15N Spectra Research Articles

Structure-Activity Relationships in Two Antimicrobial Peptides Based on Chemokine Helical Segments: RP-1 and IL-8α

Antimicrobial peptides are naturally occurring molecules, part of the innate immune system, and are of high interest as novel antibiotic therapeutics given the increasing resistance of microbes to conventional antibiotics. RP-1 and IL-8α are 18 and 19 amino acid synthetic peptides that were designed based on the sequence of the C-terminal helical segments of two chemokines: platelet factor-4 and interleukin-8. In order to characterize structure-activity relationships and to understand the selectivity of these peptides for bacterial membranes, NMR was used to determine high-resolution structures of both peptides in complex with SDS and DPC micelles. Additionally, solid state NMR experiments in oriented lipid bilayers were performed to assess structure and orientation in a bilayer environment and to indicate the impact of the peptide on bilayer organization. Both peptides structure as amphipathic α-helices with hydrophobic residues on one side and polar and positively charged residues on the opposite side. RP-1 shows very subtle structural differences when in complex with SDS (anionic) versus DPC (zwitterionic) micelles. This suggests that its specificity for prokaryotic versus eukaryotic membranes does not derive from peptide structural differences in the two systems, but rather from differences in the details of the peptide-lipid interactions. The 2H solid state NMR data are consistent with IL-8α associating peripherally with POPC bilayers, and penetrating deeper into POPC/POPG bilayers. Additional insight is gained from 31P and 15N spectra of IL-8α in these environments.

Двойной ядерный квадрупольный резонанс 14N некоторых азотсодержащих соединений

The article analyses the peculiarities of indirect observation of NQR nitrogen signals and determines the conditions for the enhancement of spin systems efficiency in static magnetic fields. It allows keeping records of the 14N spectra in the range below 1 MHz at room temperature. This method may be used for researches on low isotope matters.

Natural abundance 13C and 15N solid-state NMR analysis of paramagnetic transition-metal cyanide coordination polymers

The (13)C and (15)N MAS NMR spectra of a series of well-characterized paramagnetic metal cyanide coordination polymers are acquired at natural abundance, without the need for polarization transfer methods such as cross-polarization or INEPT. For systems where the paramagnetic centre is outside of the cyanide framework, well-resolved (13)C and (15)N spectra of cyanide ligands are obtained. Chemical shifts deviate from typical diamagnetic cyanide ranges and depend only partly on the cyanide coordination type, being dominated by their proximity to the paramagnetic center. A combination of the observed isotropic chemical shifts, their temperature dependence, and transverse relaxation time constants (T(2)) provides valuable local structural information and lays the foundation for the structural elucidation of unknown paramagnetic metal-cyanide coordination polymers. Toward this end, we apply these solid-state NMR techniques to a pair of compounds without a priori knowledge of the structures.

Evidence for Dynamics on a 100 ns Time Scale from Single- and Double-Quantum Nitrogen-14 NMR in Solid Peptides

The indirect detection of 14N spectra via protons in the manner of heteronuclear multiple-quantum correlation (HMQC) allows one to obtain single- (SQ) and double-quantum (DQ) 14N spectra in solids. A comparison of the SQ and DQ line widths as a function of temperature with simulations reveals motions in the tripeptide AAG with rates on the order of 107 s(-1) at 49 degrees C.

Molecular Structure from a Single NMR Experiment

A procedure is described for determining the structure of a small molecule from a single NMR experiment. Several standard NMR sequences are combined so that the essential structural information is obtained in just one pass. Two-dimensional (13)C-(13)C correlations ("INADEQUATE"), single- and multiple-bond (13)C-(1)H correlations, and the conventional (13)C spectrum are recorded in parallel, making use of separate receiver channels for acquisition of (13)C and (1)H signals. The natural-abundance (13)C-(13)C correlation measurements employ a high-sensitivity cryogenically cooled probe, optimized for (13)C detection. An extension of this "all-in-one" sequence with three parallel receivers permits the corresponding natural-abundance (15)N spectra to be included.

