The effect of deuterium oxide on protein turnover in Lemna minor

Lemna minor fronds transferred to a sterile culture medium containing 50% (v/v) deuterium oxide (²H₂O) rapidly undergo a loss of soluble protein with a corresponding increase in free amino acids. The loss of protein is due to two factors: (i) the inhibition of protein synthesis for 4 h followed by a slower rate of synthesis than normal, (ii) a rapid 9–10 fold increase in protein degradation. In plants grown for longer periods (3–6 days) in 50% ²H₂O medium, protein synthesis is inhibited by 20% and the rate constant of degradation is 2–3 times that measured in fronds growing in normal (H₂O containing) complete medium. The initial loss of protein is not due to the breakdown of any specific protein fraction. Investigation of several enzymes indicates that all proteins are catabolised in response to ²H₂O treatment. The implications of these results with regard to the interpretation of density-labelling experiments are discussed.     

Optimum levels of exchangeable protons in perdeuterated proteins for proton detection in MAS solid-state NMR spectroscopy

We present a systematic study of the effect of the level of exchangeable protons on the observed amide proton linewidth obtained in perdeuterated proteins. Decreasing the amount of D₂O employed in the crystallization buffer from 90 to 0%, we observe a fourfold increase in linewidth for both ¹H and ¹⁵N resonances. At the same time, we find a gradual increase in the signal-to-noise ratio (SNR) for ¹H–¹⁵N correlations in dipolar coupling based experiments for H₂O concentrations of up to 40%. Beyond 40%, a significant reduction in SNR is observed. Scalar-coupling based ¹H–¹⁵N correlation experiments yield a nearly constant SNR for samples prepared with ≤30% H₂O. Samples in which more H₂O is employed for crystallization show a significantly reduced NMR intensity. Calculation of the SNR by taking into account the reduction in ¹H T ₁ in samples containing more protons (SNR per unit time), yields a maximum SNR for samples crystallized using 30 and 40% H₂O for scalar and dipolar coupling based experiments, respectively. A sensitivity gain of 3.8 is obtained by increasing the H₂O concentration from 10 to 40% in the CP based experiment, whereas the linewidth only becomes 1.5 times broader. In general, we find that CP is more favorable compared to INEPT based transfer when the number of possible ¹H,¹H interactions increases. At low levels of deuteration (≥60% H₂O in the crystallization buffer), resonances from rigid residues are broadened beyond detection. All experiments are carried out at MAS frequency of 24 kHz employing perdeuterated samples of the chicken α-spectrin SH3 domain.     

Detection of dynamic water molecules in a microcrystalline sample of the SH3 domain of α-spectrin by MAS solid-state NMR

Water molecules are a major determinant of protein stability and are important for understanding protein–protein interactions. We present two experiments which allow to measure first the effective T₂ decay rate of individual amide proton, and second the magnetization build-up rates for a selective transfer from H₂O to H^N using spin diffusion as a mixing element. The experiments are demonstrated for a uniformly ²H, ¹⁵N labeled sample of a microcrystalline SH3 domain in which exchangeable deuterons were back-substituted with protons. In order to evaluate the NMR experimental data, as X-ray structure of the protein was determined using the same crystallization protocol as for the solid-state NMR sample. The NMR experimental data are correlated with the dipolar couplings calculated from H₂O–H^N distances which were extracted from the X-ray structure of the protein. We find that the H^N T₂ decay rates and H₂O–H^N build-up rates are sensitive to distance and dynamics of the detected water molecules with respect to the protein. We show that qualitative information about localization and dynamics of internal water molecules can be obtained in the solid-state by interpretation of the spin dynamics of a reporter amide proton.     

Dynamics of <Superscript>18</Superscript>O Incorporation from H <Subscript>2</Subscript><Superscript>18</Superscript>O into Soil Microbial DNA

