to one-third (in solution) of all glycine but is not the main pathway of glycine incorporation. Spectra of Strecker degradation products show that C2 of glycine reacts to form N-CH3, C-CHn-C, or aromatic units, but not pyrazines or pyridines. The gycine-C1 carbon incorporated into the melanoidins remains>or=90% part of COO moieties; approximately 5% of amides have also been detected. The C2-N bond stays intact for approximately 70% of the incorporated glycine. The 15N spectra show many peaks, over a 200 ppm range, documenting a multitude of different chemical environments of nitrogen, but no enamines or imines. The majority (>78%) of nitrogen, in particular most pyrrolic N, is not protonated. Because N-H predominates in amino acids and proteins, nonprotonated nitrogen may be a characteristic marker of Maillard reaction products.

Can antimicrobial peptides scavenge around a cell in less than a second?

Antimicrobial peptides, which play multiple host-defense roles, have garnered increased experimental focus because of their potential applications in the pharmaceutical and food production industries. While their mechanisms of action are richly debated, models that have been advanced share modes of peptide–lipid interactions that require peptide dynamics. Before the highly cooperative and specific events suggested in these models take place, peptides must undergo an important process of migration along the membrane surface and delivery from their site of binding on the membrane to the actual site of functional performance. This phenomenon, which contributes significantly to antimicrobial function, is poorly understood, largely due to a lack of experimental and computational tools needed to assess it. Here, we use 15N solid-state nuclear magnetic resonance to obtain molecular level data on the motions of piscidin's amphipathic helices on the surface of phospholipid bilayers. The studies presented here may help contribute to a better understanding of the speed at which the events that lead to antimicrobial response take place. Specifically, from the perspective of the kinetics of cellular processes, we discuss the possibility that piscidins and perhaps many other amphipathic antimicrobial peptides active on the membrane surface may represent a class of fast scavengers rather than static polypeptides attached to the water–lipid interface.

Morphology characterization of polyaniline nano- and microstructures

Small-angle neutron scattering (SANS), nuclear magnetic resonance (NMR), wide-angle and small-angle X-ray scattering (WAXS and SAXS) measurements were carried out to investigate the three morphological forms of polyaniline emeraldine base (PANI-EB): unstructured, microtubes, and nanofibers. Although the chemical backbone between these two materials is quite similar, their solid structures are quite different, showing differences in the molecular conformation and supramolecular packing. Detailed solid-state 13C and 15N NMR characterization of PANI nanofibers (compared to the unstructured, granular form) revealed a slight variation in the structural features of the polymer that led to some differences in the chemical environments of the respective nuclei. The presence of two extra-sharp peaks at 96.5 and 179.8 ppm is a distinct feature found exclusively in the nanofiber spectra. Moreover, the crosspolarization (CP) dynamics study disclosed the presence of a complete set of sharp NMR peaks that are responsible for the presence of a more ordered morphology in the nanofiber. Small-angle neutron scattering indicated very sharp interfaces in the PANI fibers, which are well organized and have extremely sharp domains within the length scales probed (∼10–1 nm). Overall, the X-ray scattering and spectroscopy data suggest that the nanofiber form is structurally different from the unstructured, PANI-EB powder. These differences are manifested, in part, by the additional chemistry occurring during the synthesis of the nanofibers.

Anticancer cisplatin interactions with bilayers of total lipid extract from pig brain: A 13C, 31P and 15N solid-state NMR study

Cisplatin ( cis-diamminedichloroplatinum(II)) is used in chemotherapy and it is well established that cisplatin forms platinum-DNA adducts that initiate tumor cell death. Drawbacks are side effects such as neurotoxicity and cellular cisplatin resistance and it is possible that part of these effects are linked to cisplatin interaction with lipids and the phospholipid bilayer. 13C magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of total lipid extract from pig brain with and without cisplatin show that the phosphatidylserine (PS) carboxyl resonance disappears in presence of cisplatin and that a new resonance of similar intensity appears at 185.5 ppm. Thus, indicating cisplatin interaction with the PS head-group. Static and MAS 31P NMR spectra of lipid extract with and without cisplatin show that the phospholipids to a large extent reside in a bilayer environment in pure lipid extract, and that the presence of cisplatin promotes isotropic and/or hexagonal lipid phases.