Hydroponic isotope labelling of entire plants (HILEP) for quantitative plant proteomics; an oxidative stress case study

Hydroponic isotope labelling of entire plants (HILEP) is a cost-effective method enabling metabolic labelling of whole and mature plants with a stable isotope such as 15N. By utilising hydroponic media that contain 15N inorganic salts as the sole nitrogen source, near to 100% 15N-labelling of proteins can be achieved. In this study, it is shown that HILEP, in combination with mass spectrometry, is suitable for relative protein quantitation of seven week-old Arabidopsis plants submitted to oxidative stress. Protein extracts from pooled 14N- and 15N-hydroponically grown plants were fractionated by SDS–PAGE, digested and analysed by liquid chromatography electrospray ionisation tandem mass spectrometry (LC–ESI–MS/MS). Proteins were identified and the spectra of 14N/ 15N peptide pairs were extracted using their m/ z chromatographic retention time, isotopic distributions, and the m/ z difference between the 14N and 15N peptides. Relative amounts were calculated as the ratio of the sum of the peak areas of the two distinct 14N and 15N peptide isotope envelopes. Using Mascot and the open source trans-proteomic pipeline (TPP), the data processing was automated for global proteome quantitation down to the isoform level by extracting isoform specific peptides. With this combination of metabolic labelling and mass spectrometry it was possible to show differential protein expression in the apoplast of plants submitted to oxidative stress. Moreover, it was possible to discriminate between differentially expressed isoforms belonging to the same protein family, such as isoforms of xylanases and pathogen-related glucanases (PR 2).

Identification of the oriI-Binding Site of Poliovirus 3C Protein by Nuclear Magnetic Resonance Spectroscopy

Replication of picornaviral genomes requires recognition of at least three cis-acting replication elements: oriL, oriI, and oriR. Although these elements lack an obvious consensus sequence or structure, they are all recognized by the virus-encoded 3C protein. We have studied the poliovirus 3C-oriI interaction in order to begin to decipher the code of RNA recognition by picornaviral 3C proteins. oriI is a stem-loop structure that serves as the template for uridylylation of the peptide primer VPg by the viral RNA-dependent RNA polymerase. In this report, we have used nuclear magnetic resonance (NMR) techniques to study 3C alone and in complex with two single-stranded RNA oligonucleotides derived from the oriI stem. The (1)H-(15)N spectra of 3C recorded in the presence of these RNAs revealed site-specific chemical shift perturbations. Residues that exhibit significant perturbations are primarily localized in the amino terminus and in a highly conserved loop between residues 81 and 89. In general, the RNA-binding site defined in this study is consistent with predictions based on biochemical and mutagenesis studies. Although some residues implicated in RNA binding by previous studies are perturbed in the 3C-RNA complex reported here, many are unique. These studies provide unique site-specific insight into residues of 3C that interact with RNA and set the stage for detailed structural investigation of the 3C-RNA complex by NMR. Interpretation of our results in the context of an intact oriI provides insight into the architecture of the picornavirus VPg uridylylation complex.

NMR Study of the Chemisorption and Surface Chemistry of Methylamine on Pd/SiO2

Results of an investigation of the interaction of methylamine with a Pd/SiO2 catalyst utilizing nuclear magnetic resonance (NMR) spectroscopy are presented. Two sets of experiments were conducted. In one set, a sample of catalyst was initially exposed to 13CH315NH2 at 77 K and then subjected to a lengthy equilibration period at 190 K. A 13C spectrum at 77 K obtained on the sample at this point exhibited a single symmetric line at a frequency very close to the resonance frequency for methylamine in the absence of the catalyst. The line is attributed to methylamine adsorbed without dissociation. In 13C spectra obtained at 77 K on the sample after each of a series of subsequent annealing periods at successively higher temperatures in the range 253−298 K, the line exhibited a gradual broadening on the downfield side. Deconvolution of the spectra resolved the original symmetric line from a very broad downfield line attributed to partially dehydrogenated surface species designated by the formula (CN)Hx. In the second set of experiments, 13CH315NH2 was adsorbed on a sample of catalyst at 298 K. The sample was then annealed at successively higher temperatures in the range 325−450 K. Deconvolution of the 13C spectra obtained at 298 K after the various annealing periods revealed extensive formation of dimethylamine, reaching a maximum after the annealing period at 400 K. Ammonia and methane were first detected in the 15N and 13C spectra, respectively, after the annealing period at 375 K. During the annealing periods at 425 and 450 K, the dimethylamine decomposed almost completely, leaving only methane, ammonia, and residual surface species.

