Kaempferol-3-O-glucosyl(1-2)rhamnoside from Ginkgo biloba and a reappraisal of other gluco(1-2, 1-3 and 1-4)rhamnoside structures.

A kaempferol-3-O-glucorhamnoside from Ginkgo biloba is defined as the 3-O-alpha-L-[ beta-D-glucopyranosyl(1-2)rhamnopyranoside] on the basis of 2D NMR evidence. Complete assignments of the 1H and 13C NMR spectra of this compound and of its known p-coumaroyl derivative are presented for the first time. The NMR distinctions of 1-2, 1-3 and 1-4 linked glucopyranosylrhamnopyranosides are discussed and indicate (i) that the 13C NMR assignments for one published gluco(1-3)rhamnoside are in need of modification, (ii) that the published structure of hordenine-O-[6-O-t-cinnamoyl-beta-glucosyl(1-4)-alpha-rhamnoside] from Selaginella doederleinii is not distinguished from the 1-3 linked glucorhamnoside structure, and (iii) that the 8-prenylkaempferol-3-O-[glucosyl(1-4)rhamnoside]-7-O-glucoside and the equivalent 4'-O-methylated xylosyl(1-4)rhamnoside from Epimedium pubescens and E. washanense, respectively, are (1-2)-linked.     

1H, 13C, and 15N resonance assignments of human phosphohistidine phosphatase 1 (PHPT1)

Phosphohistidine phosphatase 1 (PHPT1) is the first protein histidine phosphatase identified in vertebrates. The NMR assignments of human PHPT1 are essential for solution structure determination and NMR study of the protein interactions of PHPT1 with its potential substrates.     

Backbone 1H, 13C and 15N resonance assignments of human vaccinia-related kinase 1 (VRK1)

Vaccinia-related kinase 1 (VRK1) is one of the mitotic kinases which play key roles in cell cycle control and chromatin modifications. To understand the biological role of the kinase and gain insights into its catalytic mechanism, we performed NMR assignments of catalytically active form of VRK1 with 361 amino acids residues. Here, we present the backbone NMR resonance assignments of the kinase.     

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.     

Monoepoxy octadecadienoates and monoepoxy octadecatrienoates 1: NMR spectral characterization

Methyl esters of gamma-linolenic acid, alpha-linolenic acid and stearidonic acid were epoxidised using m-chloroperbenzoic acid to achieve nine cis-monoepoxy-C18 fatty acid methyl esters (FAMEs), including novel methyl cis-monoepoxy derivatives of stearidonic acid and a cis-6,7-epoxy derivative of gamma-linolenic acid. These nine monoepoxy FAMEs were purified by normal-phase HPLC, identified by LC-MS, 1H and 13C NMR, and characterized by mass spectrometry and NMR spectroscopy. This study is focused on structural characterization of these C18 monoepoxy FAMEs using techniques in NMR spectroscopy including 1H, 13C, 1H-1H correlated spectroscopy (COSY) and 1H-13C heteronuclear correlation (HETCOR). The proton and carbon NMR chemical shifts of the epoxide, the double bonds, and the interrupted methylenes are assigned. Also discussed is an interpretation of the 1H and 13C NMR spectra of these monoepoxides including the changes in the 13C resonance of the olefinic carbons on the neighboring double bonds resulting from epoxide formation.     

Interaction and structural study of kinin peptide bradykinin and ganglioside monosialylated 1 micelle

Partitioning of small proteins and peptides from the aqueous to membrane phase is often coupled with folding. In this work we examine the binding and folding of the kinin peptide, bradykinin (BK), in the presence of the ganglioside monosialylated 1 (GM1) micelle. Using two-dimensional NMR techniques, we have shown that at low concentration, GM1 micelle is able to induce a turn conformation to BK. A pulsed-field gradient diffusion NMR study indicated that the peptide partitions into the GM1 micelle with a DeltaG(part) of -3.14 +/- 0.03 kcal/mol. A saturation transfer difference (STD) NMR study indicated that the binding is mostly through hydrophobic residues.     

