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1.
Proline metabolism in the wild-type and in a salt-tolerant mutant of nicotiana plumbaginifolia studied by (13)C-nuclear magnetic resonance imaging
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To obtain insight into the link between proline (Pro) accumulation and the increase in osmotolerance in higher plants, we investigated the biochemical basis for the NaCl tolerance of a Nicotiana plumbaginifolia mutant (RNa) that accumulates Pro. Pro biosynthesis and catabolism were investigated in both wild-type and mutant lines. (13)C-Nuclear magnetic resonance with [5-(13)C]glutamate (Glu) as the Pro precursor was used to provide insight into the mechanism of Pro accumulation via the Glu pathway. After 24 h under 200 mM NaCl stress in the presence of [5-(13)C]Glu, a significant enrichment in [5-(13)C]Pro was observed compared with non-stress conditions in both the wild type (P2) and the mutant (RNa). Moreover, under the same conditions, [5-(13)C]Pro was clearly synthesized in higher amounts in RNa than in P2. On the other hand, measurements of enzyme activities indicate that neither the biosynthesis via the ornithine pathway, nor the catabolism via the Pro oxidation pathway were affected in the RNa mutant. Finally, the regulatory effect exerted by Pro on its biosynthesis was evaluated. In P2 plantlets, exogenous Pro markedly reduced the conversion of [5-(13)C]Glu into [5-(13)C]Pro, whereas Pro feedback inhibition was not detected in the RNa plantlets. It is proposed that the origin of tolerance in the RNa mutant is due to a mutation leading to a substantial reduction of the feedback inhibition normally exerted in a wild-type (P2) plant by Pro at the level of the Delta-pyrroline-5-carboxylate synthetase enzyme. 相似文献
2.
G Strecker S Fièvre J M Wieruszeski J C Michalski J Montreuil 《Carbohydrate research》1992,226(1):1-14
The structures of two octasaccharides, one nonasaccharide, and one undecasaccharide, isolated from human milk, have been investigated by 1H- and 13C-nuclear magnetic resonance spectroscopy. The structures of these oligosaccharides are: beta-D-Galp-(1----4)-[alpha-L-Fucp- (1----3)]-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-[alpha-L-Fucp+ ++- (1----3)]-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-D-Glc; beta-D-GALp-(1----3)-[alpha-L-Fucp-(1----4)]-beta-D-GlcpNAc-(1---- 3)-beta-D - Galp-(1----4)-[alpha-L-Fucp-(1----3)]-beta-D-GlcpNAc-(1----3)-beta -D-Galp- (1----4)-D-Glc; beta-D-Galp-(1----4)-[alpha-L-Fucp-(1----3)]-beta-D-GlcpNAc-(1---- 6)-(alpha - L-Fucp-(1----2)-beta-D-Gal-(1----3)-[alpha-L-Fucp-(1----4)]- beta-D-GlcpNAc- (1----3))-beta-D-Galp-(1----4)-D-Glc; and alpha-L-Fucp-(1----2)-beta-D-Galp-(1----3)-beta-D-GlcpNAc-(1----3) -beta-D- Galp-(1----4)-[alpha-L-Fucp-(1----3)]-beta-D-GlcpNAc-(1----6)-[alp ha-L- Fucp-(1----2)-beta-D-Galp-(1----3)-beta-D-GlcpNAc-(1----3)]-beta-D -Galp- (1----4)-D-Glc. The two octasaccharides have been previously isolated from human milk as a mixture, and in a pure form from new-born feces, but the n.m.r. data were not provided. These two octasaccharides display the di-Lewis X and the composite Lewis A-Lewis X antigenic determinant, previously described as neo-antigens of adenocarcinoma cell lines. 相似文献
3.
