Expression of deuterium-isotope-labelled protein in the yeast Pichia pastoris for NMR studies

Deuterium isotope labelling is important for NMR studies of large proteins and complexes. Many eukaryotic proteins are difficult to express in bacteria, but can be efficiently produced in the methylotrophic yeast Pichia pastoris. In order to facilitate NMR studies of the malaria parasite merozoite surface protein-1 (MSP1) complex and its interactions with antibodies, we have investigated production of the MSP1-19 protein in P. pastoris grown in deuterated media. The resulting deuteration patterns were analyzed by NMR and mass spectrometry. We have compared growth characteristics and levels of heterologous protein expression in cells adapted to growth in deuterated media (95% D₂O), compared with expression in non-adapted cells. We have also compared the relative deuteration levels and the distribution pattern of residual protiation in protein from cells grown either in 95% D₂O medium with protiated methanol as carbon source, or in 95% D₂O medium containing deuterated methanol. A high level of uniform C deuteration was demonstrated, and the consequent reduction of backbone amide signal linewidths in [¹H/¹⁵N]-correlation experiments was measured. Residual protiation at different positions in various amino acid residues, including the distribution of methyl isotopomers, was also investigated. The deuteration procedures examined here should facilitate economical expression of ²H/¹³C/¹⁵N-labelled protein samples for NMR studies of the structure and interactions of large proteins and protein complexes.     

Evidence for an arene oxide-NIH shift pathway in the transformation of naphthalene to 1-naphthol by Bacillus cereus

Bacillus cereus ATCC 14579 transformed naphthalene predominately to 1-naphthol. Experiments with [¹⁴C]naphthalene showed that over a 24 h period, B. cereus oxidized 5.2% of the added naphthalene. 1-Naphthol accounted for approximately 80% of the total metabolites. B. cereus incubated with naphthalene under the presence of ¹⁸O₂ led to the isolation of 1-naphthol that contained 94% ¹⁸O. The metabolism of [1-²H]-and [2-²H]-naphthalene by B. cereus yielded 1-naphthol which retained 95% and 94% deuterium, respectively, as determined by mass spectral analysis. NMR spectroscopic analysis of the deuterated 1-naphthol formed from [1-²H]-naphthalene indicated an NIH shift mechanism in which 19% of the deuterium migrated from the C-1 to the C-2 position. The ¹⁸O₂ and NIH shift experiments implicate naphthalene-1,2-oxide as an intermediate in the formation of 1-naphthol from naphthalene by B. cereus.Abbreviations HPLC High performance liquid chromatography - NMR nuclear magnetic resonance     

Biosynthesis of methyl chloride in the fungusPhellinus pomaceus

The biosynthetic origin of themethyl group in the methyl chloride produced by cultures ofPhellinus pomaceus (Pers.) Maire has been investigated using stable isotope labeled substrates. Feeding ofd-[6,6-²H₂] glucose,Dl-[3,3-²H₂] serine andl-[methyl-²H₃] methionine led to the production of deuterated methyl chloride in which the major labeled species contained 2, 2, and 3 deuterium atoms, respectively. The data are consistent with the methyl chloride produced by this organism being derived solely from methionine with retention of all of the methyl protons.Abbreviation SAM S-adenosylmethionine - FH₄ tetrahydrofolate - N⁵-CH₃FH₄ N⁵-methyltetrahydrofolate - B₁₂ cobalamin     

Characterization of pea histone deacetylases

The present paper is the first report on histone deacetylases from plants. Three enzyme fractions with histone deacetylase activity (HD0, HD1 and HD2) have been partially purified from pea (Pisum sativum) embryonic axes. They deacetylate biologically acetylated chicken histones and, to a lesser extent, chemically acetylated histones, this being a criterion of their true histone deacetylase nature. The three enzymes are able to accept nucleosomes as substrates. HD1 is not inhibited by n-butyrate up to 50 mM, whereas HD0 and HD2 are only slightly inhibited, thereby establishing a clear difference to animal histone deacetylases. The three activities are inhibited by acetate, Cu²⁺ and Zn²⁺ ions and mercurials, but are only scarcely affected by polyamines, in strong contrast with yeast histone deacetylase. Several criteria have been used to obtain cumulative evidence that HD0, HD1 and HD2 actually are three distinct enzymes. In vitro experiments with free histones show that HD0 deacetylates all four core histones, whereas HD1 and HD2 show a clear preference for H2A and H2B, the arginine-rich histones being deacetylated more slowly.     