Cocrystals of 3,5-Dimethyl-1H-pyrazole and Salicylic Acid: Controlled Formation of Trimers via O−H···N Hydrogen Bonds

Solid-state reactions of 3,5-dimethyl-1H-pyrazole (dmpz) and salicylic acid (sa) in different stoichiometries afford two different trimers whose structures have been determined by X-ray diffraction and analyzed by 13C and 15N solid-state nuclear magnetic resonance spectroscopy. GIAO absolute shieldings at the B3LYP/6-311++G** level have been calculated and compared with the experimental chemical shifts. Hydrogen-bond interactions between dmpz and sa provide sufficient driving force to direct molecular recognition and crystal packing.

Transport and Immobilization of 2,4,6 15N-Trinitrotoluene in Soil Microcosms Subjected to Long Term Incubation Under Aerobic Conditions

15N-labeling and solid-state 13C and 15N nuclear magnetic resonance (NMR) spectroscopy was applied to study the immobilization of 2,4,6 trinitrotoluene (TNT) into soil organic matter (SOM). Uncontaminated soil from the Ap horizon of a Luvisol was mixed with 15N-TNT (enrichment: 99 atm%) and laid over an unspiked layer of the same material. The latter covered soil from the Bt horizon. The microcosms were aerobically incubated under laboratory conditions for up to 11 months. After 1 week, within the total microcosm approximately 90% of the added 15N (15Nadd) were recovered, mostly in the top layer (87%). After 11 months, this amount decreased to 71%, indicating losses due to denitration or transamination. Within two months, half of 15Nadd had been immobilized in the residues not extractable with organic solvents and water. The amount of the sequestered 15Nadd remained fairly constant until the end of the experiment pointing towards a high stability of TNT-SOM associates. Solid-state 15N NMR revealed their formation by covalent binding, most tentatively as amides. Complete reduction of TNT to triaminotoluene (TAT) was not prerequisite. The most pronounced downwards movement of 15N-TNT occurred during the first two months. The major part of it, however, experienced quick immobilization, leaving approximately 10% of 15Nadd recovered in the leachate at the end of the experiment. Calculations indicated contributions of inorganic 15Nadd. Approximately 25% of its organic 15Nadd originated from condensed N, suggesting that in soils the transport of partly reduced TNT is in close association with the organic matter of the soil solution to which they are covalently bound.

New pyrolytic and spectroscopic data on Orgueil and Murchison insoluble organic matter: A different origin than soluble?

Pyrolysis with and without tetramethylammonium hydroxide (TMAH), vacuum pyrolysis, and solid state 15N nuclear magnetic resonance (NMR) were used to examine the macromolecular insoluble organic matter (IOM) from the Orgueil and Murchison meteorites. Conventional pyrolysis reveals a set of poorly functionalized aromatic compounds, ranging from one to four rings and with random methyl substitutions. These compounds are in agreement with spectroscopic and pyrolytic results previously reported. For the first time, TMAH thermochemolysis was used to study extraterrestrial material. The detection of aromatics bearing methyl esters and methoxy groups reveals the occurrence of ester and ether bridges between aromatic units in the macromolecular network. No nitrogen-containing compounds were detected with TMAH thermochemolysis, although they are a common feature in terrestrial samples. Along with vacuum pyrolysis results, thermochemolysis shows that nitrogen is probably sequestered in condensed structures like heterocyclic aromatic rings, unlike oxygen, which is mainly located within linkages between aromatic units. This is confirmed by solid state 15N NMR performed on IOM from Orgueil, showing that nitrogen is present in pyrrole, indole, and carbazole moieties. These data show that amino acids are neither derived from the hydrolysis of IOM nor from a common precursor. In order to reconcile the literature isotopic data and the present molecular results, it is proposed that aldehydes and ketones (1) originated during irradiation of ice in space and (2) were then mobilized during the planetesimal hydrothermalism, yielding the formation of amino acids. If correct, prebiotic molecules are the products of the subsurface chemistry of planetesimals and are thus undetectable through astronomical probes.