Local and global dynamics of the G protein-coupled receptor CXCR1

The local and global dynamics of the chemokine receptor CXCR1 are characterized using a combination of solution NMR and solid-state NMR experiments. In isotropic bicelles (q = 0.1), only 13% of the expected number of backbone amide resonances is observed in (1)H/(15)N HSQC solution NMR spectra of uniformly (15)N-labeled samples; extensive deuteration and the use of TROSY made little difference in the 800 MHz spectra. The limited number of observed amide signals is ascribed to mobile backbone sites and assigned to specific residues in the protein; 19 of the signals are from residues at the N-terminus and 25 from residues at the C-terminus. The solution NMR spectra display no evidence of local backbone motions from residues in the transmembrane helices or interhelical loops of CXCR1. This finding is reinforced by comparisons of solid-state NMR spectra of both magnetically aligned and unoriented bilayers containing either full-length or doubly N- and C-terminal truncated CXCR1 constructs. CXCR1 undergoes rapid rotational diffusion about the normal of liquid crystalline phospholipid bilayers; reductions in the frequency span and a change to axial symmetry are observed for both carbonyl carbon and amide nitrogen chemical shift powder patterns of unoriented samples containing (13)C- and (15)N-labeled CXCR1. In contrast, when the phospholipids are in the gel phase, CXCR1 does not undergo rapid global reorientation on the 10(4) Hz time scale defined by the carbonyl carbon and amide nitrogen chemical shift powder patterns.     

Membrane‐induced structure of novel human tachykinin hemokinin‐1 (hHK1)

PPT‐C encoded hemokinin‐1(hHK‐1) of Homo sapiens (TGKASQFFGLM) is a structurally distinct neuropeptide among the tachykinin family that participate in the NK‐1 receptor downstream signaling processes. Subsequently, signal transduction leads to execution of various effector functions which includes aging, immunological, and central nervous system (CNS) regulatory actions. However the conformational pattern of ligand receptor binding is unclear. The three‐dimensional structure of the hemokinin‐1 in aqueous and micellar environment has been studied by one and two‐dimensional proton nuclear magnetic resonance (2D 1H‐NMR spectroscopy) and distance geometry calculations. Data shows that hemokinin‐1 was unstructured in aqueous environment; anionic detergent SDS induces α‐helix formation. Proton NMR assignments have been carried out with the aid of correlation spectroscopy (gradient‐COSY and TOCSY) and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The inter proton distances and dihedral angle constraints obtained from the NMR data have been used in torsion angle dynamics algorithm for NMR applications (CYANA) to generate a family of structures, which have been refined using restrained energy minimization and dynamics. Helical conformation is observed from residue K3‐M11. The conformational range of the peptide revealed by NMR studies has been analyzed in terms of characteristic secondary features. Observed conformational features have been compared to that of Substance P potent NK1 agonist. Thus the report provides a structural insight to study hHK‐1‐NK1 interaction that is essential for hHK1 based signaling events. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 702–710, 2015.     

Conformational states of the nuclear GTP-binding protein Ran and its complexes with the exchange factor RCC1 and the effector protein RanBP1.