Strong coupling effects during <Emphasis Type="Italic">X</Emphasis>-pulse CPMG experiments recorded on heteronuclear <Emphasis Type="Italic">ABX</Emphasis> spin systems: artifacts and a simple solution

Simulation and experiment have been used to establish that significant artifacts can be generated in X-pulse CPMG relaxation dispersion experiments recorded on heteronuclear ABX spin-systems, such as ¹³C _i –¹³C _j –¹H, where ¹³C _i and ¹³C _j are strongly coupled. A qualitative explanation of the origin of these artifacts is presented along with a simple method to significantly reduce them. An application to the measurement of ¹H CPMG relaxation dispersion profiles in an HIV-2 TAR RNA molecule where all ribose sugars are protonated at the 2′ position, deuterated at all other sugar positions and ¹³C labeled at all sugar carbons is presented to illustrate the problems that strong ¹³C–¹³C coupling introduces and a simple solution is proposed.     

Dormancy in somatic embryos and seeds ofVitis: changes in endogenous abscisic acid during embryogeny and germination

Abscisic acid (ABA) in extracts of somatic embryos and seeds of Gloryvine (Vitis vinifera L.xV. rupestris Scheele) was measured by gas chromatography-mass spectrometry-selected ion monitoring using deuterated ABA, (±)-[C-3Me-²H₃]ABA, ([²H₃]ABA) as internal standard. The ABA content increased rapidly during embryogeny (0.035 ng/embryo at the globular stage to 0.22 ng/embryo at the mature stage). The level of ABA in the tissues of somatic embryos, expressed in ng/mg dry weight, decreased from the globular stage (0.76 ng/mg) to the mature stage (0.25 ng/mg). Chilling (4° C) induced normal germination of seeds and mature somatic embryos and precocious germination of globular, heart-shaped and torpedoshaped somatic embryos. In all cases chilling led to a marked reduction in endogenous ABA. Exogenous (±)-ABA inhibited the germination of chilled somatic embryos.Abbreviations ABA abscisic acid - [²H₃]ABA (±)-[C-3Me-²H₃]-abscisic acid - BHT 2,6-di-t-butyl-4-methylphenol - GC-MS gas chromatography-mass spectrometry - Me-ABA and Me-[²H₃]ABA methyl esters of ABA and [²H₃]ABA, respectively - SIM selected ion monitoring     

Glucose uptake in mouse brain regions under hypoxic hypoxia

Glucose uptake of individual structures within the brain was studied by dry-autoradiography with 2-deoxy-D-[¹⁴C(U)]glucose under mild hypoxic hypoxia (12% O₂88% N₂ or 9% O₂91% N₂ for 1 hr). Glucose consumption in the whole brain was estimated by combined gas chromatography-mass spectrometry (GC-MS). Mild hypoxia increased the optical density of the autoradiograph in all regions. The deuterated G-6-P (Glucose-6-phosphate) synthesized from deuterated glucose decreased significantly with 9% O₂ hypoxia (P<0.05). The ratio of the deuterated G-6-P to deuterated glucose, a more appropriate indicator of glucose utilization than the concentration of deuterated G-6-P, decreased significantly with 12% O₂ hypoxia (P<0.01). The hippocampus, white matter, colliculus superior, and corpus geniculatum laterale appeared to be particulary sensitive to hypoxia.     

Ontogenetic variation in levels of gibberellin A1 in Pisum

The levels of the biologically active gibberellin (GA), GA₁, and of its precursor, GA₂₀, were monitored at several stages during ontogeny in the apical portions of isogenic tall (Le) and dwarf (le) peas (Pisum sativum L.) using deuterated internal standards and gas chromatography-selected ion monitoring. The levels of both GAs were relatively low on emergence and on impending apical arrest. At these early and late stages of development the internodes were substantially shorter than at intermediate stages, but were capable of large responses to applied GA₃. Tall plants generally contained 10–18 times more GA₁ and possessed internodes 2–3 times longer than dwarf plants. Further, dwarf plants contained 3–5 times more GA₂₀ than tall plants. No conclusive evidence for the presence of GA₃ or GA₅ could be obtained, even with the aid of [²H₂]GA₃ and [²H₂]GA₅ internal standards. If GA₃ and GA₅ were present in tall plants, their levels were less than 0.5% and 1.4% of the level of GA₁, respectively. Comparison of the effects of gene le on GA₁ levels and internode length with the effects of ontogeny on these variables shows that the ontogenetic variation in GA₁ content was sufficient to account for much of the observed variation in internode length within the wild-type. However, evidence was also obtained for substantial differences in the potential length of different internodes even when saturating levels of exogenous GA₃ were present.Abreviations GA_n gibberellin A_n We thank Noel Davies, Omar Hasan, Leigh Johnson, Katherine McPherson and Naomi Lawrence for technical help, Professor L. Mander (Australian National University, Canberra) for deuterated GA standards and the Australian Research Council for financial assistance.     