Treatment with phenobarbital causes an increased urinary excretion of tetrahydroxylated bile acids in patients suffering from intrahepatic cholestasis. The main components were isolated from urine by means of column and thin-layer chromatography and were studied as methyl esters by nuclear magnetic resonance spectroscopy. The results obtained strongly support the contention that the main components are 1 beta-, 6 alpha- and 6 beta-hydroxylated derivatives of cholic acid. 相似文献
4.
S1 nuclease recognizes DNA conformational junctions between left-handed helical (dT-dG n. dC-dA)n and contiguous right-handed sequences 总被引:29,自引:0,他引:29
The ability of negative supercoiling to induce a left-handed helix in the recombinant plasmid pRW777, which contains a tract of 64 base pairs of almost perfect (dT-dG) . (dC-dA) from the mouse kappa immunoglobin gene, was studied. S1 nuclease recognizes and cleaves within the junction region which must exist adjacent to the (dT-dG)n . (dC-dA)n tract when in a left-handed state. The cleavage pattern indicates conformational flexibility and structural differences between the two existing junctions. The 64-base pair alternating copolymer undergoes the supercoil-induced formation of a left-handed state over the superhelical density range of -0.04 to -0.06, indicating that (dT-dG)n . (dC-dA) sequences form a left-handed helix less readily than (dC-dG)n . (dC-dG)n sequences of equivalent length. However, these supercoil densities are within the range found in vivo. Supercoil relaxation and antibody binding studies confirmed that the (dT-dG)n . (dC-dA)n tract in supercoiled pRW777 was in a left-handed helix. 相似文献
5.
Yamaguchi S Tuzi S Tanio M Naito A Lanyi JK Needleman R Saitô H 《Journal of biochemistry》2000,127(5):861-869
We compared (13)C NMR spectra of [3-(13)C]Ala- and [1-(13)C]Val-labeled bacterio-opsin (bO), produced either by bleaching bR with hydroxylamine or from a retinal-deficient strain, with those of bacteriorhodopsin (bR), in order to gain insight into the conformational changes of the protein backbone that lead to correct folding after retinal is added to bO. The observed (13)C NMR spectrum of bO produced by bleaching is not greatly different from that of bR, except for the presence of suppressed or decreased peak-intensities. From careful evaluation of the intensity differences between cross polarization magic angle spinning (CP-MAS) and dipolar decoupled-magic angle spinning (DD-MAS) spectra, it appears that the reduced peak-intensities arise from reduced efficiency of cross polarization or interference of internal motions with proton decoupling frequencies. In particular, the E-F and F-G loops and some transmembrane helices of the bleached bO have acquired internal motions whose frequencies interfere with proton decoupling frequencies. In contrast, the protein backbone of the bO from the retinal-negative cells is incompletely folded. Although it contains mainly a-helices, its very broad (13)C NMR signals indicate that its tertiary structure is different from bR. Importantly, this changed structure is identical in form to that of bleached bO from wild-type bR after it was regenerated with retinal in vitro, and bleached with hydroxylamine. We conclude that the binding of retinal is essential for the correct folding of bR after it is inserted in vitro into the lipid bilayer, and the final folded state does not revert to the partially folded form upon removal of the retinal. 相似文献
6.
The nonspecificity of dog serum albumin (DSA) for Ni(II) is mimicked by the simplest tripeptide, glycylglycyl-L-tyrosine-N-methyl amide, which forms a planar complex at high pH. In this study, the 1H and 13C nuclear magnetic resonance (nmr) spectra of the free and complexed peptide are reported. As the pH is increased for the free peptide, the deprotonation of the terminal amino group (pKa = 7.94) is reflected most strongly by the chemical shift changes of the NH2-terminal -CH2CO- unit. Large upfield and downfield shifts for the tyrosine C xi, C epsilon and C gamma carbon resonances occur on the ionization of the phenolic hydroxyl group. The planar Ni(II) complex is in slow exchange on the nmr time scale and is of 1:1 stoichiometry. The greater chemical shift changes on Ni(II) coordination are observed from the protons nearest the peptide and amino nitrogens:amide CH3 (-0.704), Tyr(3) alpha-CH (-0.667), Gly(1) alpha-CH2 (-0.382), and Gly(2) alpha-CH2 (-0.519, -0.487). In the 13C spectrum, the Gly(1) C alpha (+7.58) is most affected. The Ni(II) ion is therefore at the center of four coordinating nitrogens. Changes in the coupling constants for the Tyr(3) -CH-CH2- moiety suggests a mainly gauche conformation with the tyrosyl ring positioned above the plane of coordination and a weak bonding interaction with the Ni(II) ion is indicated. These results provide structural information regarding the reduced affinity of DSA for Ni(II). 相似文献
7.