It has been shown before by (31)P NMR that Ras bound to the nonhydrolyzable GTP analogue guanosine 5'-O-(beta, gamma-imidotriphosphate) (GppNHp) exists in two conformations which are rapidly interconverting with a rate constant of 3200 s-1 at 30 degrees C [Geyer, M., et al. (1996) Biochemistry 35, 10308-10320]. Here we show that Ran complexed with GTP also exists in two conformational states, 1 and 2, which can be directly inferred from the occurrence of two (31)P NMR resonance lines for the gamma-phosphate group of bound GTP. The exchange between the two states is slow on the NMR time scale with a value of <200 s-1 at 5 degrees C for the corresponding first-order rate constants. In wild-type Ran, the equilibrium constant K' between the two states is 0.7 at 278 K, is different for various mutants, and is strongly dependent on the temperature. The standard enthalpy DeltaH degrees and the standard entropy DeltaS degrees for the conformational transitions determined from the NMR spectra are as follows: DeltaH degrees = 37 kJ mol-1 and DeltaS degrees = 130 J mol-1 K-1 for wild-type Ran.GTP. In complex with the Ran-binding protein RanBP1, one of the Ran.GTP conformations (state 2) is stabilized. The interaction of Ran with the guanine nucleotide exchange factor protein RCC1 was also studied by (31)P NMR spectroscopy. In the presence of nucleotide, the ternary complex of Ran.nucleotide.RCC1, an intermediate in the guanine nucleotide exchange reaction, could be observed. A model for the conformational transition of Ran.GTP is proposed where the two states observed are caused by the structural flexibility of the effector loop of Ran; in solution, state 2 resembles the GTP-bound form found in the crystal structure of the Ran-RanBP complex.     

Structure and dynamics of the HIV-1 Vpu transmembrane domain revealed by solid-state NMR with magic-angle spinning

We report solid-state nuclear magnetic resonance (NMR) measurements on the peptide Vpu(1-40), comprising residues 1-40 of the 81-residue type 1 integral membrane protein Vpu encoded by the HIV-1 genome. On the basis of a combination of 13C and 15N NMR chemical shifts under magic-angle spinning (MAS), effects of local mobility on NMR signal intensities, site-specific MAS NMR line widths, and NMR-detected hydrogen-deuterium exchange, we develop a model for the structure and dynamics of the Vpu(1-40) monomer in phospholipid bilayer membranes. Our data are largely consistent with earlier structural studies of Vpu peptides by Opella and co-workers, in which solution NMR and solid-state NMR without MAS were used, but our data provide new information about local variations in the degree of mobility and structural order. In addition, our data indicate that the transmembrane alpha-helix of Vpu(1-40) extends beyond the hydrophobic core of the bilayer. We find no evidence for heterogeneity in the conformation and intermolecular contacts of the transmembrane alpha-helix, with the exception of two distinct chemical shifts observed for the C alpha and C beta atoms of A18 that may reflect distinct modes of helix-helix interaction. These results have possible implications for the supramolecular structure of Vpu oligomers that form cation-selective ion channels.     

Complete (1)H and (13)C NMR chemical shift assignments of mono-, di-, and trisaccharides as basis for NMR chemical shift predictions of polysaccharides using the computer program casper

The computer program casper uses (1)H and (13)C NMR chemical shift data of mono- to trisaccharides for the prediction of chemical shifts of oligo- and polysaccharides. In order to improve the quality of these predictions the (1)H and (13)C, as well as (31)P when applicable, NMR chemical shifts of 30 mono-, di-, and trisaccharides were assigned. The reducing sugars gave two distinct sets of NMR resonances due to the α- and β-anomeric forms. In total 35 (1)H and (13)C NMR chemical shift data sets were obtained from the oligosaccharides. One- and two-dimensional NMR experiments were used for the chemical shift assignments and special techniques were employed in some cases such as 2D (1)H,(13)C-HSQC Hadamard Transform methodology which was acquired approximately 45 times faster than a regular t(1) incremented (1)H,(13)C-HSQC experiment and a 1D (1)H,(1)H-CSSF-TOCSY experiment which was able to distinguish spin-systems in which the target protons were only 3.3Hz apart. The (1)H NMR chemical shifts were subsequently refined using total line-shape analysis with the PERCH NMR software. The acquired NMR data were then utilized in the casper program (http://www.casper.organ.su.se/casper/) for NMR chemical shift predictions of the O-antigen polysaccharides from Klebsiella O5, Shigella flexneri serotype X, and Salmonella arizonae O62. The data were compared to experimental data of the polysaccharides from the two former strains and the lipopolysaccharide of the latter strain showing excellent agreement between predicted and experimental (1)H and (13)C NMR chemical shifts.     

