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1.
Summary The multiplet structure of cross peaks in double-quantum-filtered COSY NMR spectra is analysed for those resonances that include passive heteronuclear couplings. Interestingly, the cross peak involving the sugar-ring protons H2 and H3 in nucleic acids display an E.COSY-type appearance exclusively when the backbone torsion angle (C4-C3-O3-P) adopts a gauche(-) conformation. This observation allows an unambiguous analysis of the conformation around , without the knowledge of 3Jcp coupling constants.  相似文献   

2.
Spin-State-Selective Excitation (S3E), which forexample selectively excites amide proton resonances corresponding toexclusively either the or the spin state of the covalentlybound 15N atom is employed for E.COSY-type extraction ofheteronuclear J coupling constants. Instead of having one spectrum with twopeaks (corresponding to the or spin state of15N), S3E generates two spectra, each with onlyone peak for each 15N nucleus. These two spectra are generatedfrom the same data set, so that there is no reduction in sensitivitycompared to conventional 1JNH-resolved methods.Another interesting feature in comparison with conventional methods is that1JNH can be suppressed during the evolutionperiod, meaning that no heteronuclear multiplet structure is visible in the1 frequency dimension. The S3E pulsesequence element is combined with NOESY for measurement of3JN-H and JN-Hcoupling constants in either a hetero- or a homonuclear correlated version.Experimental confirmation is obtained using the protein RAP 17-;97(N-terminal domain of 2-macroglobulin ReceptorAssociated Protein).  相似文献   

3.
Summary A method for estimating CH-CH coupling constants from the shape and fine structure of NH-CH fingerprint-region cross peaks of COSY spectra is presented. Spectral simulations have been used to analyse the effect of variations in 3JNH-CH, 3JCH-CH, linewidths and digital resolution on the appearance of NH-CH COSY cross peaks. On the basis of these simulations a set of rules for broadly categorising experimental NH-CH cross peaks according to CH-CH coupling constants has been devised. The method has been applied to the analysis of NH-CH cross peaks of hen lysozyme. The results are compared to previous measurements of CH-CH coupling constants using E.COSY techniques.  相似文献   

4.
Summary The surface tension and foaminess of (a) unlimited, (b) substrate limited, and (c) oxygen transfer limited growth media of Hansenula polymorpha were measured using methanol, ethanol or glucose as a substrate.The time dependence of can be described by the Avrami-Überreiter relationship: log (2.3 log V)=n log t+log b, where V = (Oeq/(teq, and O, t and eq are at tM=0, tM=t and tM (equilibrium value).The constants n and b are functions of the fermentation time tF as long as the growth is unlimited but they are constant in the state of limited growth. With glucose substrate, the foaminess can be presented as a definite function of the time, tDG, which is necessary to attain eq. With alcohol as a substrate no definite (tDG) function was found.Symbols b constant in Eq. (1) - n constant in Eq. (1) - S substrate concentration - T temperature - tM time h (measured from the beginning of the determination of the surface tension ) - tF cultivation time h (measured from the time of inoculation) - tDG time (min) necessary to attain the equilibrium surface tension ) - X dry biomass concentration (gl–1) - V (Oeq)/(teq) - VS equilibrium volume of the foam (cm3) - VG volumetric gas flow rate during the estimation of (cm3 s–1) - vvm volumetric gas flow rate with regard to the volume of the medium (min–1) - wSG superficial gas velocity (cm s–1) - m maximum specific growth rate (h–1) - VS/VG foaminess (s) - surface tension, mMm–1 (milli Newton m–1) - O at tM=0 - eq equilibrium surface tension ( at tM) - t at tM=t - HP probes from Hansenula polymorpha cultivation - NLG non limited growth - OTLG oxygen transfer limited growth - SLG substrate limited growth  相似文献   