8.
Aubert S Bligny R Douce R Gout E Ratcliffe RG Roberts JK 《Journal of experimental botany》2001,52(354):37-45
The relative contribution of glutamate dehydrogenase (GDH) and the aminotransferase activity to mitochondrial glutamate metabolism was investigated in dilute suspensions of purified mitochondria from potato (Solanum tuberosum) tubers. Measurements of glutamate-dependent oxygen consumption by mitochondria in different metabolic states were complemented by novel in situ NMR assays of specific enzymes that metabolize glutamate. First, a new assay for aminotransferase activity, based on the exchange of deuterium between deuterated water and glutamate, provided a method for establishing the effectiveness of the aminotransferase inhibitor amino-oxyacetate in situ, and thus allowed the contribution of the aminotransferase activity to glutamate oxidation to be assessed unambiguously. Secondly, the activity of GDH in the mitochondria was monitored in a coupled assay in which glutamine synthetase was used to trap the ammonium released by the oxidative deamination of glutamate. Thirdly, the reversibility of the GDH reaction was investigated by monitoring the isotopic exchange between glutamate and [(15)N]ammonium. These novel approaches show that the oxidative deamination of glutamate can make a significant contribution to mitochondrial glutamate metabolism and that GDH can support the aminotransferases in funneling carbon from glutamate into the TCA cycle. 相似文献
9.
10.
A 13C-nmr study of the salt-induced helix–coil transition of the basic polypeptides poly(L -lysine) [(Lys)n], poly(L -arginine) [(Arg)n], and poly (L -ornithine) [(Orn)n] was performed to serve as a reference of the helical portion of histones and other proteins. As is the case with pH-induced helix–coil transition, the downfield displacement of the Cα and carbonyl carbon signals are observed in the helical state. The upfield shift of the Cβ signals, on the other hand, is noted in the salt-induced transition. Regardless of the differences in the side chains and also the salts used, very similar helix-induced chemical shifts are obtained for (Lys)n and (Arg)n. However, the displacement of the Cα, Cβ, and carbonyl carbons of (Orn)n in the presence of 4M NaClO4 is found to be almost 50% of that of (Lys)n and (Arg)n. This is explained by the fact that the maximum helical content is about 50%, consistent with the ORD result. Further, the motion of the backbone and side chains of the helical from was estimated by measuring the spin-lattice relaxation time (T1), nuclear Overhauser enhancement (NOE), and line width. In the case of (Lys)n, the motion of the side chains is charged very little in comparison with that of the random coil. Indicating that the aggregation of the salt-induced helix is small in contrast to that of the pH-induced helix. For (Arg)n, however, the precipitate of the helical polymers is mainly due to aggregation. 相似文献
11.
Gérard Strecker Jean-Michel Wieruszeski Jean-Claude Michalski Jean Montreuil 《Glycoconjugate journal》1989,6(2):169-182
The structure of a nonasaccharide and of two decasaccharides isolated from human milk has been investigated by using methylation, fast atom bombardment mass spectrometry and 1H-/13C-nuclear magnetic resonance spectroscopy. The structures of these oligosaccharides were: trifucosyllacto-N-hexaose; Fuc alpha 1-2Gal beta 1-3(Fuc alpha 1-4)GlcNAc beta 1-3[Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-6]Gal beta 1-4Glc, difucosyllacto-N-octaoses; Gal beta 1-3(Fuc alpha 1-4)GlcNAc beta 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-6[Gal beta 1-3GlcNAc beta 1-3]Gal beta 1-4Glc and Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-6[Fuc alpha 1-3 Gal beta 1-3GlcNAc beta 1-3]Gal beta 1-4Glc. The two decasaccharides possess a new type of core structure proposed to be named iso-lacto-N-octaose. 相似文献
12.
