Notice

¹⁷O NMR studies of ¹⁷O enriched water solutions containing superoxide dismutase have been performed between pH 7.5 and 11.7. Whereas T₁ measurements do not reveal any interaction between ¹⁷O and the paramagnetic copper center, the linewidth results appreciably increased with increasing pH with an apparent pK_a of 11.3. Comparison with ¹H NMR relaxation studies allows to interpret the present data as due to binding to the copper ion of an OH^? anion at high pH. The binding position should be of “equatorial” type, not involving the binding position of the coordinated water.     

Synthesis and iron-binding properties of quinolobactin,a siderophore from a pyoverdine-deficient <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis>

Quinolobactin is a new siderophore produced by a pyoverdine deficient mutant of Pseudomonas fluorescens. A simple and efficient synthesis of quinolobactin is described, starting from xanthurenic acid. The protonation constants of quinolobactin were determined by potentiometric titrations as pK_a2=5.50 ± 0.07, pK_a1=10.30 ± 0.05. The equilibria of the metal complexes were studied by means of spectrophotometric and potentiometric titrations. The overall stability constants of the quinolobactin-Fe^III complexes was found to be log ₁₁₁=18.60 ± 0.10, log ₁₂₁=32.60 ± 0.20, log ₁₂₀=28.20 ± 0.25 resulting in a pFe^III value of 18.2 at pH 7.4. The UV-visible spectral parameters of the [FeL₂] are in agreement with a complex containing two ligands coordinated to one Fe³⁺ cation through the oxygen and nitrogen quinoline atoms.     

Phosphophoryn,a biomineralization template protein: pH-dependent protein folding experiments

The protein folding behavior of a polyelectrolyte protein, bovine dentine phosphophoryn (BDPP), in the pH range of 1.82–11.0 has been investigated. One- and two-dimensional nmr spectroscopy has been utilized to obtain proton spin assignments for amino acid residues in D₂O and in H₂O. One-dimensional ³¹P-nmr experiments verify the existence of three separate classes of O-phosphoserine (PSer) resonances in BDPP (α, β, χ), representing three distinct PSer residue populations at pH 6.94. By means of pH titration and ¹H-nmr, five populations of Asp residues can be identified. Three of these populations exhibit secondary inflection points on their pH titration curves that correspond to an observed pK_a of 6.17–6.95. The presence or absence of secondary inflection points for Asp populations and the ³¹P-nmr chemical shift dispersion for the three PSer residue populations indicate that BDPP may be comprised of homologous (Asp-Asp)_n. (PSer-PSer)_n, and heterologous (PSer-Asp)_n sequences arranged into polyelectrolyte cluster regions. The pH titration also revealed that certain populations of Ser, Gly, and Pro residues in BDPP exhibit pH-dependent resonance frequency shifts. The “apparent” pK, for the transition points of these frequency shifts corresponds to either the pK of Pser monophosphatc ester and/or the pK_a of Asp COOH group of BDPP polyelectrolyte regions. On the basis of these transition points, we can assign four types of Ser, Gly, or Pro-containing “intervening” regions in BDPP, based on their sensitivity to protonation and deprotonation events occurring at (Asp)_n, (PSer)_n, or (PSer-Asp)_n anionic cluster regions that flank the intervening regions. Our ¹H-ninr experiments also reveal that BDPP assumes a folded conformation at low pH. As the pH increases, this conformation undergoes several unfolding transitions as the BDPP molecule assumes more open conformations in response to increased electrostatic repulsion between polyelectrolyte anionic regions in the protein. These folding-unfolding transitions are mediated by the intervening regions, which act as “hinges” to allow the polyelectrolyte regions to fold relative to one another. © 1994 John Wiley & Sons, Inc.     

Random-coil chemical shifts of phosphorylated amino acids

The ¹H, ¹³C, ¹⁵N and ³¹ P random-coil chemical shifts and phosphate pK_a values of the phosphorylated amino acids pSer, pThr and pTyr in the protected peptide Ac-Gly-Gly-X-Gly-Gly-NH₂ have been obtained in water at 25°C over the pH range 2 to 9. Analysis of ROESY spectra indicates that the peptides are unstructured. Phosphorylation induces changes in random-coil chemical shifts, some of which are comparable to those caused by secondary structure formation, and are therefore significant in structural analyses based on the chemical shift.     