Heavy water (H₂¹⁸O) has been used to label DNA of soil microorganisms in stable isotope probing experiments, yet no measurements have been reported for the ¹⁸O content of DNA from soil incubated with heavy water. Here we present the first measurements of atom% ¹⁸O for DNA extracted from soil incubated with the addition of H₂¹⁸O. Four experiments were conducted to test how the atom% ¹⁸O of DNA, extracted from Ponderosa Pine forest soil incubated with heavy water, was affected by the following variables: (1) time, (2) nutrients, (3) soil moisture, and (4) atom% ¹⁸O of added H₂O. In the time series experiment, the atom% ¹⁸O of DNA increased linearly (R ² = 0.994, p < 0.01) over the first 72 h of incubation. In the nutrient addition experiment, there was a positive correlation (R ² = 0.991, p = 0.006) between the log₁₀ of the amount of tryptic soy broth, a complex nutrient broth, added to soil and the log₁₀ of the atom% ¹⁸O of DNA. For the experiment where soil moisture was manipulated, the atom% ¹⁸O of DNA increased with higher soil moisture until soil moisture reached 30%, above which ¹⁸O enrichment of DNA declined as soils became more saturated. When the atom% ¹⁸O for H₂O added was varied, there was a positive linear relationship between the atom% ¹⁸O of the added water and the atom% ¹⁸O of the DNA. Results indicate that quantification of ¹⁸O incorporated into DNA from H₂¹⁸O has potential to be used as a proxy for microbial growth in soil.     

Site-specific solvent exposure analysis of a membrane protein using unnatural amino acids and F nuclear magnetic resonance

Membrane proteins play an essential role in cellular metabolism, transportation and signal transduction across cell membranes. The scarcity of membrane protein structures has thus far prevented a full understanding of their molecular mechanisms. Preliminary topology studies and residue solvent exposure analysis have the potential to provide valuable information on membrane proteins of unknown structure. Here, a ¹⁹F-containing unnatural amino acid (trimethylfluoro-phenylalanine, tfmF) was applied to accomplish site-specific ¹⁹F spin incorporation at different sites in diacylglycerol kinase (DAGK, an Escherichia coli membrane protein) for site-specific solvent exposure analysis. Due to isotope effect on ¹⁹F spins, a standard curve for ¹⁹F-tfmF chemical shifts was drawn for varying solvent H₂O/D₂O ratios. Further site-specific ¹⁹F solvent isotope shift analysis was conducted for DAGK to distinguish residues in water-soluble loops, interfacial areas or hydrophobic membrane regions. This site-specific solvent exposure analysis method could be applied for further topological analysis of other membrane proteins.     

The effects of water-stress on leaf H2 18O enrichment

Summary Water-stress experiments withPhaseolus vulgaris L. were undertaken to determine the transpiration rate dependency of the naturally occurring leaf H₂ ¹⁸O fractionation process. Water-stress leaf H₂ ¹⁸O levels were observed to be unexpectedly higher than controls. Speculations on the cause of this phenomenon are discussed. Since transpiration rate variations should theoretically affect only the rate and not the extent of leaf H₂ ¹⁸O fractionation, the respective time courses for water-stressed and control leaf H₂ ¹⁸O accumulations were compared. Water-stressed leaves displayed a slower rate of isotopic enrichment relative to controls, as was predicted from their reduced transpiration rates. In an absolute sense, however, both control and water-stress leaf H₂ ¹⁸O fractionation rates were markedly greater than projected values from the existing model. Consequently, transpiration rates cannot be derived accurately at present from the observed rates of leaf H₂ ¹⁸O discrimination. Several modifications of the theory are also considered.     

A Novel Cytotoxic Cerium Complex: Aquatrichloridobis(1,10‐phenanthroline)cerium(III) (KP776). Synthesis,Characterization, Behavior in H2O,Binding towards Biomolecules,and Antiproliferative Activity

The lanthanide complex aquatrichloridobis(1,10‐phenanthroline)cerium(III) [Ce(phen)₂(H₂O)Cl₃] (KP776) was fully characterized by elemental analysis, IR‐, and ¹H‐ and ¹³C‐NMR spectroscopy, as well as TG/DTA measurements, and its behavior in H₂O, important for the application as a chemotherapeutic, was studied. In addition, the binding of KP776 to nucleotides and single serum proteins was investigated by capillary electrophoresis, whereas binding to proteins in human plasma was observed by ICP‐MS. The compound shows promising anticancer properties in vitro: proliferation of human cancer cell lines is strongly inhibited with IC₅₀ values in the very low micromolar range.     

The thermal stability of turnip yellow mosaic virus under hydrostatic pressure

The thermal stability of an isometric plant virus, Turnip Yellow Mosaic Virus (TYMV), has been investigated at low and high hydrostatic pressure, using small angle neutron scattering. Contrast variation allowed us to separately observe the structural changes of the protein capsid and the RNA core. The experiments were performed in 0.05M Tris buffer at pD = 8.0 and in 0.05M bis-Tris buffer at pD = 6.0 containing different H₂O/D₂O mixtures (40% and 70% D₂O). It was found that hydrostatic pressure enhances the stability of TYMV. The thermally induced uncoating of RNA as well as structural transitions of the protein capsid are shifted to higher temperature upon increasing the pressure from 5 × 10⁶ Pa to 2 × 10⁸ Pa.     