Summary Four enhanced carbonyl carbon resonances were observed whenStreptomyces subtilisin inhibitor (SSI) was labeled by incorporating specifically labeled [1-13C]Cys. The13C signals were assigned by the15N,13C double-labeling method along with site-specific mutagenesis. Changes in the spectrum of the labeled protein ([C]SSI) were induced by reducing the disulfide bonds with various amounts of dithiothreitol (DTT). The results indicate that, in the absence of denaturant, the Cys71-Cys101 disulfide bond of each SSI subunit can be reduced selectively. This disulfide bond, which is in the vicinity of the reactive site scissile bond Met73-Val74, is more accessible to solvent than the other disulfide bond. Cys35-Cys50, which is embedded in the interior of SSI. This half-reduced SSI had 65% of the inhibitory activity of native SSI and maintained a conformation similar to that of the fully oxidized SSI. Reoxidation of the half reduced-folded SSI by air regenerates fully active SSI which is indistinguishable with intact SSI by NMR. In the presence of 3 M guanidine hydrochloride (GuHCl), however, both disulfide bonds of each SSI subunit were readily reduced by DTT. The fully reduced-unfolded SSI spontaneously refolded into a native-like structure (fully reduced-folded state), as evidenced by the Cys carbonyl carbon chemical shifts, upon removing GuHCl and DTT from the reaction mixture. The time course of disulfide bond regeneration from this state by air oxidation was monitored by following the NMR spectral changes and the results indicated that the disulfide bond between Cys71 and Cys101 regenerates at a much faster rate than that between Cys35 and Cys50.Nomenclature of the various states of SSI that are observed in the present study
Fully oxidized-folded
native or intact (without GuHCl or DTT)
-
half reduced-folded
(Cys71-Cys101 reduced; DTT without GuHCl)
-
inversely half reduced-folded
(Cys35-Cys50 reduced; a reoxidation intermediate from fully reduced-folded state)
-
fully reduced-unfolded
(reduced by DTT in the presence of GuHCl)
-
fully reduced-folded
(an intermediate state obtained by removing DTT and GuHCl from the fully reduced-unfolded SSI reaction mixture) 相似文献
13.
The helix–coil transition of poly-N5-(3-hydroxypropyl)-L -glutamine (PHPLG) has been studied in methanol–water by CD and cmr spectroscopy. For polydisperse PHPLG, two separate peaks arising from residues in helical and random-coil conformations are observed during the transition for both main-chain carbons. These results are discussed and compared to those observed in the case of a polymer sample obtained by racemization of PHPLG in 0.1 M NaOH and of PHPLG samples of controlled molecular weight and dispersity. The dominant influence of the molecular-weight heterogeneity on the double-peak phenomenon has been verified. The linewidths and chemical shift of the 13C resonances are discussed in terms of side-chain–main-chain interactions and side-chain solvation. 相似文献
14.