Biological 1H NMR spectroscopy

Proton nuclear magnetic resonance spectroscopy (1H NMR) is a powerful analytical method used to identify and quantitate chemical compounds. In recent years, it has been used to study rates of metabolism in microbes, isolated perfused tissues, intact animals, and human beings. This review highlights some of the more recent biological applications of 1H NMR in the study of metabolic pathophysiology in animals and man. 1H NMR can rapidly analyze complex mixtures of metabolites found in body fluid and biopsy specimens. In vivo 1H NMR methods can measure intracellular pH, a wide variety of metabolites, tissue perfusion, and rates of metabolism of endogenous and exogenous compounds. Using 13C labeled compounds or magnetization transfer techniques metabolic fluxes may be measured in vivo during virtually all normal and abnormal physiological conditions.     

One- and two-dimensional proton NMR studies of cys-102 S-methylated yeast isozyme-1 ferricytochrome c.

The effect of S-methylating cysteine-102 (cys-102) (SH----SSCH3) of yeast isozyme-1 (iso-1) ferricytochrome c has been studied using proton NMR spectroscopy. COSY, NOESY, and one-dimensional nuclear Overhauser effect (NOE) difference spectroscopies have all been used. The NMR spectrum of this derivative is very similar to that of native yeast iso-1 ferricytochrome c. The advantage of using the cys-102 S-methylated derivative is that it is unable to spontaneously dimerize in solution, like native iso-1 monomer does. This makes the derivative a simple, ideal protein for long NMR experiments. This work yields many proton resonance assignments for S-methylated yeast iso-1 monomer and confirms all of the assignments for iso-1 monomer that were previously made using only the one-dimensional NOE method.     

Fingerprinting Analysis of Rhizoma Chuanxiong of Commercial Types using 1H Nuclear Magnetic Resonance Spectroscopy and High Performance Liquid Chromatography Method

The 1H nuclear magnetic resonance (1H NMR) fingerprints of fractionated non-polar extracts (control substance for a plant drug (CSPD) A) from Rhizoma chuanxiong, the rhizomes of Ligusticum chuanxiong Hort., of seven specimens from different sources were measured on Fourier Transform (FT)-NMR spectrometer and assigned by comparing them with the 1H NMR spectra of the isolated pure compounds. The 1H NMR fingerprints showed exclusively characteristic resonance signals of the major special constituents of the plant. Although the differences in the relative intensity of the 1H NMR signals due to a discrepancy in the ratio of the major constituents among these samples could be confirmed by high performance liquid chromatography analysis, the general features of the 1H NMR fingerprint established for an authentic sample of the rhizomes of L. chuanxiong exhibited exclusive data from those special compounds and can be used for authenticating L. Chuanxiong species.     

Mapping hypoxia-induced bioenergetic rearrangements and metabolic signaling by 18O-assisted 31P NMR and 1H NMR spectroscopy

Brief hypoxia or ischemia perturbs energy metabolism inducing paradoxically a stress-tolerant state, yet metabolic signals that trigger cytoprotection remain poorly understood. To evaluate bioenergetic rearrangements, control and hypoxic hearts were analyzed with 18O-assisted 31P NMR and 1H NMR spectroscopy. The 18O-induced isotope shift in the 31P NMR spectrum of CrP, betaADP and betaATP was used to quantify phosphotransfer fluxes through creatine kinase and adenylate kinase. This analysis was supplemented with determination of energetically relevant metabolites in the phosphomonoester (PME) region of 31P NMR spectra, and in both aromatic and aliphatic regions of 1H NMR spectra. In control conditions, creatine kinase was the major phosphotransfer pathway processing high-energy phosphoryls between sites of ATP consumption and ATP production. In hypoxia, creatine kinase flux was dramatically reduced with a compensatory increase in adenylate kinase flux, which supported heart energetics by regenerating and transferring beta- and gamma-phosphoryls of ATP. Activation of adenylate kinase led to a build-up of AMP, IMP and adenosine, molecules involved in cardioprotective signaling. 31P and 1H NMR spectral analysis further revealed NADH and H+ scavenging by alpha-glycerophosphate dehydrogenase (alphaGPDH) and lactate dehydrogenase contributing to maintained glycolysis under hypoxia. Hypoxia-induced accumulation of alpha-glycerophosphate and nucleoside 5'-monophosphates, through alphaGPDH and adenylate kinase reactions, respectively, was mapped within the increased PME signal in the 31P NMR spectrum. Thus, 18O-assisted 31P NMR combined with 1H NMR provide a powerful approach in capturing rearrangements in cardiac bioenergetics, and associated metabolic signaling that underlie the cardiac adaptive response to stress.     

