Determination of the backbone torsion angle ɛ in nucleic acids

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 ³J_cp coupling constants.     

Integration of spin-state-selective excitation into 2D NMR correlation experiments with heteronuclear ZQ/2Q π rotations for1 JXH

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 ¹J_NH-resolved methods.Another interesting feature in comparison with conventional methods is that¹J_NH 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 of³J_N-H and J_N-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).     

A method for the estimation of χ1 torsion angles in proteins

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 ³J_NH-CH, ³J_CH-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.     

Foam behavior of biological media

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 = (_O – _eq/(_t – _eq, and _O, _t and _eq are at t_M=0, t_M=t and t_M (equilibrium value).The constants n and b are functions of the fermentation time t_F 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, t_DG, which is necessary to attain _eq. With alcohol as a substrate no definite (t_DG) function was found.Symbols b constant in Eq. (1) - n constant in Eq. (1) - S substrate concentration - T temperature - t_M time h (measured from the beginning of the determination of the surface tension ) - t_F cultivation time h (measured from the time of inoculation) - t_DG time (min) necessary to attain the equilibrium surface tension ) - X dry biomass concentration (gl^–1) - V (_O – _eq)/(_t – _eq) - V_S equilibrium volume of the foam (cm³) - V_G 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) - w_SG superficial gas velocity (cm s^–1) - _m maximum specific growth rate (h^–1) - V_S/V_G foaminess (s) - surface tension, mMm^–1 (milli Newton m^–1) - _O at t_M=0 - _eq equilibrium surface tension ( at t_M) - _t at t_M=t - HP probes from Hansenula polymorpha cultivation - NLG non limited growth - OTLG oxygen transfer limited growth - SLG substrate limited growth     

Measurement of Three-bond, 13C′-13Cβ J Couplings in Human Ubiquitin by a Triple Resonance, E. COSY-type NMR Technique

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.     

Determination of Hα,Hβ and Hβ,C′ coupling constants in13C-labeled proteins

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 C_aliphatic-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.     

Determination of 3J(H infi supN,C infi sup′ ) coupling constants in proteins with the C′-FIDS method

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(N_i,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(H^N,C) couplings in biomacromolecules of the size currently under NMR spectroscopic investigation.     

Endogenous gibberellin A1 level and α-amylase activity in germinating rice seeds

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     

Polymerization of nucleotide analogues I: Reaction of nucleoside 5′-phosphorimidazolides with 2′-amino-2′-deoxyuridine

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 - U_{N 3} 2-azido-2-deoxyuridine - U_{NH 2} 2-amino-2-deoxyuridine - ImpA adenosine 5-phosphorimidazolide - ImpU uridine 5-phosphorimidazolide - ImpU_{N 3} 2-azido-2-deoxyuridine 5-phosphorimidazolide - ImpU_{NH 2} 2-amino-2-deoxyuridine 5-phosphorimidazolide - pA adenosine 5-phosphate - pU uridine 5-phosphate - pU_{N 3} 2-azido-2-deoxyuridine 5-phosphate - pU_{NH 2} 2-amino-2-deoxyuridine 5-phosphate - UpA uridylyl-[35]-adenosine - UpU uridylyl-[35]-uridine - U_NpA adenylyl-[52]-2-amino-2-deoxy-uridine - U_NpU uridylyl-[52]-2-amino-2-deoxyuridine (pU_N)_n n=2,3,4 [25]-linked oligomers of pU_{NH 2} poly(A) polyadenylic acid - Im imidazole - MeIm l-methylimidazole     

Bis-intercalative dinuclear platinum(II) 6-phenyl-2,2′-bipyridine complexes exhibit enhanced DNA affinity but similar cytotoxicity compared to the mononuclear unit

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 (2–4), 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 2–4 exhibited multiple binding modes of mono/bisintercalation and groove binding, as revealed by viscosity and fluorescence titration measurements. While complexes 2–4 bound to DNA with significantly enhanced affinities as compared to 1, compounds 1 and 2–4 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     

HNCCH-TOCSY,a triple resonance experiment for the correlation of backbone 13Cα and 15N resonances with aliphatic side-chain proton resonances and for measuring vicinal 13CO, 1Hβ coupling constants in proteins

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 ³J_{H co} coupling constants can be evaluated. The pulse sequence is applied to oxidized Desulfovibrio vulgaris flavodoxin.     

Inactivation of DNA polymerases by adenosine 2′,3′-riboepoxide 5′-triphosphate allows estimation of the primers affinity

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 K_d values for oligonucleotide primers of different length. The K_d values are smaller by a factor of 2.5 than the K_m values for the same primers determined in the reaction of DNA polymerization in the case of DNA polymerase . The K_d and K_m values are nearly the same for Klenow fragment. Such approach to the determination of K_m/K_d 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      

Determination of sugar conformations by NMR in larger DNA duplexes using both dipolar and scalar data: Application to d(CATGTGACGTCACATG)2

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 T_1p (=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 P_s 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 P_s 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 (f_s) of 0.86±0.05. There was no statistically significant difference between f_s for the purines and the pyrimidines, although there was a small tendency for P_s 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 f_s 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.     

Local helix content in an alanine-rich peptide as determined by the complete set of 3JHNα coupling constants

Summary Alanine-rich peptides serve as models for exploring the factors that control helix structure in peptides and proteins. Scalar CH-NH couplings (³J_HN) 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 ³J_HN values is, therefore, very challenging. Szyperski and co-workers [(1992) J. Magn. Reson., 99, 552–560] have recently developed a method for determining ³J_HN from NOESY spectra. Because ³J_HN 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 ³J_HN can be obtained for every residue in the helical peptide Ac-(AAAAK)₃A-NH₂. The resulting ³J_HN 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 ³J_HN 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.     

Measurement of two-bond JCOHα coupling constants in proteins uniformly enriched with13C

Summary A simple E.COSY type technique is described for measurement of two-bond J_COH coupling constants in proteins that are uniformly enriched with¹³C. The method has been used to measure²J_COH for 132 residues in the proteins calmodulin and staphylococcal nuclease having non-overlapping H–C correlations. Measured²J_COH 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 of²J_COH, but also indicates that parameters other than have a significant effect on the value of the coupling.     

Phylogeny and evolutionary rates of G protein α subunit genes

Summary Rooted phylogenetic trees for a total of 34 genes encoding the stimulatory (s), inhibitory (i), transducin (t), G_x (x), G_z (z), G₁₁ (11), G₁₂ (12), G₁₃ (13), G₁₆ (16), G_q (q), and other (o) G protein a subunits have been constructed. The analysis shows that the G₁₂ (12 and 13), G_q (11, 16, and q), and G_s (s genes) groups form one cluster, and the G_x (x and z genes), G_i (i genes), G_t (t1 and t2), and G_o (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     

Interrelation between HeLa-S3 cell transfection and hemolysis in red blood cell suspension using pulsed ultrasound of various duty cycles

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.     

Contrasting leaf and ‘ecosystem’ CO2 and H2O exchange in Avena fatua monoculture: Growth at ambient and elevated CO2

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.     

Template-directed reactions of aminonucleosides

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 A_NH2 9-(2-amino-2-deoxy--D-xylofuranosyl) adenine - A_NHMe 3-methylamino-3-deoxyadenosine - ImpA adenosine 5-phosphorimidazolide - A³ pA adenylyl-[35]-adenosine - A² pA adenylyl-[25]-adenosine - U_NPA adenylyl-[52]-2-amino-2-deoxyuridine - xylo A_NPA 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