In order to check our current knowledge on the principles involved in beta-hairpin formation, we have modified the sequence of a 3:5 beta-hairpin forming peptide with two different purposes, first to increase the stability of the formed 3:5 beta-hairpin, and second to convert the 3:5 beta-hairpin into a 2:2 beta-hairpin. The conformational behavior of the designed peptides was investigated in aqueous solution and in 30% trifluoroethanol (TFE) by analysis of the following nuclear magnetic resonance (NMR) parameters: nuclear Overhauser effect (NOE) data, and C(alpha)H, (13)C(alpha), and (13)C(beta) conformational shifts. From the differences in the ability to adopt beta-hairpin structures in these peptides, we have arrived to the following conclusions: (i) beta-Hairpin population increases with the statistical propensity of residues to occupy each turn position. (ii) The loop length, and in turn, the beta-hairpin type, can be modified as a function of the type of turn favored by the loop sequence. These two conclusions reinforce previous results about the importance of beta-turn sequence in beta-hairpin folding. (iii) Side-chain packing on each face of the beta-sheet may play a major role in beta-hairpin stability; hence simplified analysis in terms of isolated pair interactions and intrinsic beta-sheet propensities is insufficient. (iv) Contributions to beta-hairpin stability of turn and strand sequences are not completely independent. (v) The burial of hydrophobic surface upon beta-hairpin formation that, in turn, depends on side-chain packing also contributes to beta-hairpin stability. (vi) As previously observed, TFE stabilizes beta-hairpin structures, but the extent of the contribution of different factors to beta-hairpin formation is sometimes different in aqueous solution and in 30% TFE. 相似文献
15.
The conformation and dynamic structure of single-stranded poly(inosinic acid), poly(I), in aqueous solution at neutral pH have been investigated by nmr of four nuclei at different frequencies: 1H (90 and 250 MHz), 2H (13.8 MHz), 13C (75.4 MHz), and 31P (36.4 and 111.6 MHz). Measurements of the proton-proton coupling constants and of the 1H and 13C chemical shifts versus temperature show that the ribose is flexible and that base-base stacking is not very significant for concentrations varying from 0.04 to 0.10M in the monomer unit. On the other hand, the proton T1 ratios between the sugar protons, T1 (H1′)/T1 (H3′), indicate a predominance of the anti orientation of the base around the glycosidic bond. The local motions of the ribose and the base were studied at different temperatures by measurements of nuclear Overhauser enhancement (NOE) of protonated carbons, the ratio of the proton relaxation times measured at two frequencies (90 and 250 MHz), and the deuterium quadrupolar transverse relaxation time T2. For a given temperature between 22 and 62°C, the 13C-{1H} NOE value is practically the same for seven protonated carbons (C2, C8, C1′, C2′, C3′, C4′, C5′). This is also true for the T1 ratio of the corresponding protons. Thus, the motion of the ribose–base unit can be considered as isotropic and characterized by a single correlation time, τc, for all protons and carbons. The τc values determined from either the 13C-{1H} NOE or proton T1 ratios, T1(90 MHz)/T1(250 MHz), and/or deuterium transverse relaxation time T2 agree well. The molecular motion of the sugar-phosphate backbone (O-P-O) and the chemical-shift anisotropy (CSA) were deduced from T1 (31P) and 31P-{1H} NOE measurements at two frequencies. The CSA contribution to the phosphorus relaxation is about 12% at 36.4 MHz and 72% at 111.6 MHz, corresponding to a value of 118 ppm for the CSA (σ = σ∥ ? σ?). Activation energies of 2–6 kcal/mol for the motion of the ribose–base unit and the sugarphosphate backbone were evaluated from the proton and phosphorus relaxation data. 相似文献
16.