5.
A [CO]HN(CA)CB-E.COSY pulse scheme is described for measurement of three-bond couplings, 3JCC, between carbonyl and aliphatic C carbons in ubiquitin, uniformly enriched with 13C and 15N. A Karplus relation, 3JCC = 1.28 cos2( - 120°) -1.02 cos( - 120°) +0.30 Hz, is obtained by correlating the 3JCC values measured for human ubiquitin with backbone angles from its crystal structure. As predicted, the new Karplus parametrization yields 3JCC values slightly larger than previously obtained by quantitative J correlation [Hu, J.-S. and Bax, A. (1997) J. Am. Chem. Soc., 119, 6360-6368], but considerably smaller than what has been reported on the basis of other E.COSY-type measurements carried out on flavodoxin.  相似文献   

6.
Summary A sensitive method to assign H protons stereospecifically as well as to determine rotamer populations about 1, in two 3D experiments is presented. The SOFT-HCCH-COSY experiment allowed us to measure the3J(H,C) couplings, using constant time evolution of C in t2 and Caliphatic-selective decoupling during t3. The SOFT-HCCH-E.COSY experiment allowed us to measure the3J(H,H) couplings, using constant time evolution of C in t2, a small flip angle1H excitation pulse in the second mixing time, and double-band-selective decoupling (aliphatic and carbonyl carbons) during t3. The method was applied to ribonuclease T1.  相似文献   

7.
Summary We introduce the C-FIDS-1H,15N-HSQC experiment, a new method for the determination of 3J(H infi supN ,C infi sup ) coupling constants in proteins, yielding information about the torsional angle . It relies on the 1H,15N-HSQC or HNCO experiment, two of the the most sensitive heteronuclear correlation experiments for isotopically labeled proteins. A set of three 1H,15N-HSQC or HNCO spectra are recorded: a reference experiment in which the carbonyl spins are decoupled during t1 and t2, a second experiment in which they are decoupled exclusively during t1 and a third one in which they are coupled in t1 as well as t2. The last experiment yields an E.COSY-type pattern from which the 2J(H infi supN ,C infi-1 sup ) and 1J(Ni,C infi-1 sup ) coupling constants can be extracted. By comparison of the coupled multiplet (obtained from the second experiment) with the decoupled multiplet (obtained from the first experiment) convoluted with the 2J(H infi supN ,C infi-1 sup ) coupling, the 3J(H infi supN ,C infi sup ) coupling can be found in a one-parameter fitting procedure. The method is demonstrated for the protein rhodniin, containing 103 amino acids. Systematic errors due to differential relaxation are small for nJ(HN,C) couplings in biomacromolecules of the size currently under NMR spectroscopic investigation.  相似文献   

8.
The role of endogenous gibberellin A1 (GA1) in the induction of -amylase activity was investigated during germination of rice (Oryza sativa L.) seeds. The level of endogenous GA1 and the -amylase activity in the seeds of normal rice, cv. Nipponbare, increased simultaneously from 3 days after the imbibition of water. The -amylase activities in the dwarf rice, cv. Waito-C and Tan-ginbozu, were less than that in the normal rice. The level of endogenous GA1 and -amylase activity were decreased in proportion to the concentration of a growth retardant, uniconazole. The retardation in -amylase activity caused by the treatment of uniconazole was recovered by the application of exogenous GA1. These results indicate that the endogenous GA1 biosynthesized de novo regulates -amylase production in germinating rice seeds.Abbreviations GA(s) gibberellin(s) - ABA abscisic acid - AE fraction acidic ethyl acetate-soluble fraction - HPLC high performance liquid chromatography - R t retention time - GC-SIM gas chromatography-selected ion monitoring  相似文献   

9.
    