Fire-induced transformation of C- and N- forms in different organic soil fractions from a Dystric Cambisol under a Mediterranean pine forest ( Pinus pinaster)

High intensity forest fires in Mediterranean ecosystems probably have long-term effects on the organic matter (OM) of the forest soils. Therefore, we analyzed the quality and quantity of humic materials extracted from the A horizons (0–15 cm) of a fire-affected (FA) and a control fire-unaffected (FU) Dystric Cambisol from the Sierra de Aznalcóllar (Spain). 13C and 15N solid-state nuclear magnetic resonance (NMR) spectra of the samples from the FA and FU sites confirmed that aromatic and newly formed heterocyclic N-forms are important fire-induced products. Wildfire resulted in a doubling of organic C and N concentration in the A horizon (0–15 cm) of FA. Solid-state 13C NMR spectroscopy revealed that all C compound classes were enriched, including O- and N-alkyl C, with the highest for aromatic C (enrichment factor: 2.9). This suggests that the inputs from charred biomass and black carbon particles incorporated into the A horizon contained considerable amounts of unburned or partly charred remains. This conclusion is supported by the identification of diagnostic lignin-derived pyrolysis products in the hydrofluoric acid (HF) treated soils by analytical pyrolysis gas chromatography mass spectrometry (Py-GC/MS). Most of the partially charred material in addition to slightly altered plant necromass accumulated in the NaOH insoluble fraction (RES). Although the yield of alkali soluble OM from site FA was higher than that from site FU, the qualitative fire-induced alterations in C composition were slight. This may indicate that within this fraction, the OM was derived mainly from slightly charred or unburned materials. Fire also changed the chemical composition of the N-fraction of all humic extracts. These accumulated as pyrrole-type N compounds, although amide N was found to be the dominant form. This suggests that not all peptide-structures of the necromass were transformed by the fire. It is also possible that those amides are part of the melanoidized compounds, known to be formed during thermal treatment of mixtures containing sugars and amino acids. The fact that pyrrole-type N forms were not detected in the solid-state 15N NMR spectra of FU supports their pyrogenic origin. Apart from the importance of the alkali-insoluble fractions of wildfire-affected soils as a source of molecular descriptors of the thermal effect, pyrrole identified in soil organic matter (SOM) by means of solid-state 15N NMR spectroscopy could be a molecular marker for the presence of pyromorphic humic material in the soil.

Solid-state nuclear magnetic resonance studies of HIV and influenza fusion peptide orientations in membrane bilayers using stacked glass plate samples

The human immunodeficiency virus (HIV) and influenza virus fusion peptides are ∼20-residue sequences which catalyze the fusion of viral and host cell membranes. The orientations of these peptides in lipid bilayers have been probed with 15N solid-state nuclear magnetic resonance (NMR) spectroscopy of samples containing membranes oriented between stacked glass plates. Each of the peptides adopts at least two distinct conformations in membranes (predominantly helical or beta strand) and the conformational distribution is determined in part by the membrane headgroup and cholesterol composition. In the helical conformation, the 15N spectra suggest that the influenza peptide adopts an orientation approximately parallel to the membrane surface while the HIV peptide adopts an orientation closer to the membrane bilayer normal. For the beta strand conformation, there appears to be a broader peptide orientational distribution. Overall, the data suggest that the solid-state NMR experiments can test models which correlate peptide orientation with their fusogenic function.

Stabilization of N-compounds in soil and organic-matter-rich sediments—what is the difference?