Untersuchungen zum Wirkungsmechanismus von Indolyl-3-Essigsäure mit Hilfe von schwerem Wasser

Summary The auxin-induced cell elongation and the formation of indoleacetyl-aspartic acid (IAAsp) of pea epicotyl sections and Agrostemma hypocotyl sections are inhibited by heavy water. The formation of IAAsp requires a specific enzyme. The lack of IAAsp in D₂O-treated plant tissues may be due to an influence of D₂O on the induction or on the synthesis of that enzyme. Treatment of plant sections with synthetic IAAsp has no effect on the growth of the sections in D₂O. Indole-3-acetic acid (IAA) increases the incorporation of ³²P-orthophosphate into ribosomal and soluble RNA of pea epicotyl sections in H₂O but not in D₂O. The synthesis of ribosomal RNA is decreased by heavy water.The effects of IAA and D₂O on the soluble proteins of pea sections have been studied by PAA-gel electrophoresis. D₂O does not change the pattern of protein bands in comparison with the H₂O-control, but prevents the probably IAA-induced alteration of the Rf-value of one protein band on the pherogram. It is assumed that the inhibition of auxin-induced reactions in the D₂O-medium is due to the stabilizing effect of heavy water on allosteric proteins. The results of this work support the hypothesis that IAA acts as allosteric effector.     

1H(C) and 1H(N) total NOE correlations in a single 3D NMR experiment. 15N and 13C time-sharing in t1 and t2 dimensions for simultaneous data acquisition

Simultaneous data acquisition in time-sharing (TS) multi-dimensional NMR experiments has been shown an effective means to reduce experimental time, and thus to accelerate structure determination of proteins. This has been accomplished by spin evolution time-sharing of the X and Y heteronuclei, such as ¹⁵N and ¹³C, in one of the time dimensions. In this work, we report a new 3D TS experiment, which allows simultaneous ¹³C and ¹⁵N spin labeling coherence in both t ₁ and t ₂ dimensions to give four NOESY spectra in a single 3D experiment. These spectra represent total NOE correlations between ¹H_N and ¹H_C resonances. This strategy of double time-sharing (2TS) results in an overall four-fold reduction in experimental time compared with its conventional counterpart. This 3D 2TS CN-CN-H HSQC-NOESY-HSQC pulse sequence also demonstrates improvements in water suppression, ¹⁵N spectral resolution and sensitivity, which were developed based on 2D TS CN-H HSQC and 3D TS H-CN-H NOESY-HSQC experiments. Combining the 3D TS and the 3D 2TS NOESY experiments, NOE assignment ambiguities and errors are considerably reduced. These results will be useful for rapid protein structure determination to complement the effort of discerning the functions of diverse genomic proteins.     

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.     

Dependence of co-operativity in the reaction of haemoglobin with oxygen on deuterium oxide concentration and temperature

Concentrated deionized solutions of haemoglobin in water were diluted with unbuffered pure ²H₂O and left to stand for 15 to 70 hours. Oxygen equilibrium curves were measured at different temperatures and concentrations of ²H₂O. In 98.5% ²H₂O Hill's constant retained its normal value at temperatures below 11 °C, but was reduced by 0.4 above 11 °C. This temperature effect was reversible. Lowering the ²H₂O concentrations raised the transition temperature between the states of low and high co-operativity of the reaction. The shape of the transition curve remained unchanged. Further experiments allowed a phase diagram to be constructed which shows the boundaries between the two states: one of low co-operativity at high concentration of ²H₂O and high temperature, and another of high co-operativity at low values of these variables. Reversibility of the isotope effect even at ²H₂O concentration of 98.5% excludes a purely steric interpretation. Possible dynamic and co-operative interactions between the protein molecule and its surrounding water molecules are discussed.     