The limited proteolytic pattern of transducin,G t , and its purified subunits with chymotrypsin were analyzed and the cleavage sites on the t subunit were identified. The t subunit in the GTPS bound form was cleaved into a major 38 kD fragment, whereas t -GDP was progressively digested into 38, 23, 21, and 15 kD fragments. The t subunit was not very sensitive to proteolytic digestion with chymotrypsin. The t subunit was not cleaved and only a small portion of t was digested into several fragments. In order to determine which proteolytic fragment of t still contained the carboxyl terminal region, chymotrypsinization was carried out usingG t previously32P-labeled at Cys347 by petrussis toxin-catalyzed ADP-ribosylation. The32P-label was mainly associated with the t subunit and a 15 kD fragment. The 23 and 21 kD fragments were not32P-labeled. Analysis of amino terminal sequences of 38, 21, and 15 kD proteolytic bands allowed the identification of the major cleavage sites. Chymotrypsin had two cleavage sites in the amino terminal region of t , at Leu15 and Leu19. Chymotrypsin removed 15–19 amino acid residues from the amino terminus of t , generating two peptides (38 kD) which comigrates in gel electrophoresis. Chymotrypsin also cleaved at Trp207 in a conformation-dependent manner. Trp207 of t -GTPS was resistant to proteolysis but t -GDP and the 38 kD fragments of t -GDP produced the 23 and 21 kD fragments, respectively, and a 15 kD fragment containing the carboxyl terminus. This proves that the environment of Trp207 changes when GTP or GTPS is bound, leading to its inaccessibility to chymotrypsin.  相似文献   

10.
Summary 2-Amino-2-deoxyuridine reacts efficiently with nucleoside 5-phosphorimidazolides in aqueous solution. The dinucleoside monophosphate analogues were obtained in yields exceeding 80% under conditions in which little reaction occurs with the natural nucleosides.In a similar way, the 5-phosphorimidazolide of 2-amino-2-deoxyuridine undergoes self-condensation in aqueous solution to give a complex mixture of oligomers.The phosphoramidate bond in the dinucleoside monophosphate analogues is stable for several days at room temperature and pH 7. The mechanisms of their hydrolysis under acidic and alkaline conditions are described.Abbreviations A adenosine - C cytidine - G guanosine - U uridine - T thymidine - UN 3 2-azido-2-deoxyuridine - UNH 2 2-amino-2-deoxyuridine - ImpA adenosine 5-phosphorimidazolide - ImpU uridine 5-phosphorimidazolide - ImpUN 3 2-azido-2-deoxyuridine 5-phosphorimidazolide - ImpUNH 2 2-amino-2-deoxyuridine 5-phosphorimidazolide - pA adenosine 5-phosphate - pU uridine 5-phosphate - pUN 3 2-azido-2-deoxyuridine 5-phosphate - pUNH 2 2-amino-2-deoxyuridine 5-phosphate - UpA uridylyl-[35]-adenosine - UpU uridylyl-[35]-uridine - UNpA adenylyl-[52]-2-amino-2-deoxy-uridine - UNpU uridylyl-[52]-2-amino-2-deoxyuridine (pUN)n n=2,3,4 [25]-linked oligomers of pUNH 2 poly(A) polyadenylic acid - Im imidazole - MeIm l-methylimidazole  相似文献   

11.
The interactions between a series of platinum complexes, including (pyridyl)(6-phenyl-2,2-bipyridine)platinum(II) hexafluorophosphate (1), three dinuclear bis[(6-phenyl-2,2-bipyridine)platinum(II)] complexes (24), and (4-aminopyridine)(4,6-diphenyl-2,2-bipyridine)platinum(II) perchlorate (5) with DNA have been investigated. All Pt(II) complexes, except 5, were demonstrated to be DNA intercalators, based on viscosity measurements. Absorption and fluorescence titration results indicated that the addition of a phenyl ring to the 6-phenyl-2,2-bipyridine ligand dramatically reduced the DNA binding of the Pt(II) complex 5. The dinuclear complexes 24 exhibited multiple binding modes of mono/bisintercalation and groove binding, as revealed by viscosity and fluorescence titration measurements. While complexes 24 bound to DNA with significantly enhanced affinities as compared to 1, compounds 1 and 24 showed similar IC50 values against a panel of cancer cell lines. In addition, these complexes showed similar cellular uptakes. The results indicated that the cytotoxicity of these (6-phenyl-2,2-bipyridine)platinum compounds may not be mediated through DNA binding but may involve interacting mechanisms with cellular components other than DNA.Electronic Supplementary Material Supplementary material is available for this article if you access the article at . A link in the frame on the left on that page takes you directly to the supplementary material.Abbreviations AO acridine orange - ampy 4-aminopyridine - bipy 2,2-bipyridine - bp base pair - CNN 6-phenyl-2,2-bipyridine - ctDNA calf thymus DNA - EB ethidium bromide - EI electron ionization - en ethylenediamine - FAB fast atomic bombardment - H33342 Hoechst 33342 - IC50 inhibitory concentration 50% - ICP-AES inductively coupled plasma–atomic emission spectroscopy - MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-tetrazolium bromide - 4-PhCNN 4,6-diphenyl-2,2-bipyridine - phen 1,10-phenanthroline - terpy 2,2:6,2-terpyridine - Tris tris(hydroxymethyl)aminomethane  相似文献   