Restriction of lipid motion in membranes triggered by β-sheet aggregation of the anti-apoptotic BH4 domain

The regulative BH4 domain of human Bcl-2 protein exerts its anti-apoptic activity via the mitochondrion. In the present study, we investigated the molecular interactions of this domain with negatively charged liposomes mimicking the outer mitochondrial membrane. To model the overproduction of Bcl-2 found in cancer processes, we studied the impact of elevated concentrations of its regulative BH4 segment on these mitochondrial membranes from the peptide and lipid perspective. Combined solid state (2)H-NMR and differential scanning calorimetry revealed the coexistence of small sized fluid and rigid membrane domains over a large temperature range, which is confirmed by (31)P-NMR at 30 degrees C. The latter are stabilized, in a cholesterol-like manner, by the presence of a BH4 peptide. In the same time scale, the reduction of the headgroup order is seen in the static (14)N and (31)P-NMR spectra when BH4 inserts into the bilayers. Indeed, attenuated total reflection spectroscopy indicated a dominant aggregated beta-sheet secondary structure of BH4 with a 42 degrees tilt relative to the membrane surface. These results are discussed in terms of membrane stabilization versus apoptotic mechanisms at the outer mitochondrial membrane location.

Dynamic Structure of Disulfide-Removed Linear Analogs of Tachyplesin-I in the Lipid Bilayer from Solid-State NMR

Tachyplesin-I (TP-I) is a 17-residue beta-hairpin antimicrobial peptide containing two disulfide bonds. Linear analogs of TP-I where the four Cys residues were replaced by aromatic and aliphatic residues, TPX4, were found to have varying degrees of activities, with the aromatic analogs similarly potent as TP-I. Understanding the different activities of the linear analogs should give insight into the mechanism of action of TP-I. To this end, we have investigated the dynamic structures of the active TPF4 and the inactive TPA4 in bacteria-mimetic anionic POPE/POPG bilayers and compared them with the wild-type TP-I using solid-state NMR spectroscopy. 13C isotropic chemical shifts and backbone (phi, psi) torsion angles indicate that both TPF4 and TPA4 adopt beta-strand conformations without a beta-turn at key residues. 1H spin diffusion from lipid chains to the peptide indicates that the inactive TPA4 binds to the membrane-water interface, similar to the active TP-I. Thus, neither the conformation nor the depth of insertion of the three peptides correlates with their antimicrobial activities. In contrast, the mobility of the three peptides correlates well with their activities: the active TP-I and TPF4 are both highly mobile in the liquid-crystalline phase of the membrane while the inactive TPA4 is completely immobilized. The different mobilities are manifested in the temperature-dependent 13C and 15N spectra, 13C-1H and 15N-1H dipolar couplings and 1H rotating-frame spin-lattice relaxation times. The dynamics of TP-I and TPF4 are both segmental and global. Combined, these data suggest that TP-I and TPF4 disrupt the membrane by large-amplitude motion in the plane of the membrane. The loss of this motion in TPA4 due to aggregation significantly weakens its activity because a higher peptide concentration is required to disturb lipid packing. Thus molecular motion, rather than structure, appears to be the key determinant for the membrane-disruptive activities of tachyplesins.

Solid-State NMR Investigation of the Possible Existence of “Nanoblocks” in the Clear Solution Synthesis of MFI Materials

The structure of the intermediate species in the clear solution synthesis of the MFI framework (zeolite ZSM 5) has been investigated using the characteristic 13C, 14N, 15N, and 2D spectra of the tetrapropylammonium (TPA) template ions as probes as well as the 29Si spectra of the silicate species present in samples that can be isolated by centrifugation. Comparison with the corresponding spectra of the final products that can be characterized by X-ray diffraction indicates that there is no evidence for the involvement of nanospecies, as has been proposed, and that crystallization is most probably from an amorphous gel. This conclusion is supported by the lack of deuterium rotational echo double-resonance dephasing of the 29Si spectra by deuterated TPA of the earliest intermediate species obtained, while it is clearly observed in the final product. These observations indicate that any TPA ions present in the gel phase are not in intimate contact with the silicon nuclei as they would be if in the local MFI e...

Field-cycled dynamic nuclear polarization (FC-DNP) of 14N and 15N nitroxide radicals at low magnetic field

Theoretical Overhauser-detected EPR spectra of 14N and 15N nitroxide systems in low magnetic field by field-cycled dynamic nuclear polarization (FC-DNP) were described by a combination of DNP theory and a model of FC-DNP. Spectra were simulated at magnetic fields between 0 and 8 mT. The simulations were able to predict both the EPR peak positions and their amplitudes, corresponding to those from FC-DNP experiments with 14N and 15N TEMPOL solutions. EPR irradiation was in the 45–133 MHz range while NMR signal detection occurred at a field of 59 mT. At this frequency range, four π transitions of a 14N system and three π transitions of a 15N system were observed. The simulation programmes were also used to predict the spectral amplitudes of the FC-DNP with EPR irradiation power in the 1–15 W range. Theoretical FC-DNP systems were in good agreement with experimental results; however, at low magnetic fields the inhomogeneity of our magnet system resulted in the EPR peaks being left-shifted and somewhat broader than those from the theoretical prediction.