Chemical modification ofPseudomonas fluorescens malonyl-CoA synthetase by diethylpyrocarbonate: Kinetic evidence for an essential histidyl residue on alpha subunit

Malonyl-CoA synthetase fromPseudomonas fluorescens was inactivated by diethylpyrocarbonate (DEP) with the second-order rate constant of 775 M^–1 min^–1 atpH 7.0, 25°C, showing a concomitant increase in absorbance at 242 nm due to the formation of N-carbethoxyhistidyl derivatives. The inactivated enzyme at low concentration of DEP (<0.2 mM) could be completely reactivated by hydroxylamine but not completely reactivated at high concentration (>0.5 mM), indicating that there may be another functional group modified by DEP. Complete inactivation of malonyl-CoA synthetase required the modification of seven residues per molecule of enzyme; however, only one is calculated to be essential for enzyme activity by a statistical analysis of the residual enzyme activity.pH dependence of inactivation indicated the involvement of a residue with apK _a of 6.7, which is closely related to that of histidyl residue of proteins. Whena subunit treated with DEP was mixed with subunits complex, the enzyme activity completely disappeared, whereas when subunit complex treated with the reagent was mixed witha subunit, the activity remained. Inactivation of the enzyme by the reagent was protected by the presence of malonate and ATP. These results indicate that a catalytically essential histidyl residue is located at or near the malonate and ATP binding region ona subunit of the enzyme.     

Delayed fluorescence study on P*QA→ P+QA − charge separation energetics linked to protons and salt in reaction centers from Rhodobacter sphaeroides

The energetics of the first stable charge separated state, P⁺Q_A– relative to that of P–Q_A was examined in isolated RC from Rhodobacter sphaeroides by delayed fluorescence. The temperature dependence of the delayed fluorescence indicates that the charge separation is a highly enthalpy-driven process (H = – 818 ± 20 meV at pH 8) and the free energy gap between P–Q_A and P⁺Q_A– drops with increasing pH (40 ± 4 meV between pH 6 and 10). The pH-dependence of the free energy change of the P⁺Q_A– state runs parallel to the (integrated) net proton uptake due to the PQ_A/P⁺Q_A– redox change in a wide pH range and under different ionic conditions. Elevation of the ionic strength increases the delayed fluorescence intensity and decreases the (dark and light) pK_a values as well as the light-induced pK_a changes of the protonatable groups of the protein. The observed dependence of the energetics of P⁺Q_A– on the concentration and composition of mobile ions is discussed in terms of binding and screening of protonatable groups and surface charges as dominant modes of electrostatic interaction between RC and salt.     

Nucleosides,XLIV1 Synthesis,Properties and Biological Activity of Indazole Nucleosides

Abstract

Various new haloindazole-1-β-D-ribofuranosides (10-17,20,21) and a 2-β-D-ribofuranoside (18) have been synthesized by the fusion method and by direct halogenations, respectively. The new nucleosides have been characterized by UV and ¹H NMR spectra as well as pK_a determinations. Indazole ribofuranosides behave in aqueous acid like purine and benzimidazole nucleosides showing the same mechanism of cleavage of the glycosidic bonds. Toxicity studies against various cell populations indicate only little biological activities.     

Inhibition of an outwardly rectifying anion channel by HEPES and related buffers

Summary The effect of pH buffers and related compounds on the conductance of an outwardly rectifying anion channel has been studies using the patch-clamp technique. Single-channel current-voltage relationships were determined in solutions buffered by trace amounts of bicarbonate and in solutions containing N-substituted taurines (HEPES, MES, BES, TES) and glycines (glycylglycine, bicine and tricine), Tris andbis-Tris at millimolar concentrations. HEPES (pK_a=7.55) reduced the conductance of the channel when present on either side of the membrane. Significant inhibition was observed with 0.6mm HEPES on the cytoplasmic side (HEPES _i ) and this effect increased with [HEPES _i ] so that conductance at the reversal potential was diminished 25% with 10mm HEPES _i )and 70% at very high [HEPES _i ]. HEPES _i block was relieved by applying positive voltage but positive currents were not consistent with a Woodhulltype blocking scheme in that calculated dissociation constants and electrical distances depended on HEPES concentration. Results obtained by varying total HEPES _i concentration at constant [HEPES^–] and vice versa suggest both the anionic and zwitterionic (protonated) forms of HEPES inhibit. Structure-activity studies with related compounds indicate the sulfonate group and heterocyclic aliphatic groups are both required for inhibition from the cytoplasmic side. TES (pK_a=7.54), substituted glycine buffers (pK_a=8.1–8.4) andbis-Tris (pK_a=6.46) had no measurable effect on conductance and appear suitable for use with this channel.     