12.
Summary A 3D triple resonance experiment has been designed to provide intraresidual and sequential correlations between amide nitrogens and -carbons in uniformly 13C15N-labeled proteins. In-phase 13C magnetization is transferred to the aliphatic side-chain protons via the side-chain carbons using a CC-TOCSY mixing sequence. Thus, the experiment alleviates the resonance assignment process by providing information about the amino acid type as well as establishing sequential connectivities. Leaving the carbonyl spins untouched throughout the transfer from 13C to 1H leads to E.COSY-type cross peaks, from which the 3JH co coupling constants can be evaluated. The pulse sequence is applied to oxidized Desulfovibrio vulgaris flavodoxin.  相似文献   

13.
Template-primer dependent inactivation of human DNA polymerase and Klenow fragment of E. coli DNA polymerase I by adenosine 2,3-riboepoxide 5-triphosphate was used for quantitative analysis of the Kd values for oligonucleotide primers of different length. The Kd values are smaller by a factor of 2.5 than the Km values for the same primers determined in the reaction of DNA polymerization in the case of DNA polymerase . The Kd and Km values are nearly the same for Klenow fragment. Such approach to the determination of Km/Kd ratio can likely be used for detailed quantitative analysis of DNA polymerases.Abbreviations epATP adenosine 2,3-riboepoxide 5-triphosphate - KF Klenow fragment of E. coli DNA polymerase I - Pol I E. coli DNA polymerase I - Pol human placenta DNA polymerase   相似文献   

14.
Summary Different methods for determining sugar conformations in large oligonucleotides have been evaluated using both J-coupling and NOE data. In order to simulate COSY spectra, reliable estimates of line widths are required. We have measured T1p (=T2) values for a large number of protons of the hexadecamer d(CATGTGACGTCACATG)2 using a new two-dimensional NMR experiment (T1RHOSY) to provide baseline information for the simulations. Both DQF-COSY and P.E.COSY cross-peaks have been systematically simulated as a function of line width, digitisation and signal-to-noise ratio. We find that for longer correlation times (5 ns), where line widths are comparable to or larger than active couplings, only {ie190-1} is reasonably accurately determined (within ±1 Hz). Under these conditions, additional information is needed to determine the sugar conformation. We have used apparent distances H1-2-H4 and H2-H4, which provide a range of Ps over an interval of ca. 20°. Complete analysis of time courses for intraresidue NOEs, with and without coupling constants, has also been evaluated for determining nucleotide conformations. Whereas Ps is poorly determined in the absence of both intrasugar NOEs and coupling constants, the range of solutions is decreased when intrasugar NOEs and {ie190-2} are also available. DQF-COSY, P.E.COSY and NOESY spectra at different mixing times of the hexadecamer d(CATGTGACGTCACATG)2 were recorded at three temperatures. A detailed analysis of the NOEs and coupling constants provided estimates of the sugar conformations in the hexadecamer. At 50 °C, the sugar conformations are well determined by the scalar and dipolar data, with pseudorotation phase angles of 126–162° and mole fractions of the S conformation (fs) of 0.86±0.05. There was no statistically significant difference between fs for the purines and the pyrimidines, although there was a small tendency for Ps of the purines to be larger than those of the pyrimidines. At 25 °C, the sugar conformations were much less well determined, although the estimates of fs were the same within experimental error as at 50 °C. The experimental and theoretical results provide guidelines for the limits of conformational analysis of nucleic acids based on homonuclear NMR methods.  相似文献   