Determinations of indole-3-acetic acid in xylem sap of Ricinus communis L. using mass fragmentography

Mass fragmentography employing a deuterated internal standard was used to make quantitative analyses of indole-3-acetic acid in xylem sap collected from Ricinus communis L. When contamination of the sap by microorganisms was reduced by frequent collection, levels of IAA were found to be less than 0.5 ng ml^-1. It is therefore proposed that the transpiration stream does not play a significant role in the transport of IAA within the plant.Abbreviations IAA indole-3-acetic acid - GC-MS gas chromatography-mass spectrometry - BSTFA bis-Trimethylsilytrifluoroacetamide - TMCS trimethylchlorosilane - BSA bis-Trimethylsilylacetamide - TMS₂-IAA bis-trimethylsilyl derivative of IAA     

SYNTHETIC STUDIES TO IMPROVE THE DEUTERIUM LABELLING IN NUCLEOSIDES FOR FACILITATING STRUCTURAL STUDIES OF LARGE RNAS BY HIGH-FIELD NMR SPECTROSCOPY

Synthetic studies to prepare ribonucleosides deuterated at C2′ and the application of the developed procedures for the synthesis of ² H ₅ -ribonucleosides from 1,2-O-isopropylidene-3-O-benzyl-ribofuranose-3,4,5,5′- ² H ₄ have been reported.     

Synthesis of methyl [18-2H3], [17-2H2], [16-2H2], [14-2H2] and [12-2H2] cis-9-octadecenoates

Methyl [17-²H₂]oleate was prepared by stepwise reduction from 17-oxooleate in 24% yield. Methyl [18-²H₃], [16-²H₂], [14-²H₂] and [12-²H₂] oleates were synthesized from appropriately deuterated octylbromides by conversion to deuterated 7-hexadecyn-1-ols and chain extention to deuterated stearolates followed by semihydrogenation; overall yields were about 17%.     

The effect of selective deuteration on magnetization transfer in larger proteins

Summary The effects of selective deuteration on calculated NOESY intensities have been analyzed for the structure of theE. coli trp aporepressor, a 25 kDa protein. It is shown that selectively deuteratedtrp aporepressor proteins display larger calculated NOESY intensities than those for the same interproton distances in the natural abundance protein. The relatively larger magnetization transfer is demonstrated by a comparison of the NOE build-up curves for specific proton pairs, and for the calculated NOE intensities of short-range NOEs to backbone amide protons. This increase in intensity is especially pronounced for the NH¹–NH¹⁺¹ cross peaks in the -helical regions, and particularly for amide protons of two sequential deuterated residues. The effect is shown to be further intensified for longer mixing times. It is also shown that in all cases, each amide proton exhibits stronger NOEs to its own side chain, with an enhanced effect for deuterated derivatives. This theoretical analysis demonstrates that an evaluation of the relative NOE intensities for different selectively deuterated analogs may be an important tool in assigning NMR spectra of large proteins. These results also serve as a guide for the interpretation of NOEs in terms of distances for structure calculations based on data using selectively deuterated proteins.     

High resolution <Superscript>13</Superscript>C-detected solid-state NMR spectroscopy of a deuterated protein

High resolution ¹³C-detected solid-state NMR spectra of the deuterated beta-1 immunoglobulin binding domain of the protein G (GB1) have been collected to show that all ¹⁵N, ¹³C′, ¹³Cα and ¹³Cβ sites are resolved in ¹³C–¹³C and ¹⁵N–¹³C spectra, with significant improvement in T ₂ relaxation times and resolution at high magnetic field (750 MHz). The comparison of echo T ₂ values between deuterated and protonated GB1 at various spinning rates and under different decoupling schemes indicates that ¹³Cα T ₂′ times increase by almost a factor of two upon deuteration at all spinning rates and under moderate decoupling strength, and thus the deuteration enables application of scalar-based correlation experiments that are challenging from the standpoint of transverse relaxation, with moderate proton decoupling. Additionally, deuteration in large proteins is a useful strategy to selectively detect polar residues that are often important for protein function and protein–protein interactions.     