Effect of isotope substitution and controlled dehydration on the photoinduced electron transport reactions of quinone acceptors and multiheme cytochrome C in bacterial photosynthetic reaction center

Isotope substitution of H₂O by ²H₂O causes an increase in the rate of dark recombination between photooxidized bacteriochlorophyll (P⁺) and reduced primary quinone acceptor in Rhodobacter sphaeroides reaction centers (RC) at room temperature. The isotopic effect declines upon decreasing the temperature. Dehydration of RC complexes of Ectothiorhodospira shaposhnikovii chromatophores containing multiheme cytochrome c causes a decrease in the efficiency of transfer of a photomobilized electron between the primary and secondary quinone acceptors and from cytochrome to P⁺. In the case of H₂O medium these effects are observed at a lower hydration than in ²H₂O-containing medium. In the E. shaposhnikovii chromatophores subjected to dehydration in H₂O, the rate of electron transfer from the nearest high-potential cytochrome heme to P⁺ is virtually independent of hydration within the P/P₀ range from 0.1 to 0.5. In samples hydrated in ²H₂O this rate is approximately 1.5 times lower than in H₂O. However, the isotopic effect of this reaction disappears upon dehydration. The intramolecular electron transfer between two high-potential hemes of cytochrome c in samples with ²H₂O is inhibited within this range of P/P₀, whereas in RC samples with H₂O there is a trend toward gradual inhibition of the interheme electron transfer with dehydration. The experimental results are discussed in terms of the effects of isotope substitution and dehydration on relaxation processes and charge state of RC on implementation of the reactive states of RC providing electron transfer control.     

Accumulation of small heat shock proteins, including mitochondrial HSP22, induced by oxidative stress and adaptive response in tomato cells

Changes in gene expression, by application of H₂O₂, O₂°^–generating agents (methyl viologen, digitonin) and gamma irradiation to tomato suspension cultures, were investigated and compared to the well-described heat shock response. Two-dimensional gel protein mapping analyses gave the first indication that at least small heat shock proteins (smHSP) accumulated in response to application of H₂O₂ and gamma irradiation, but not to O₂°^– generating agents. While some proteins seemed to be induced specifically by each treatment, only part of the heat shock response was observed. On the basis of Northern hybridization experiments performed with four heterologous cDNA, corresponding to classes I–IV of pea smHSP, it could be concluded that significant amounts of class I and II smHSP mRNA are induced by H₂O₂ and by irradiation. Taken together, these results demonstrate that in plants some HSP genes are inducible by oxidative stresses, as in micro-organisms and other eukaryotic cells. HSP22, the main stress protein that accumulates following H₂O₂ action or gamma irradiation, was also purified. Sequence homology of amino terminal and internal sequences, and immunoreactivity with Chenopodium rubrum mitochondrial smHSP antibody, indicated that the protein belongs to the recently discovered class of plant mitochondrial smHSP. Heat shock or a mild H₂O₂ pretreatment was also shown to lead to plant cell protection against oxidative injury. Therefore, the synthesis of these stress proteins can be considered as an adaptive mechanism in which mitochondrial protection could be essential.     

Further quasi-classical trajectory studies on the C++ H2O reaction

Further trajectory studies on the C⁺ + H₂O reaction have been performed using a potential energy surface described through a finite element method in its p version. In former trajectory studies [Y. Ishikawa, T. Ikegami and R.C. Binning Jr., Direct ab initio molecular dynamics study of C⁺+H₂O: angular distribution of products and distribution of product kinetic energies, Chem. Phys. Lett. 370 (2003), pp. 490–495; J.R. Flores, Quasichemical trajectories on a finite element density functional potential energy surface: the C⁺+H₂O reaction revisited, J. Chem. Phys. 125 (2006), 164309], tunnelling was not taken into account. The present results together with the analysis of the electronic excited states [J.R. Flores and A.B. González, The role of the excited electronic states in the C⁺+H₂O reaction, J. Chem. Phys. 128 (2008), 144310] are useful to interpret the mechanism of the title reaction, which has been the subject of crossed beam experiments [D.M. Sonnenfroh, R.A. Curtiss and J.M. Farrar, Collision complex formation in the reaction of C⁺ with H₂O, J. Chem. Phys. 83 (1985), pp. 3958–3964] and can be considered a prototypical ion–molecule reaction.     

Structure of the O-polysaccharide from the lipopolysaccharide of Providencia alcalifaciens O60

An O-polysaccharide (O-antigen) was isolated by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O60 and studied by sugar and methylation analyses as well as ¹H and ¹³C NMR spectroscopy, including 2D ROESY and ¹H,¹³C HMBC experiments in D₂O and a ROESY experiment in a 9:1 H₂O–D₂O mixture to reveal correlations for NH protons. It was found that the polysaccharide is built up of linear pentasaccharide repeating units containing an amide of d-glucuronic acid with l-serine and has the following structure:The O-antigen studied is structurally and serologically closely related to the O-antigen of Proteus vulgaris O44.     