15.
Summary Alanine-rich peptides serve as models for exploring the factors that control helix structure in peptides and proteins. Scalar CH-NH couplings (3JHN) are an extremely useful measure of local helix content; however, the large alanine content in these peptides leads to significant signal overlap in the CH region of 1H 2D NMR spectra. Quantitative determination of all possible 3JHN values is, therefore, very challenging. Szyperski and co-workers [(1992) J. Magn. Reson., 99, 552–560] have recently developed a method for determining 3JHN from NOESY spectra. Because 3JHN may be determined from 2D peaks outside of the CH region, there is a much greater likelihood of identifying resolved resonances and measuring the associated coupling constants. It is demonstrated here that 3JHN can be obtained for every residue in the helical peptide Ac-(AAAAK)3A-NH2. The resulting 3JHN profile clearly identifies a helical structure in the middle of the peptide and further suggests that the respective helix termini unfold via distinct pathways.Abbreviations 3JHN three-bond CH-NH scalar coupling constant - NOE nuclear Overhauser enhancement - NOESY two-dimensional nuclear Overhauser spectroscopy - COSY two-dimensional correlated spectroscopy - DQF-COSY two-dimensional double-quantum-filtered correlated spectroscopy - TOCSY two-dimensional total correlation spectroscopy To whom correspondence should be addressed.Deceased March 5, 1996.  相似文献   

16.
Summary Rooted phylogenetic trees for a total of 34 genes encoding the stimulatory (s), inhibitory (i), transducin (t), Gx (x), Gz (z), G11 (11), G12 (12), G13 (13), G16 (16), Gq (q), and other (o) G protein a subunits have been constructed. The analysis shows that the G12 (12 and 13), Gq (11, 16, and q), and Gs (s genes) groups form one cluster, and the Gx (x and z genes), Gi (i genes), Gt (t1 and t2), and Go (o genes) groups form another cluster. During mammalian evolution, the rates of synonymous substitutions for these genes were estimated to be between 1.77 × 10–9/site/year and 5.63 × 10–9/site/year, whereas those of nonsynonymous substitutions were between 0.008 × 10–9/site/year and 0.067 × 10–9/site/year. These evolutionary rates are similar to those for histone genes, suggesting equally important biological functions of the G protein a subunits. Offprint requests to: S. Yokoyama  相似文献   

17.
Summary A simple E.COSY type technique is described for measurement of two-bond JCOH coupling constants in proteins that are uniformly enriched with13C. The method has been used to measure2JCOH for 132 residues in the proteins calmodulin and staphylococcal nuclease having non-overlapping H–C correlations. Measured2JCOH coupling constants fall in the 0 to –9.5 Hz range. A separate experiment, measuring the accuracy of these values, indicates a root-mean-square error of 1 Hz. Comparison of the J couplings with the dihedral back bone angles from crystallographic studies confirms a weak but statistically significant correlation between the dihedral angle and the magnitude of2JCOH, but also indicates that parameters other than have a significant effect on the value of the coupling.  相似文献   

18.
We have studied the in vitro transfection of a plasmid DNA with the lacZ gene to HeLa-S3 cells and hemolysis in a red blood cell (RBC) suspension under pulsed ultrasound with duty cycles of 10, 20 and 30% using a digital sonifier at a frequency of 20 kHz and an intensity of 6.2 W/cm2 on the surface of a horn tip. Cultured HeLa-S3 cells in suspension were exposed to pulsed ultrasound for an apparent exposure time t from 0 to 60 s. HeLa-S3 viability decreased as a single exponential function of the total exposure time t=t with a common time constant =3.8 s for three duty cycles. Transfection was evaluated by counting the number of -galactosidase(-Gal)-positive cells relative to the total number of cells. Pulsed ultrasound provided an enhanced transfer of the -Gal plasmid to HeLa-S3 cells, 3.4-fold as compared with that in the case of the control. The optimal transfection efficiencies were 0.75, 0.80 and 0.74% near t= with =10, 20 and 30%, respectively. The number ratio of -Gal-positive cells to the surviving cells after exposure increased with t according to a modified logistic equation. The degree of hemolysis also increased exponentially with t at a time constant =0/ for the RBC suspension in physiological saline at a hematocrit concentration of 0.5% with 0=0.9 s. Thus the total exposure time for the optimal transfection efficiency was , that is, nearly four times of 0. Hemolysis in the RBC suspension may be a useful model for determining optimal transfection by pulsed ultrasound of various duty cycles.  相似文献   