Histone-induced conformational changes in DNA as probed by quasi-elastic light scattering.

Quasi-elastic light scattering as measured by intensity fluctuation (self-beat) spectroscopy in the time domain can be profitably used to follow both the translational diffusion D and the dominant internal flexing mode τ_int of DNA and its complexes with various histones in aqueous salt solutions. Without histones, DNA is found to have D = 1.6 × 10^?8 cm²/sec and τ_int ? 5 × 10^?4 sec in 0.8 M NaCl, 2 M urea at 20°C. Total histone as well as fraction F2A induce supercoiling (D = 2.6 × 10^?8 cm²/sec, τ_int ? 2.8 × 10^?4 sec) whereas fraction F1 induces uncoiling (D = 1.0 × 10^?8 cm²/sec, τ_int ? 9.4 × 10^?4 sec). Upon increasing the salt concentration to 1.5 M the DNA–histone complex dissociates (D = 1.8 × 10^?8 cm²/sec). Upon decreasing the salt concentration to far below 0.8 M, the DNA–histone complex eventually precipitates as a chromatin gel.     

Denitrification of nitrate to nitrogen gas by washed cells ofRhizobium japonicum and by bacteroids fromGlycine max

Nitrate, nitrite and nitrous oxide were denitrified to N₂ gas by washed cells ofRhizobium japonicum CC706 as well as by bacteroids prepared from root nodules ofGlycine max (L.) Merr. (CV. Clark 63). Radiolabelled N₂ was produced from either K¹⁵NO₃ or Na¹⁵NO₂ by washed cells ofRh. japonicum CC705 grown with either nitrate only (5 mM) or nitrate (5 mM) plus glutamate (10 mM). Nitrogen gas was also produced from N₂O. Similar results were obtained with bacteroids ofG. max. The stoichiometry for the utilization of¹⁵NO ₃ ^- or¹⁵NO ₂ ^- and the produciton of¹⁵N₂ was 2:1 and for N₂O utilization and N₂ production it was 1:1. Some of the¹⁵N₂ gas produced by denitrification of¹⁵NO ₃ ^- in bacteroids was recycled via nitrogenase into cell nitrogen.     

Differences in structure and stability between normal and deuterated proteins (phycocyanin)

Differential scanning microcalorimetry was used to investigate the enthalpy (ΔH_d) and the temperature (t_d) of thermal denaturation of normal and deuterated phycocyanins isolated from two blue-green algae, Plectonema calothricoides and Phormidium luridum. Values of t_d in deuterated proteins are about 5°C lower than those in normal proteins. The magnitudes of ΔH_d in deuterated proteins are 18–36% lower than in normal proteins. The heatcapacity change (ΔC_p) in protein unfolding is essentially the same (2 kcal/mol/K) for deuterated and normal proteins within the experimental error. At close to physiological temperature (27°C), the differences in thermodynamic functions in the native and denatured states are much higher in normal proteins than in deuterated proteins. CD was employed to evaluate both the secondary structures and urea denaturation of these two types of proteins. In P. luridum, deuterated protein is about 8% higher in α-helix content; in P. calothricoides it is not significantly higher. Deuterated proteins are less resistant to the denaturant urea than are normal proteins: the denaturant concentration at the midpoint of the denaturation curve is 0.6–1.2 mol/L lower in the deuterated proteins. The apparent free energies of unfolding of deuterated proteins at zero denaturant concentration are 1.1–1.5 kcal/mol less than for normal proteins.     

北亚热带3种典型森林群落对降水中氮、磷、硫的截留及分配特征

森林群落在净化空气、截留沉降污染物、改善地表水质等方面具有重要作用。本研究以北亚热带地区3种典型森林群落（毛竹林、杉木林、青冈阔叶林）为研究对象,通过分析沉降污染物（NH₄⁺-N、NO₃^--N、NO₂^--N、TP和SO₄^2-）在大气降水、林内穿透雨、树干茎流、枯透水和地表径流中的浓度和通量变化特征,探讨不同森林群落对氮、磷、硫的截留净化作用和分配特征。结果表明,该区域大气降水中NH₄⁺-N、NO₃^--N、NO₂^--N、TP和SO₄^2-年均浓度分别为1.06、0.61、0.04、0.07、1.84 mg/L,其年均pH为5.88;各森林群落林冠层能够调升降雨的pH且全年稳定,对TP和NH₄⁺-N均有吸附作用,截留率分别为79.09%-84.68%和30.88%-69.36%;而枯落物层则是林下氮、磷、硫的主要释放源,对NH₄⁺-N、NO₃^--N、TP和SO₄^2-均具有淋溶作用;此外,由地表径流（输出）与大气降水（输入）的对比分析可知,各林地对沉降污染物中氮、磷、硫的截留率均超过98%;3种森林群落对沉降污染物中氮、磷、硫的截留能力依次为：青冈阔叶林 > 毛竹林 > 杉木林,阔叶林对沉降污染物的净化能力要高于毛竹林及针叶的杉木林。     