Solvent isotope effect on the differences in structure and stability between normal and deuterated proteins

Differential scanning microcalorimetry was used to investigate the enthalpy (ΔH_d) and the temperature (t_d) of thermal denaturation of normal (nondeuterated) (H-PC) and deuterated (D-PC) phycocyanins in D₂O solvent. Values of t_d in D-PC are about 5–7°C lower than those in H-PC. The magnitudes of ΔH_d in D-PC are only 21–32% of those in H-PC. During the protein unfolding, the heat-capacity changes (ΔC_p) in D-PC are also lower than those in H-PC. CD was employed to evaluate the secondary structure and the urea denaturation of these proteins in D₂O solvent. These proteins have about the same α-helix content. D-PC is less resistant to the denaturant urea than is H-PC. In general, the apparent free-energy change in the process of protein unfolding at zero denaturant concentration is higher in H-PC than in D-PC. Comparisons of the present results for D₂O solvent with those previously reported for H₂O reveal that solvent isotope effect essentially does not change the α-helix content in H-PC and D-PC. However, D-PC or H-PC has a higher random-coil content in its secondary structure in D₂O than in H₂O. Substitution of H₂O with D₂O as the solvent increases t_d in both D-PC and H-PC, lowers ΔH_d in H-PC, and greatly lowers ΔH_d in D-PC. The deuterium solvent isotope effect does not change ΔC_p in H-PC but lowers ΔC_p in D-PC. In the urea denaturation, the magnitudes of (C_u)_1/2 in H-PC and D-PC are not affected by such a solvent effect, whereas those of ΔG are greatly increased. These results are correlated with the structure and stability of the proteins.     

Copper complexes with bioactive ligands

Biological properties of new copper(II) complexes of 2-methylthionicotinate (2-MeSNic) of composition Cu(2-MeSNic)₂(MeNia)₂·4H₂O (where MeNia isN-methylnicotinamide), Cu(2-MeSNic)₂(Nia)₂·2H₂O (where Nia is nicotinamide) and Cu(2-MeSNic)₂(₂ (where L is isonicotinamide (iNia) or ethyl nicotinate (EtNic)) are reported. Gram⁻-bacteria (Escherichia coli) are more resistant against Cu(II) complexes than Gram⁺-bacteria (Staphylococcus aureus)—significant antistaphylococcal activity was found with Cu(2-MeSNic)₂(MeNia)₂·4H₂O (IC₅₀ 1.3 mmol/L).Caddida parapsilosis was most inhibited by Cu(2-MeSNic)₂·H₂O and Cu(2-MeSNic)₂(MeNia)₂·4H₂O (IC₅₀ 1.4 mmol/L and 1.5 mmol/L, respectively). Biosynthesis of nucleic acids influenced by Cu(2-MeSNic)₂-(Nia)₂·2H₂O indicated by incorporation of¹⁴C-adenine (IC_50(Ade) 0.31 mmol/L) is more sensitive than biosynthesis of proteins indicated by incorporation of¹⁴C-leucine (IC_50(Leu) 9.94 mmol/L). Cu(II) complexes with expressed antimicrobial activity showed no mutagenic activity.     

EPR spectroscopy and catalase activity of manganese-bound DNA-binding protein from nutrient starved cells

DNA-binding proteins from nutrient-starved cells (DPS) protect cells from oxidative stress by removing H₂O₂ and iron. A new class of DPS-like proteins has recently been identified, with DPS-like protein from Sulfolobus solfataricus (SsDPS) being the best characterized to date. SsDPS protects cells from oxidative stress and is upregulated in response to H₂O₂ but also in response to iron depletion. The ferroxidase active site of SsDPS is structurally similar to the active sites of manganese catalase and rat liver arginase. The present work shows that the ferroxidase center in SsDPS binds two Mn²⁺ ions with K _D = (1/K ₁ K ₂)^1/2 = 48(3) μM. The binding constant of the second Mn²⁺ is significantly higher than that of the first, inducing dinuclear Mn(II) cluster formation for all but the lowest concentrations of added Mn²⁺. In competition experiments, equimolar amounts of Fe²⁺ were unable to displace the bound manganese. EPR spectroscopy of the Mn₂ ²⁺ cluster showed signals comparable to those of other characterized dimanganese clusters. The exchange coupling for the cluster was determined, J = −1.4(3) cm⁻¹ (H = −2JS ₁ S ₂), and is within the range expected for a μ_1,1-carboxylato bridge between the manganese ions. Manganese-bound SsDPS showed catalase activity at a rate 10–100 times slower than for manganese catalases. EPR spectra of SsDPS after addition of H₂O₂ showed the appearance of an intermediate in the reaction with H₂O₂.