A conformational study of alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->O)-L-Ser by NMR 1H,1H T-ROESY experiments and molecular-dynamics simulations

The conformational preference of alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->O)-L-Ser has been investigated by one-dimensional (1)H,(1)H T-ROESY experiments and molecular-dynamics simulations with CHARMM22 type of force fields and water as explicit solvent. Proton-proton distances were obtained from the simulations and subsequently experimentally determined distances could be derived. Measurements were performed on the title compound as well as on selectively deuterium-substituted analogues synthesized as part of this study to alleviate possible NMR spectroscopic difficulties. A very good agreement was present between the separate NMR experiments. In the subsequent analysis a key nuclear Overhauser effect between the anomeric protons in the two sugar residues was used to assess the conformational dynamics revealed by the molecular simulations. The combined results support a model in which two states are significantly populated as a result of flexibility around the bond defined by the glycosidic torsion angle psi.     

Overcoming the overlap problem in the assignment of 1H NMR spectra of larger proteins by use of three-dimensional heteronuclear 1H-15N Hartmann-Hahn-multiple quantum coherence and nuclear Overhauser-multiple quantum coherence spectroscopy: application to interleukin 1 beta

The application of three-dimensional (3D) heteronuclear NMR spectroscopy to the sequential assignment of the 1H NMR spectra of larger proteins is presented, using uniformly labeled (approximately 95%) [15N]interleukin 1 beta, a protein of 153 residues and molecular mass of 17.4 kDa, as an example. The two-dimensional (2D) 600-MHz spectra of interleukin 1 beta are too complex for complete analysis, owing to extensive cross-peak overlap and chemical shift degeneracy. We show that the combined use of 3D 1H-15N Hartmann-Hahn-multiple quantum coherence (HOHAHA-HMQC) and nuclear Overhauser-multiple quantum coherence (NOESY-HMQC) spectroscopy, designed to provide the necessary through-bond and through-space correlations for sequential assignment, provides a practical general-purpose method for resolving ambiguities which severely limit the analysis of conventional 2D NMR spectra. The absence of overlapping cross-peaks in these 3D spectra allows the unambiguous identification of C alpha H(i)-NH(i+1) and NH(i)-NH(i+1) through-space nuclear Overhauser connectivities necessary for connecting a particular C alpha H(i)-NH(i) through-bond correlation with its associated through-space sequential cross-peak The problem of amide NH chemical shift degeneracy in the 1H NMR spectrum is therefore effectively removed, and the assignment procedure simply involves inspecting a series of 2D 1H-1H slices edited by the chemical shift of the directly bonded 15N atom. Connections between residues can be identified almost without any knowledge of the spin system types involved, though this type of information is clearly required for the eventual placement of the connected residues within the primary sequence.     

Heteronuclear nuclear magnetic resonance assignments, structure and dynamics of SUMO-1, a human ubiquitin-like protein

The structure of a ubiquitin-like protein, small ubiquitin-related modifier-1 (SUMO-1), was earlier determined using homonuclear nuclear magnetic resonance (NMR) spectroscopy, since the spectral quality of the protein was not suitable for heteronuclear NMR data collection. In this study, a slightly different construct of the SUMO-1 gene was used for protein over-expression. The protein purified from this construct showed high spectral qualities, therefore, multi-dimensional heteronuclear NMR data for a dynamic study and structural determination were acquired. The structure of SUMO-1 obtained in this study differs in several respects from the structure obtained from homonuclear NMR data. Furthermore, structural differences were observed between the new SUMO-1 and ubiquitin structures. These differences may be important for SUMO-1-specific recognition in cells. Additionally, relaxation parameters indicate that SUMO-1 undergoes highly anisotropic tumbling in solution and that the long amino (N)-terminal sequence of SUMO-1 is highly dynamic with increasing flexibility towards the end.     