NMR assignments of 1H, 13C and 15N spectra of methionine sulfoxide reductase B1 from Mus musculus

Isotopically labeled, 15N and 15N/13C forms of recombinant methionine-r-sulfoxide reductase 1 (MsrB1, SelR) from Mus musculus were produced, in which catalytic selenocysteine was replaced with cysteine. We report here the 1H, 13C and 15N NMR assignment of the reduced form of this mammalian protein.

The Membrane Alignment of Helical Peptides from Non-oriented15N Chemical Shift Solid-State NMR Spectroscopy

The analysis of the structure, topology, and dynamics of membrane polypeptides provides important clues for the understanding of their functional mechanisms. The interactions of these peptides and proteins are often characterized by a high degree of mobility, and solid-state NMR spectroscopy is a method that allows the monitoring not only of the structure but also of the dynamics of membrane polypeptides. Previously, it has been demonstrated that the 15N chemical shift obtained from uniaxially oriented samples directly correlates with the alignment of the NH vector relative to the magnetic field direction and therefore also the tilt angle of helical polypeptides. Theoretical considerations indicate that under conditions of fast diffusion around the membrane normal the proton-decoupled 15N spectrum obtained from polypeptides associated with non-oriented membranes should also be a good indicator of the helix tilt angle. Here we provide experimental proof of this novel concept and discuss its advantages and ...

A New Series of Glycopeptide Antibiotics Incorporating a Squaric Acid Moiety

The aglycones of the antibiotics eremomycin, vancomycin and ristocetin (3, 4 and 6, respectively) were prepared by deglycosidation of the parent antibiotics with hydrogen fluoride, and complete assignation of their 1H, 13C and 15N spectra was performed. The squaric acid amide esters (11-14), were prepared from dimethyl squarate. The corresponding asymmetric diamides (16-19, 22, 23) were also synthesized using 4-phenylbenzylamine and triglycine. The advantage of the method is the high regioselectivity and that no protecting group strategy is required. Electrospray mass spectroscopic method was elaborated for the determination of the site of substitution of the modified antibiotics. The antibacterial activity of the prepared compounds is discussed in detail.

Identification of Hydrogen Bonds to the Rieske Cluster through the Weakly Coupled Nitrogens Detected by Electron Spin Echo Envelope Modulation Spectroscopy

The interaction of the reduced[2Fe-2S] cluster of isolated Rieske fragment from the bc1 complex of Rhodobacter sphaeroides with nitrogens (14N and 15N) from the local protein environment has been studied by X- and S-band pulsed EPR spectroscopy. The two-dimensional electron spin echo envelope modulation spectra of uniformly 15N-labeled protein show two well resolved cross-peaks with weak couplings of approximately 0.3-0.4 and 1.1 MHz in addition to couplings in the range of 6-8 MHz from two coordinating Ndelta of histidine ligands. The quadrupole coupling constants for weakly coupled nitrogens determined from S-band electron spin echo envelope modulation spectra identify them as Nepsilon of histidine ligands and peptide nitrogen (Np), respectively. Analysis of the line intensities in orientation-selected S-band spectra indicated that Np is the backbone N-atom of Leu-132 residue. The hyperfine couplings from Nepsilon and Np demonstrate the predominantly isotropic character resulting from the transfer of unpaired spin density onto the 2s orbitals of the nitrogens. Spectra also show that other peptide nitrogens in the protein environment must carry a 5-10 times smaller amount of spin density than the Np of Leu-132 residue. The appearance of the excess unpaired spin density on the Np of Leu-132 residue indicates its involvement in hydrogen bond formation with the bridging sulfur of the Rieske cluster. The configuration of the hydrogen bond therefore provides a preferred path for spin density transfer. Observation of similar splittings in the 15N spectra of other Rieske-type proteins and [2Fe-2S] ferredoxins suggests that a hydrogen bond between the bridging sulfur and peptide nitrogen is a common structural feature of [2Fe-2S] clusters.