An efficient route to substituted 1-silacyclopent-2-enes and 1-silacyclohex-2-enes via consecutive 1,2-hydroboration and 1,1-organoboration

The reaction of alkyn-1-yl(vinyl)silanes R₂Si(CCR¹)CHCH₂ [R = Me (1), Ph (2); R¹ = ^tBu (a), Ph (b), SiMe₃ (c)] with 9-borabicyclo[3.3.1]nonane in a 1:1 ratio affords the 1-silacyclopent-2-ene derivatives 4a-c (R = Me) and 5a-c (R = Ph) as a result of selective intermolecular 1,2-hydroboration of the vinyl group, followed by intramolecular 1,1-organoboration of the alkynyl substituent. The analogous reaction sequence converts the alkyn-1-yl(allyl)dimethylsilanes 3a,c into the 1-silacyclohex-2-ene derivatives 7a,c. All reactions were monitored by ²⁹Si NMR spectroscopy and the structural assignment of the final products was based on multinuclear magnetic resonance data (¹H, ¹¹B, ¹³C and ²⁹Si NMR). The molecular structure of 6a was determined by X-ray analysis.     

Large contributions of coupled protonation equilibria to the observed enthalpy and heat capacity changes for ssDNA binding to Escherichia coli SSB protein

Many macromolecular interactions, including protein‐nucleic acid interactions, are accompanied by a substantial negative heat capacity change, the molecular origins of which have generated substantial interest. We have shown previously that temperature‐dependent unstacking of the bases within oligo(dA) upon binding to the Escherichia coli SSB tetramer dominates the binding enthalpy, ΔH_obs, and accounts for as much as a half of the observed heat capacity change, ΔC_p. However, there is still a substantial ΔC_p associated with SSB binding to ssDNA, such as oligo(dT), that does not undergo substantial base stacking. In an attempt to determine the origins of this heat capacity change, we have examined by isothermal titration calorimetry (ITC) the equilibrium binding of dT(pT)₃₄ to SSB over a broad pH range (pH 5.0–10.0) at 0.02 M, 0.2 M NaCl and 1 M NaCl (25°C), and as a function of temperature at pH 8.1. A net protonation of the SSB protein occurs upon dT(pT)₃₄ binding over this entire pH range, with contributions from at least three sets of protonation sites (pK_a1 = 5.9–6.6, pK_a2 = 8.2–8.4, and pK_a3 = 10.2–10.3) and these protonation equilibria contribute substantially to the observed ΔH and ΔC_p for the SSB‐dT(pT)₃₄ interaction. The contribution of this coupled protonation (∼ −260 to −320 cal mol⁻¹ K⁻¹) accounts for as much as half of the total ΔC_p. The values of the “intrinsic” ΔC_p,0 range from −210 ± 33 cal mol⁻¹ °K⁻¹ to −237 ± 36 cal mol⁻¹K⁻¹, independent of [NaCl]. These results indicate that the coupling of a temperature‐dependent protonation equilibria to a macromolecular interaction can result in a large negative ΔC_p, and this finding needs to be considered in interpretations of the molecular origins of heat capacity changes associated with ligand‐macromolecular interactions, as well as protein folding. Proteins 2000;Suppl 4:8–22. © 2000 Wiley‐Liss, Inc.     