Metabolic fingerprinting of wild type and transgenic tobacco plants by 1H NMR and multivariate analysis technique

The metabolomic analysis of wild type and constitutive salicylic acid producing tobacco plants (CSA tobacco, Nicotiana tabacum 'Samsun' NN) plants overexpressing salicylate biosynthetic genes was carried out by 1H NMR spectrometry and multivariate analysis techniques. The principle component analysis (PCA) of the 1H NMR spectra showed a clear discrimination between those samples by PC1 and PC2. The discrimination of non-inoculated, TMV-virus inoculated, and systemic leaves or veins could also be obtained by PCA analysis. Major peaks in 1H NMR spectra contributing to the discrimination were assigned as those of chlorogenic acid, malic acid, and sugars. This method allows an efficient differentiation between wild type and transgenic plants without any pre-purification steps.     

Structure and calcium-binding properties of Frq1, a novel calcium sensor in the yeast Saccharomyces cerevisiae

The FRQ1 gene is essential for growth of budding yeast and encodes a 190-residue, N-myristoylated (myr) calcium-binding protein. Frq1 belongs to the recoverin/frequenin branch of the EF-hand superfamily and regulates a yeast phosphatidylinositol 4-kinase isoform. Conformational changes in Frq1 due to N-myristoylation and Ca(2+) binding were assessed by nuclear magnetic resonance (NMR), fluorescence, and equilibrium Ca(2+)-binding measurements. For this purpose, Frq1 and myr-Frq1 were expressed in and purified from Escherichia coli. At saturation, Frq1 bound three Ca(2+) ions at independent sites, which correspond to the second, third, and fourth EF-hand motifs in the protein. Affinity of the second site (K(d) = 10 microM) was much weaker than that of the third and fourth sites (K(d) = 0.4 microM). Myr-Frq1 bound Ca(2+) with a K(d)app of 3 microM and a positive Hill coefficient (n = 1.25), suggesting that the N-myristoyl group confers some degree of cooperativity in Ca(2+) binding, as seen previously in recoverin. Both the NMR and fluorescence spectra of Frq1 exhibited very large Ca(2+)-dependent differences, indicating major conformational changes induced upon Ca(2+) binding. Nearly complete sequence-specific NMR assignments were obtained for the entire carboxy-terminal domain (residues K100-I190). Assignments were made for 20% of the residues in the amino-terminal domain; unassigned residues exhibited very broad NMR signals, most likely due to Frq1 dimerization. NMR chemical shifts and nuclear Overhauser effect (NOE) patterns of Ca(2+)-bound Frq1 were very similar to those of Ca(2+)-bound recoverin, suggesting that the overall structure of Frq1 resembles that of recoverin. A model of the three-dimensional structure of Ca(2+)-bound Frq1 is presented based on the NMR data and homology to recoverin. N-myristoylation of Frq1 had little or no effect on its NMR and fluorescence spectra, suggesting that the myristoyl moiety does not significantly alter Frq1 structure. Correspondingly, the NMR chemical shifts for the myristoyl group in both Ca(2+)-free and Ca(2+)-bound myr-Frq1 were nearly identical to those of free myristate in solution, indicating that the fatty acyl chain is solvent-exposed and not sequestered within the hydrophobic core of the protein, unlike the myristoyl group in Ca(2+)-free recoverin. Subcellular fractionation experiments showed that both the N-myristoyl group and Ca(2+)-binding contribute to the ability of Frq1 to associate with membranes.