14N nuclear quadrupole resonance of some sulfa drugs

The 14N nuclear quadrupole double resonance spectra of different polymorphs of sulfanilamide, sulfadiazine, sulfamerazine and sulfamethazine have been measured and the 14N quadrupole coupling tensors have been determined. The obtained 14N spectra are compared with those of other sulfa drugs like sulfathiazole. It is shown that different polymorphs can be easily discriminated. The application of this technique for non-destructive analysis, polymorph determination and quality control in the production of pharmaceuticals is stressed.

Structure and Multinuclear Solid-State NMR of a Highly Birefringent Lead−Gold Cyanide Coordination Polymer

The coordination polymer Pb(H2O)[Au(CN)2]2 (1) was synthesized by the reaction of KAu(CN)2 and Pb(NO3)2. The structure contains 1-D chains of lead(II)-OH2 linked via Au(CN)2(-) moieties, generating a 2-D slab; weak aurophilic interactions of 3.506(2) and 3.4885(5) A occur within and between slabs. The geometry about each lead(II) is bicapped trigonal prismatic, having six N-bound cyanides at the prism vertices and waters at two of the faces. Dehydration at 175 degrees C yields microcrystalline Pb[Au(CN)2]2 (2), which, along with 1, was examined by 13C, 15N, 1H, and 207Pb solid-state NMR methods. Two 15N resonances are assigned to the mu2-bridging and hydrogen-bonding cyanides in 1. Upon dehydration, the 207Pb NMR spectrum becomes axially symmetric and yields a reduced shielding span, indicating higher site symmetry, while the 13C and 15N spectra reveal a single cyanide. Although no single-crystal X-ray structure of 2 could be obtained, a structure is proposed on the basis of the NMR and X-ray powder data, consisting of a lead(II) center in a distorted square-prismatic environment, with cyanides present at each corner. The birefringence of single crystals of 1 is found to be 7.0 x 10(-2) at room temperature. This value is large compared to that of most optical materials and can be attributed to the anisotropy of the 2-D slabs of 1, with all CN bonds aligned in the same direction by the polarizable lead(II) center.

Interaction of the replication terminator protein of Bacillus subtilis with DNA probed by NMR spectroscopy

Termination of DNA replication in Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the dimeric 29 kDa replication terminator protein (RTP) and DNA terminator sites. We have used NMR spectroscopy to probe the changes in 1H– 15N correlation spectra of a 15N-labelled RTP.C110S mutant upon the addition of a 21 base pair symmetrical DNA binding site. Assignment of the 1H– 15N correlations was achieved using a suite of triple resonance NMR experiments with 15N, 13C,70% 2H enriched protein recorded at 800 MHz and using TROSY pulse sequences. Perturbations to 1H– 15N spectra revealed that the N-termini, α3-helices and several loops are affected by the binding interaction. An analysis of this data in light of the crystallographically determined apo- and DNA-bound forms of RTP.C110S revealed that the NMR spectral perturbations correlate more closely to protein structural changes upon complex formation rather than to interactions at the protein–DNA interface.

Solution NMR Structure Investigation for Releasing Mechanism of Neocarzinostatin Chromophore from the Holoprotein

Holo-neocarzinostatin (holo-NCS) is a complex protein carrying the anti-tumor active enediyne ring chromophore by a scaffold consisting of an immunoglobulin-like seven-stranded anti-parallel beta-barrel. Because of the labile chromophore reflecting its extremely strong DNA cleavage activity and complete stabilization in the complex, holo-NCS has attracted much attention in clinical use as well as for drug delivery systems. Despite many structural analyses for holo-NCS, the chromophore-releasing mechanism to trigger prompt attacks on the target DNA is still unclear. We determined the three-dimensional structure of the protein and the internal motion by multinuclear NMR to investigate the releasing mechanism. The internal motion studied by 13C NMR methine relaxation experiments showed that the complex has a rigid structure for its loops as well as the beta-barrel in aqueous solution. This agrees with the refined NMR solution structure, which has good convergence in the loop regions. We also showed that the chromophore displayed a similar internal motion as the protein moiety. The structural comparison between the refined solution structure and x-ray crystal structure indicated characteristic differences. Based on the findings, we proposed the chromophore-releasing mechanism by a three-state equilibrium, which sufficiently describes both the strong binding and the prompt releasing of the chromophore. We demonstrated that we could bridge the dynamic properties and the static structure features with simple kinetic assumptions to solve the biochemical function.