Intramolecular domain-domain interactions and intermolecular self-association in bovine prothrombin. A potentiometric and laser light-scattering study

The interaction of bovine prothrombin with Ca²⁺ and Mg²⁺ ions was investigated by following H⁺ release as a function of metal ion concentration at pH 6 and pH 7.4 at high and low ionic strength. Prothrombin Ca²⁺ and Mg²⁺ binding is characterized by high- and low-affinity sites. M²⁺ binding at these sites is associated with intramolecular conformational changes and also with intermolecular self-association. The pH dependence of H⁺ release by M²⁺ is bell shaped and consistent with controlling pK_a values of 4.8 and 6.5. At pH 6 and low ionic strength, both Ca²⁺ and Mg²⁺ titrations following H⁺ release clearly show independent low- and high-affinity binding sites. Laser light scattering reveals that at pH 7.4 and low ionic strength, and at pH 6.0 and high ionic strength, the prothrombin molecular weight is between 73 and 98 kD. At pH 7.4 and high ionic strength, prothrombin is monomeric in the absence of metal ions, but appears to dimerize in the presence of M²⁺. At pH 6.0 and low ionic strength prothrombin exists as a dimer in the absence of metal ions and is tetrameric in the presence of Ca²⁺ and remains dimeric in the presence of Mg²⁺. These results and those for metal ion-dependent H⁺ release indicate that H⁺ release occurs concomitantly with association processes involving prothrombin.Abbreviations GLA -carboxyglutamic acid; fragment 1. amino terminal residues 1–156 of bovine prothrombin - MES 2-(N-morpholino) ethanesulfonic acid - MOPS 3-(N-morpholino) propanesulfonic acid - PS/PC phosphatidylserine/phosphatidylcholine vesicles - ionic strength     

Diastereoselective synthesis of a “chiral-at-Ru” secondary phosphine complex

Synthesis of the half-sandwich ruthenium complex [RuCl(η⁵-indenyl){P(Bu^t)(Ph)H}(PPh₃)], 2, containing an unsymmetrically-substituted secondary phosphine, is described. A 60:40 kinetic distribution of the resulting diastereomers 2a and 2b shifts in solution at room temperature to give predominantly 2a. The relative stereochemistries at ruthenium and the secondary phosphine in each diastereomer have been assigned based on ¹H NOESY NMR and crystallographic data.     

The amphiphilic peptide adenoregulin enhances agonist binding to A1-adenosine receptors and [35S]GTPγS to brain membranes

Summary 1. Adenoregulin is an amphilic peptide isolated from skin mucus of the tree frog,Phyllomedusa bicolor. Synthetic adenoregulin enhanced the binding of agonists to several G-protein-coupled receptors in rat brain membranes.2. The maximal enhancement of agonist binding, and in parentheses, the concentration of adenoregulin affording maximal enhancement were as follows: 60% (20 µM) for A₁-adenosine receptors, 30% (100 µM) for A_2a-adenosine receptors, 20% (2 µM) for ₂-adrenergic receptors, and 30% (100 µM) for 5HT_1A receptors. High affinity agonist binding for A_1-, _2-, and 5HT_1A-receptors was virtually abolished by GTPS in the presence of adenoregulin, but was only partially abolished in its absence. Magnesium ions increased the binding of agonists to receptors and reduced the enhancement elicited by adenoregulin.3. The effect of adenoregulin on binding of N⁶-cyclohexyladenosine ([³H]CHA) to A₁-receptors was relatively slow and was irreversible. Adenoregulin increased the B_max value for [³H]CHA binding sites, and the proportion of high affinity states, and slowed the rate of [³H]CHA dissociation. Binding of the A₁-selective antagonist, [³H]DPCPX, was maximally enhanced by only 13% at 2 µM adenoregulin. Basal and A₁-adenosine receptor-stimulated binding of [³⁵S]GTPS were maximally enhanced 45% and 23%, respectively, by 50 µM adenoregulin. In CHAPS-solubilized membranes from rat cortex, the binding of both [³H]CHA and [³H]DPCPX were enhanced by adenoregulin. Binding of [³H]CHA to membranes from DDT₁ MF-2 cells was maximally enhanced 17% at 20 µM adenoregulin. In intact DDT₁ MF-2 cells, 20 µM adenoregulin did not potentiate the inhibition of cyclic AMP accumulation mediatedvia the adenosine A₁ receptor.4. It is proposed that adenoregulin enhances agonist binding through a mechanism involving enhancement of guanyl nucleotide exchange at G-proteins, resulting in a conversion of receptors into a high affinity state complexed with guanyl nucleotide-free G-protein.     