The production and utilization of nitric oxide by a new,denitrifying strain of Pseudomonas aeruginosa

When a new strain of Pseudomonas aeruginosa was grown aerobically and then transferred to anaerobic conditions, cells reduced NO ₃ ^– quantitatively to NO ₂ ^– in NO ₃ ^– -respiration. In the absence of nitrate, NO ₂ ^– was immediately reduced to NO or N₂O but not to N₂ indicating that NO ₂ ^– -reductase but not N₂O-reductase was active. The formation of the products NO or N₂O depended on the pH in the medium and the concentration of NO ₂ ^– present. When P. aeruginosa was grown anaerobically for at least three davs N₂O-reductase was also active. Such cells reduced NO to N₂ via N₂O. The new strain generated a H⁺-gradient and grew by reducing N₂O to N₂ but not by converting NO to N₂O. For comparison, Azospirillum brasilense Sp7 showed the same pattern of NO-reduction. In contrast, Paracoccus denitrificans formed 3.5 H⁺/NO during the reduction of NO to N₂O in oxidant pulse experiments but could not grow in the presence of NO. Thus the NO-reduction pattern in P. denitrificans on one side and P. aeruginosa and A. brasilense on the other was very different. The mechanistic implications of such differences are discussed.     

Evidence from triton X-100 polyacrylamide gel electrophoresis that histone f2a2, not f2b, is phosphorylated in Chinese hamster cells

Chinese hamster cells (line CHO) were labeled in suspension culture with ³H-lysine and ³²PH₃PO₄. Preparative polyacrylamide gel electrophoresis of histone fractions from these cells was performed in the presence of 8 $\text{M}$ urea, 6 mM Triton X-100, and 0.9 N acetic acid. This method separates histones f2a2 and f2b by a Large distance, thus making it possible to resolve the controversy concerning which histone -- f2b or f2a2 -- is phosphorylated. It is shown that the two most highly phosphorylated histones in interphase CHO cells are f1 and f2a2. Histones f2b and f3 are shown to contain no significant incorporation of ³²PO₄ in interphase cells, while histone f2a1 contains a small but detectable amount of incorporated ³²PO₄. Binding of the nonionic detergent Triton X-100 to hydrophobic centers appears to be greatest for histones f2a2 and f3, thus significantly retarding the mobility of these two histones during electrophoresis.     

Effect of Linker Length on Avidin Binding to Biotinylated Gramicidin A

Biotinylated gramicidins are an important component of the AMBRI^® “ion channel switch™” biosensor. These gramicidin A (gA) analogues have a biotin attached to the C-terminus of gA via a number of aminocaproyl linker groups (X). The structure of gA5XB has been determined in deuterated sodium dodecyl sulfate micelles and is similar to native gA and other modified gA analogues. The biotin and aminocaproyl groups were mobile and located in the aqueous phase and when avidin was added, NMR and MS studies showed that gA5XB bound more effectively to avidin than gA2XB. The length and flexibility of the linker appears to be important for biotin–avidin binding and, in the AMBRI^® biosensor, gA5XB is a more effective gated ion channel than gA2XB. The conformation and dynamics of the aminocaproyl linker groups were investigated using ²H solid-state NMR. Deuterated aminocaproyl linkers were coupled to gA and incorporated into oriented bilayers in order to analyse the order and dynamics of the aminocaproyl linker. The small ²H splittings and the T ₁ relaxation times indicated that the aminocaproyl linker is undergoing fast rotation in phospholipid bilayers. Native d ₄ -gA as well as d ₄ -gA2XB, where the ethanolamine has been deuterated, were also incorporated into oriented bilayers. Solid-state ²H NMR data showed that the addition of the linker group restricted the mobility of the ethanolamine. However, these modifications to the C-terminus of gA did not interfere with ion channel function and clarify how the biotinylated gA analogues perform in the lipid bilayer as part of the AMBRI^® biosensor.Australian Peptide Conference Issue.