Most of the organic nitrogen in soils and sediments ultimately derives from living organisms where it is mainly present as peptides and amino acids. These biomolecules are considered to have a biologically labile chemical structure and are expected to be quickly mineralized during early stages of organic matter stabilization. In spite of this, nitrogen is still found in aged soils, recent and even fossilized sediments. To elucidate the nature of this recalcitrant nitrogen and the processes that are involved in its formation, solid-state 15N nuclear magnetic resonance (NMR) spectroscopy was recently introduced into geosciences and applied to various environments differing in the origin of their organic matter precursors as well as in chemical and physical conditions of the environment. Results obtained with this approach indicate that survival of peptide-like structures is a ubiquitous phenomenon, although the mechanisms for their stabilization may differ in different ecological systems. However, a conspicuous change in organic nitrogen composition is observed in fossilized sediments and for organic matter formed by vegetation fires. Cyclization and rearrangement of peptide structures result in the formation of heteroaromatic N during fossilization, which was not detected for recent sediments and soils. From this, it may be concluded that such compounds are only formed in environments in which abiotic transformation of biogenic precursors dominates over biotic degradation.

NMR Studies of Chloroquine−Ferriprotoporphyrin IX Complex

By examining natural-abundance 13C and 15N solid-state nuclear magnetic resonance (NMR) spectra of acid-precipitated chloroquine−ferriprotoporphyrin IX (CQ−FPIX) aggregates, the first direct evidence is provided for CQ−FPIX complexes in the solid state, formed in a physiologic aqueous solution that mimics the environment inside the digestive vacuole of the malaria parasite. The 13C spectra demonstrate a loss of signal from aromatic sites, whereas 15N experiments indicate the disappearance of the signal from the N atom of the quinoline ring. These signal losses are due to the paramagnetic Fe(III) center of FPIX. In addition, contact shifts are also observed for both 13C and 15N spectra, suggesting the formation of a covalent complex between CQ and FPIX in the solid state.

Formation of Heteroaromatic Nitrogen after Prolonged Humification of Vascular Plant Remains as Revealed by Nuclear Magnetic Resonance Spectroscopy

In the search for the mechanisms involved in the immobilization of organic nitrogen in humified remains of vascular plants, the efforts of the present investigation were directed toward the examination of the transformation of nitrogenous compounds during the peat and coal stage by means of solid-state nuclear magnetic resonance (NMR) spectroscopy. While accumulation of heteroaromatic-N is not detected in most of the studied peat layers, a clear shoulder in the chemical shift region of pyrrole- or indole-N is observed in the solid-state 15N NMR spectrum of material from the deepest (and thus oldest) peat layer underlying the sapropel from Mangrove Lake, Bermuda (10000 years). This points to the assumption that transformation of nitrogen occurs between an advanced stage of peatification and an early stage of coalification. The observed sudden alteration in nitrogen functionality indicates that continuous accumulation of newly synthesized or selectively preserved biogenic structures is not responsible for the presence of heteroaromatic-N in these fossilized deposits. It seems rather likely that abiotic conditions, occurring during advanced sediment maturation, have an effect on the observed N transformation. With increasing coalification, pyrrole-type-N becomes the dominant form in the macromolecular coal network. Pyridine-type-N was only detected in a coal of anthracite rank.

Anaerobic/Aerobic Composting of Soil Contaminated with 2,4,6-Trinitrotoluene

A loam soil from Pennsylvania without a history of exposure to explosives was incubated with 5 g kg−1 of 15N-labeled 2,4,6-trinitrotoluene (TNT) and 200 μCi kg−1 of 14C-TNT for 3 days and then amended with compost at a 1:2 soil to compost ratio. The compost was prepared by mixing 40% alfalfa hay, 40% grass hay, 10% spent mushroom compost, and 10% municipal biosolids. The mixture of soil and compost was inoculated with methanogens from cattle manure, amended with glucose and starch, and incubated for 37 days under anaerobic conditions. The anaerobic incubation was followed by 26 days of forced aerobic incubation. At the end of the aerobic phase, most of the radioactivity was associated with organic matter; only 8.7% could be extracted with water and methanol, but no TNT was present in the extracts as determined by high-performance liquid chromatography. The unextractable radioactivity was associated with humic acid (40.0±1.0%), fulvic acid (14.3±1.4%), and humin (28.2±0.5%). Radioactive materials associated with humic acid and humin were analyzed by solid-state 15N-nuclear magnetic resonance (NMR) spectrometry. The NMR spectra indicated that nitro groups of TNT had been reduced to amino groups thatwere subsequently involved in the formation of covalent bonds with soil organic matter.