Na+ and H+ dependent Mn2+ binding to phosphatidylserine vesicles as a test of the Gouy-Chapman-Stern theory

Summary Mn²⁺ binding to phosphatidylserine (PS) vesicles was measured by EPR as a function of [Na⁺] and pH. At nearly physiological monovalent salt concentration the apparent Mn²⁺ affinity (K _a) increased monitonically over the pH range 5.7–8.35, withK _a roughly [H⁺]^–1 above pH 7.3. It was found, moreover, thatK _a fell off more rapidly with added NaCl at pH 6.1 than at pH 7.87. Qualitatively, these results are consistent with two types of Mn²⁺-PS binding: (i) simple adsorption and (ii) adsorption with the release of an amino proton from PS. The existence of Mn²⁺-induced H⁺ displacement from PS was verified through titration measurements, employing a pH electrode.When H⁺ displacement is taken into account, the variation inK _a with [Na⁺] observed at pH 6.1 is found to be in reasonably good agreement with that expected from the Gouy-Chapman-Stern theory of ionic binding to charged surfaces.     

Gas Exchange of Carrot Leaves in Response to Elevated CO2 Concentration

Short-term responses of four carrot (Daucus carota) cultivars: Cascade, Caro Choice (CC), Oranza, and Red Core Chantenay (RCC) to CO₂ concentrations (C _a) were studied in a controlled environment. Leaf net photosynthetic rate (P _N), intercellular CO₂ (C _i), stomatal conductance (g _s), and transpiration rate (E) were measured at C _a from 50 to 1 050 mol mol^–1. The cultivars responded similarly to C _a and did not differ in all the variables measured. The P _N increased with C _a until saturation at 650 mol mol^–1 (C _i= 350–400 mol mol^–1), thereafter P _N increased slightly. On average, increasing C _a from 350 to 650 and from 350 to 1 050 mol mol^–1 increased P _N by 43 and 52 %, respectively. The P _N vs. C _i curves were fitted to a non-rectangular hyperbola model. The cultivars did not differ in the parameters estimated from the model. Carboxylation efficiencies ranged from 68 to 91 mol m^–2 s^–1 and maximum P _N were 15.50, 13.52, 13.31, and 14.96 mol m^–2 s^–1 for Cascade, CC, Oranza, and RCC, respectively. Dark respiration rate varied from 2.80 mol m^–2 s^–1 for Oranza to 3.96 mol m^–2 s^–1 for Cascade and the CO₂ compensation concentration was between 42 and 46 mol mol^–1. The g _s and E increased to a peak at C _a= 350 mol mol^–1 and then decreased by 17 and 15 %, respectively when C _a was increased to 650 mol mol^–1. An increase from 350 to 1 050 mol mol^–1 reduced g _s and E by 53 and 47 %, respectively. Changes in g _s and P _N maintained the C _i:C _a ratio. The water use efficiency increased linearly with C _a due to increases in P _N in addition to the decline in E at high C _a. Hence CO₂ enrichment increases P _N and decreases g _s, and can improve carrot productivity and water conservation.     

Neutral mixed-ligand complexes of 2,2′-bipyridinedichloroplatinum(II) with dianionic aromatic chelating ligands as potential photosensitizers

The electronic spectra of NCS^? and I^? adducts of cobalt(II) human carbonic anhydrase I are pH dependent at pH values below 7. The pK_a of such equilibrium is dependent on the anion concentration and varies between 4.6 and 6.6. The ¹H NMR spectra show that the three histidine residues are bound to the metal ion over the entire pH range investigated. It is supposed that a Glu residue triggers the change in stereochemistry around the metal ion. It is possible that such a Glu residue is Glu 106 present in the active cavity.     