19.
Elevated CO2 (ambient + 35 Pa) increased shoot dry mass production in Avena fatua by 68% at maturity. This increase in shoot biomass was paralleled by an 81% increase in average net CO2 uptake (A) per unit of leaf area and a 65% increase in average A at the ecosystem level per unit of ground area. Elevated CO2 also increased ecosystem A per unit of biomass. However, the products of total leaf area and light-saturated leaf A divided by the ground surface area over time appeared to lie on a single response curve for both CO2 treatments. The approximate slope of the response suggests that the integrated light saturated capacity for leaf photosynthesis is 10-fold greater than the ecosystem rate. Ecosystem respiration (night) per unit of ground area, which includes soil and plant respiration, ranged from-20 (at day 19) to-18 (at day 40) mol m-2 s-1 for both elevated and ambient CO2 Avena. Ecosystem below-ground respiration at the time of seedling emergence was -10 mol m-2 s-1, while that occuring after shoot removal at the termination of the experiment ranged from -5 to-6 mol m-2 s-1. Hence, no significant differences between elevated and ambient CO2 treatments were found in any respiration measure on a ground area basis, though ecosystem respiration on a shoot biomass basis was clearly reduced by elevated CO2. Significant differences existed between leaf and ecosystem water flux. In general, leaf transpiration (E) decreased over the course of the experiment, possibly in response to leaf aging, while ecosystem rates of evapotranspiration (ET) remained constant, probably because falling leaf rates were offset by an increasing total leaf biomass. Transpiration was lower in plants grown at elevated CO2, though variation was high because of variability in leaf age and ambient light conditions and differences were not significant. In contrast, ecosystem evapotranspiration (ET) was significantly decreased by elevated CO2 on 5 out of 8 measurement dates. Photosynthetic water use efficiencies (A/E at the leaf level, A/ET at the ecosystem level) were increased by elevated CO2. Increases were due to both increased A at leaf and ecosystem level and decreased leaf E and ecosystem ET.  相似文献   

20.
Summary We have studied the reactions between adenosine 5-phosphorimidazolide and 9-(2-amino-2-deoxyxylofuranosyl) adenine (I) or 3-methylamino-3-deoxyadenosine (II), both with and without a poly (U) template. We find that both amino compounds react much more rapidly than does adenosine, in the absence of a template. The rate of reaction is greatly enhanced by a poly (U) template in the case of I, but the enhancement is slight in the case of II.Abbreviations A adenosine - xylo ANH2 9-(2-amino-2-deoxy--D-xylofuranosyl) adenine - ANHMe 3-methylamino-3-deoxyadenosine - ImpA adenosine 5-phosphorimidazolide - A3 pA adenylyl-[35]-adenosine - A2 pA adenylyl-[25]-adenosine - UNPA adenylyl-[52]-2-amino-2-deoxyuridine - xylo ANPA 9-[adenylyl-(52)-2-amino-2-deoxy--D-xylofuranosyl]adenine - A(NMe)pA adenylyl-[53]-3-methylamino-3-deoxyadenosine - pA adenosine 5phosphate - AppA P1, P2-diadenosine 5pyrophosphate - (pA)n n = 2, 3 [2-5]-linked oligomers of pA - A2 pA2 pA [2-5]-linked trinucleoside diphosphate of A - poly (U) polyuridylic acid  相似文献   

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