Biekofsky RR Martin SR McCormick JE Masino L Fefeu S Bayley PM Feeney J 《Biochemistry》2002,41(21):6850-6859
Calmodulin, the Ca(2+)-dependent activator of many cellular processes, contains two well-defined structural domains, each of which binds two Ca(2+) ions. In its Ca(2+)-free (apo) form, it provides an attractive model for studying mechanisms of protein unfolding, exhibiting two separable, reversible processes, indicating two structurally autonomous folding units. (1)H-(15)N HSQC NMR in principle offers a detailed picture of the behavior of individual residues during protein unfolding transitions, but is limited by the lack of dispersion of resonances in the unfolded state. In this work, we have used selective [(15)N]Ile labeling of four distinctive positions in each calmodulin domain to monitor the relative thermal stability of the folding units in wild-type apocalmodulin and in mutants in which either the N- or C-domain is destabilized. These mutations lead to a characteristic perturbation of the stability (T(m)) of the nonmutated domain relative to that of wild-type apocalmodulin. The ability to monitor specific (15)N-labeled residues, well-distributed throughout the domain, provides strong evidence for the autonomy of a given folding unit, as well as providing accurate measurements of the unfolding parameters T(m) and DeltaH(m). The thermodynamic parameters are interpreted in terms of interactions between one folded and one unfolded domain of apocalmodulin, where stabilization on the order of a few kilocalories per mole is sufficient to cause significant changes in the observed unfolding behavior of a given folding unit. The selective (15)N labeling approach is thus a general method that can provide detailed information about structural intermediates populated in complex protein unfolding processes. 相似文献
17.
Zeins, the maize storage proteins, are the most abundant proteins in the corn endosperm, and are synthesized on the rough endoplasmatic reticulum and deposited in discrete organelles called protein bodies. Several authors, using circular dichroism and optical rotatory dispersion, have concluded that these proteins have a high alpha-helical content in alcoholic solution. In this work we have studied these proteins, within the protein bodies themselves and after extraction from the corn grains with 70% ethanol, using NMR (nuclear magnetic resonance) spectroscopy. We conclusively demonstrate the presence of free fatty acids within both the protein bodies and also in the alcohol extracted alpha zeins. We present evidence for a direct interaction between the free fatty acids and the alpha zein proteins within the protein body and suggest possible mechanisms by which such an association has arisen during the evolution of the maize endosperm. 相似文献
18.
A conformational study of adenylyl-(3'',5'')-adenosine and adenylyl-(2'',5'')-adenosine in aqueous solution by carbon-13 magnetic resonance spectroscopy. 总被引:4,自引:4,他引:0
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The solution conformation of adenylyl-(3',5')-adenosine and adenylyl-(2',5')-adenosine in both the stacked and unstacked states was studied by carbon-13 magnetic resonance spectroscopy. Large chemical shift differences between the base carbons in the dimers and those in the corresponding monomers are attributed in part to the influence of base-base interaction. Carbon-phosphorus couplings across three bonds revealed the preferred populations for certain backbone rotamers, demonstrating that significant changes in conformation about the "c(3')-O and C(5')-O bonds do not occur in the temperature or salt-induced unstacking of adenylyl-(3',5')-adenosine. However, rotations about the C(2')-O and C(5')-O bonds occur in the temperature-mediated unstacking of adenylyl-(2',5')-adenosine. 相似文献
19.
Latosińska JN Latosińska M Tomczak MA Seliger J Zagar V 《Journal of molecular modeling》2011,17(7):1781-1800
The quinolinol derivatives clioquinol (5-chloro-7-iodo-8-quinolinol, Quinoform) and cloxiquine (5-chloro-8-quinolinol) were
studied experimentally in the solid state via 35Cl NQR, 1H-17O and 1H-14N NQDR spectroscopies, and theoretically by density functional theory (DFT). The supramolecular synthon pattern of O–H···N
hydrogen bonds linking dimers and π–π stacking interactions were described within the QTAIM (quantum theory of atoms in molecules)
/DFT (density functional theory) formalism. Both proton donor and acceptor sites in O–H···N bonds were characterized using
1H-17O and 1H-14N NQDR spectroscopies and QTAIM. The possibility of the existence of O–H···H–O dihydrogen bonds was excluded. The weak intermolecular
interactions in the crystals of clioquinol and cloxiquine were detected and examined. The results obtained in this work suggest
that considerable differences in the NQR parameters for the planar and twisted supramolecular synthons permit differentiation
between specific polymorphic forms, and indicate that the more planar supramolecular synthons are accompanied by a greater
number of weaker hydrogen bonds linking them and stronger π···π stacking interactions. 相似文献