Biogeochemical Properties of Bacteriogenic Iron Oxides

Bacteriogenic iron oxides (BIOS) are composite materials that consist of intact and partly degraded remains of bacterial cells intermixed with variable amounts of poorly ordered hydrous ferric oxide (HFO) minerals. They form in response to chemical or bacterial oxidation of Fe²⁺, which gives rise to Fe³⁺. Once formed, Fe³⁺ tends to undergo hydrolysis to precipitate in association with bacterial cells. In acidic systems where the chemical oxidation of Fe²⁺ is slow, bacteria are capable of accelerating the reaction by several orders of magnitude. At circumneutral pH, the chemical oxidation of Fe²⁺ is fast. This requires Fe²⁺ oxidizing bacteria to exploit steep redox gradients where low pO₂ slows the abiotic reaction enough to allow the bacteria to compete kinetically. Because of their reactive surface properties, BIOS behave as potent sorbents of dissolved metal ions. Strong enrichments of Al, Cu, Cr, Mn, Sr, and Zn in the solid versus aqueous phase (log 10 K_d values range from 1.9 to 4.2) are common; however, the metal sorption properties of BIOS are not additive owing to surface chemical interactions between the constituent HFO and bacteria. These interactions have been investigated using acid-base tritrations, which show that the concentration of high pK_a sites is reduced in BIOS compared to HFO. At the same time, hydroxylamine insoluble material (i.e., residual bacterial fraction) is enriched in low pK_a sites relative to both BIOS and HFO. These differences indicate that low pK_a or acidic sites associated with bacteria in BIOS interact specifically with high pK_a or basic sites on intermixed HFO.     

Macrocyclic dinuclear gold(I) and silver(I) NHCs complexes

The ligands bis-(imidazolium) hexafluorophosphate (Himy = -C₃N₂H₃-, imidazolium; R = 1-naphthylmethylene, 1a; 9-anthracenylmethylene, 1b) with an oxoether chain were easily prepared by the reaction of substituted imidazole with the diglycol diiodide, followed by exchange of anions with . 1a and 1b reacted with Ag₂O in DMSO or CH₃CN to yield [2 + 2] dinuclear Ag(I) NHCs macrocyclic complexes 2a and 2b, which showed much different conformation in solid corresponding to the R- substituent. Carbene transmetalation reactions of 2a-b with Au(SMe₂)Cl give dinuclear Au(I) analogs 3a and 3b. The new NHCs complexes were characterized by elemental analyses, ¹H NMR, ¹³C NMR and the structures of 2a-b and 3a were confirmed by X-ray diffraction determination.     

Benchmarking pK<Subscript>a</Subscript> prediction methods for Lys115 in acetoacetate decarboxylase

Three different pK_a prediction methods were used to calculate the pK_a of Lys115 in acetoacetate decarboxylase (AADase): the empirical method PROPKA, the multiconformation continuum electrostatics (MCCE) method, and the molecular dynamics/thermodynamic integration (MD/TI) method with implicit solvent. As expected, accurate pK_a prediction of Lys115 depends on the protonation patterns of other ionizable groups, especially the nearby Glu76. However, since the prediction methods do not explicitly sample the protonation patterns of nearby residues, this must be done manually. When Glu76 is deprotonated, all three methods give an incorrect pK_a value for Lys115. If protonated, Glu76 is used in an MD/TI calculation, the pK_a of Lys115 is predicted to be 5.3, which agrees well with the experimental value of 5.9. This result agrees with previous site-directed mutagenesis studies, where the mutation of Glu76 (negative charge when deprotonated) to Gln (neutral) causes no change in K_m, suggesting that Glu76 has no effect on the pK_a shift of Lys115. Thus, we postulate that the pK_a of Glu76 is also shifted so that Glu76 is protonated (neutral) in AADase.

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Graphical abstract Simulated abundances of protonated species as pH is varied

     

Characterization of an artificial dimer of ribonuclease H using 1H NMR spectroscopy

Summary The protein fusion technique was applied in the synthesis of an artificial dimer of ribonuclease H (305 residues). ¹H NMR spectroscopy was used to analyze the structure of this dimer. Spectral profiles and pK_a values of the histidine residues obtained using ¹H NMR indicate that the dimer retains the secondary and tertiary structures of the intact monomer. Selective spin-lattice relaxation measurements suggest that the two monomeric units in the dimer are in tight contact. Furthermore, the 2D ¹H NMR and paramagnetic relaxation filter results show that the two monomers bind together through interactions between the N- and C-terminal sites of the linked regions.